Temporal determinants of long-term retention of olfactory memory in the cricket Gryllus bimaculatus
1 Research Institute for Electronic Science, Hokkaido University, Sapporo
060-0812, Japan
2 PRESTO, Japan Science and Technology Corporation (JST), Japan
* Author for correspondence (e-mail: makoto{at}ncp8.es.hokudai.ac.jp )
Accepted 5 March 2002
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Summary |
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Key words: differential conditioning, memory, olfaction, insect, cricket, Gryllus bimaculatus, anaesthetic-resistant memory, long-term memory
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Introduction |
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We found in a study using an operant conditioning paradigm that the cricket
Gryllus bimaculatus can learn an olfactory stimulus
(Matsumoto and Mizunami,
2000). Crickets that had been given only one session of training
to associate peppermint odour with a water reward and vanilla odour with
saline solution exhibited a significantly increased preference for peppermint.
Olfactory memory formed by a single training session was retained for at least
24 h, and that formed by three training sessions was retained for at least 7
days.
In the present study, we examined variables that govern olfactory long-term
memory retention to determine the basic properties of olfactory memory
formation in crickets. We changed our previous operant conditioning paradigm
to a classical one because this enabled us to control precisely the timing
between the presentation of the unconditioned stimulus (US) and the
conditioned stimulus (CS), which has a profound influence on the formation and
retention of associative memory in many systems of associative learning
(Carew and Sahley, 1986;
Rescorla, 1988
). We focused on
memory retention 2 h, 1 day and 4 days after training, which represent middle-
to long-term memory retention in some insects (fruit flies Drosophila
melanogaster, Tully et al.,
1994
; honeybees, Gerber et al.,
1998
; Menzel,
1999
), because we wanted to focus our interest on the variables
that determine long-term memory in insects. We examined (i) the effects of
elementary aversive and appetitive conditioning trials and of differential
conditioning trials, (ii) the effects of the interval between the CS and US,
(iii) the effects of the interval between CS/US pairing trials, and (iv) the
effects of anaesthetic treatment with CO2. The results are
discussed with reference to data for honeybees and fruit flies.
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Materials and methods |
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Training and testing
For conditioning, peppermint and vanilla odours were used as the
conditioned stimuli (CS) and water and 20 % sodium chloride solution as the
appetitive and aversive unconditioned stimuli (US), respectively. Because
crickets have an innate preference for vanilla odour over peppermint odour
(see Results and Matsumoto and Mizunami,
2000), conditioning was designed to associate peppermint odour
with the appetitive US (reward) and vanilla odour with the aversive US
(punishment). Hypodermic syringes (1 ml) were used for conditioning
(Fig. 1A). A small filter paper
(3 mmx3 mm) was attached to the needle of the syringe 10 mm from its
tip. The syringe used for the appetitive conditioning trial was filled with
water, and the filter paper attached to the needle was soaked with peppermint
essence. The syringe used for the aversive conditioning trial was filled with
saline solution, and the filter paper was soaked with vanilla essence. For
odour presentation, the filter paper was placed within 1 cm of the cricket's
head. At 2 s after the onset of odour presentation, a drop of water or saline
solution was placed in front of the mouth of the cricket for 2 s. The air in
the beaker was then ventilated for 2 s using a hand-held vacuum cleaner. After
training, each cricket was fed a diet of insect pellets ad libitum in
a beaker until the odour preference test was given.
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The apparatus used for the odour preference test was slightly modified from
that described previously (Matsumoto and
Mizunami, 2000). Briefly, the apparatus consisted of three
chambers, a `test chamber' and two removable `waiting chambers', one of which
was placed at the `waiting position' and the other at the `entrance position'
(Fig. 1B). There was a sliding
door between the waiting chamber at the entrance position and the test
chamber. On the floor of the test chamber, there were two circular holes (H in
Fig. 1B) that connected the
chamber with two of the three sources of odour. Each odour source consisted of
a cylindrical plastic container covered with a fine gauze net. The three
containers were mounted on a rotatable holder. Two odour sources could be
located simultaneously just below the holes at the `offer position' by
rotating the holder.
Before the preference test, a cricket was transferred from the beaker to the waiting chamber at the waiting position and left for approximately 4 min to become accustomed to its surroundings. The waiting chamber was then slid into the entrance position, and the door to the test chamber was opened. When the cricket entered the test chamber, the door was closed and the test started. Two minutes later, the relative positions of the vanilla and peppermint sources were changed by rotating the container holder. An odour source was considered to have been visited when the cricket probed the top net with its mouth. The time spent visiting each odour source was measured cumulatively. If the total time of a visit to either source was less than 20 s, the data were rejected. The preference test lasted for 4 min. At the end of training, the sliding door was opened and the cricket was gently pushed into the waiting chamber and then was transferred to a beaker. Daily testing and training began at 11:00 h and lasted a maximum of 4 h. Following testing, crickets were fed a diet of insect pellets ad libitum until the next retention test. When crickets were subjected to retention tests 1 day and 4 days after training, a few drops of water were given after the 1 day retention test and also on the subsequent day, but not thereafter. Each cricket was used only once.
Conditioning procedure
We used five conditioning procedures. In the first, crickets were trained
to associate peppermint odour with water, which we refer to as the appetitive
conditioning procedure or the P+ (peppermint-rewarded) conditioning trial. In
the second procedure, crickets were trained to associate vanilla odour with
saline solution, which we refer to as the aversive or the V-
(vanilla-punished) conditioning trial. In the third procedure, an appetitive
conditioning trial was followed by an aversive conditioning trial, which we
refer to as (a set of) differential conditioning trials with
peppermint-rewarded and vanilla-punished or P+/V- conditioning trials. In the
fourth procedure, an aversive conditioning trial was followed by the
presentation of peppermint odour alone, without pairing with the US, which we
refer to as (a set of) differential conditioning trials with vanilla-punished
and peppermint-unpunished or V-/P0 conditioning trials. In the
fifth procedure, an appetitive conditioning trial was followed by presentation
of vanilla odour alone, which we refer to as (a set of) differential
conditioning trials with peppermint-rewarded and vanilla-unrewarded or
P+/V0 conditioning trials.
Experiment 1: effects of two-trial conditioning
The effects of two-trial conditioning were tested in three groups of
crickets. The first group was subjected to a set of P+/V- conditioning trials
(group 1, N=29), the second group was subjected to two V-
conditioning trials (group 2, N=15) and the third group was subjected
to two P+ conditioning trials (group 3, N=18). The inter-trial
interval (ITI) was 5 min for all groups. The odour preference of individual
crickets was tested before (PT-0) and 2 h (PT-1), 1 day (PT-2) and 4 days
(PT-3) after training; thus, each cricket underwent three cumulative retention
tests. We have shown that the retention tests, given three times, have no
significant extinction effect (Matsumoto
and Mizunami, 2000).
Experiment 2: effects of four-trial conditioning
The effects of four-trial conditioning were tested in five groups of
crickets. Three groups of crickets were subjected to two sets of P+/V- (group
1, N=45), V-/P0 (group 4, N=22) or
P+/V0 (group 5, N=23) conditioning trials. The other two
groups were subjected to four V- (group 2, N=22) or P+ (group 3,
N=26) conditioning trials. The ITI was 5 min for all groups. The
odour preference of individual crickets was tested before and 2 h, 1 day and 4
days after training.
Experiment 3: effects of different numbers of differential
conditioning trials
To study the effects of different numbers of trials, three groups of
crickets were subjected to one (group 1, N=29), two (group 2,
N=45) or three (group 3, N=24) sets of P+/V- conditioning
trials, and another group was subjected to two P+ conditioning trials followed
by two V- conditioning trials (group 4, N=22). The ITI was 5 min. The
odour preference of individual crickets was tested before and 2 h, 1 day and 4
days after training.
Experiment 4: non-associative controls and the effects of
inter-stimulus interval
Different procedures were used for eight groups of crickets.
Non-associative control procedures were used for three groups. The first group
received CS presentations without pairing with the US in the sequence
peppermint, vanilla, peppermint and then vanilla (CS alone group,
N=26); the second group received US presentations without pairing
with the CS in the sequence water, saline, water and then saline (US alone
group, N=21); and the third group received unpaired presentations of
the CS and US, in the sequence peppermint, water, vanilla, saline, peppermint,
saline, vanilla and then water (CS/US unpaired group, N=27). The
other five groups were subjected to two sets of P+/V- conditioning trials (see
Fig. 1C) with different time
relationships between the US and CS. In the coincident group, the CS was
presented immediately before the onset of US presentation (N=25). In
the backward 4s group, the CS was presented 4s after the onset of US
presentation (N=25). In the forward conditioning groups, the CS was
presented 5, 10 and 20s before the onset of US presentation (forward 5s group,
N=25; forward 10s group, N=23; forward 20s group,
N=23). The ITI was 2.5 min for the unpaired group and 5 min for the
other groups. The odour preference of individual crickets was tested before
and 2 h after training.
Experiment 5: effects of inter-trial intervals
To observe the effects of ITIs, five groups of crickets were subjected to
two sets of P+/V- conditioning trials with ITIs of 30s (group 1,
N=39), 1 min (group 2, N=40), 2 min (group 3,
N=39), 5 min (group 4, N=45) and 10 min (group 5,
N=47). The odour preference of individual crickets was tested before
and 2 h, 1 day and 4 days after training.
Experiment 6: effects of anaesthetic treatment with
CO2
To determine the effects of anaesthetic treatment, 12 groups of crickets
were anaesthetized with CO2 for 60s at different times after two
sets of P+/V- conditioning trials (N=21-27). The ITI was 2 min. One
group (control group) received training but no anaesthetic treatment
(N=38). Another group received anaesthetic treatment 60 min before
training (N=17). After treating the crickets with CO2 for
60s, the air in the beaker was ventilated for 10s. Crickets began to move
their legs approximately 2 min after the cessation of CO2 treatment
and recovered normal locomotion within 7 min. The odour preference of
individual crickets was tested before and 5 h after training.
Data analysis
The initial odour preference of a given cricket group was evaluated by
comparing the time that an individual spent visiting the peppermint source
(tp) with the time spent visiting the vanilla source
(tv) using the MannWhitney U-test (M-W).
To compare initial odour preferences in different cricket groups, the relative
odour preference of individuals was measured using the peppermint preference
index (PPI) (%), defined as
100tp/(tp+tv);
PPIs were compared using the KruskalWallis test (K-W). The PPI was also
used to evaluate the change in odour preference before and after training of a
given cricket group: the preference for peppermint after training
(PPIafter) was compared with that before (PPIbefore)
training using Wilcoxon's test (WCX). To compare the levels of learning
performance in different tests, the learning performance of individuals was
measured using the performance index (PI) (%), defined as
PPIafterPPIbefore. Wilcoxon's test (WCX) was used
to compare the levels of performance in different tests of a given cricket
group, and the MannWhitney U-test (M-W) was used to compare
those of different cricket groups.
Since initial preferences for peppermint did not differ significantly among groups of crickets used in any experiments (see Results), we also used PPIafter to compare the levels of performance of different groups of crickets. Because all statistical comparisons using PPIafter resulted in the same conclusions as those using PI, except for slight differences in the levels of significance, we confine our description to the results of comparisons using PI to avoid redundancy.
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Results |
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Experiment 1: effects of two-trial conditioning procedures
Three groups of crickets were subjected to two trials of P+/V- (group 1 in
Fig. 2A), V- (group 2) or P+
(group 3) conditioning (for notation, see Materials and methods). At 2 h after
training, all groups exhibited significant levels of conditioning: their
preference for peppermint was significantly greater than that before training
(Fig. 2B) (WCX,
P<0.001, groups 1 and 3; P<0.01, group 2). The level
of performance (performance index, PI) 2 h after training did not differ
significantly among these three groups
(Fig. 2C) (M-W,
P>0.05).
No significant decay in the level of performance from 2 h to 1 day or to 4
days was found in crickets that had received two-trial P+/V- or P+
conditioning (Fig. 2C) (WCX,
P>0.05). However, no significant level of olfactory retention was
found 1 day or 4 days after training in crickets that had received two trials
of V- conditioning: their preference for peppermint did not differ
significantly from that before training (WCX, P>0.05). This
appears to reflect a natural decay of retention with time because we have
shown previously that the extinction effect of the retention test is very
small (Matsumoto and Mizunami,
2000). The results indicate that only twopairing trials lead to
robust retention with no significant decay from 2 h to 4 days after training
in differential conditioning or in elementary appetitive conditioning.
Experiment 2: effects of four-trial conditioning procedures
Five groups of crickets were subjected to four trials of V- (group 2 in
Fig. 3A), P+ (group 3), P+/V-
(group 1), V-/P0 (group 4) and P+/V0 (group 5)
conditioning. At 2 h after training, all groups exhibited significant levels
of conditioning: their preference for peppermint were significantly greater
than that before training (WCX, P<0.001). At 2 h after training,
the level of performance of the P+/V- conditioning group (group 1, in
Fig. 3B) was significantly
greater than those of the other four groups (M-W, P<0.05 compared
with group 2; P<0.01 compared with group 3; P<0.001
compared with groups 4 and 5); and the levels of performance of groups 2-5
were not significantly different (M-W, P>0.05).
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The V- conditioning group exhibited significant decay in performance from 2 h to 4 days after training (WCX, P<0.01) and exhibited no significant olfactory retention 4 days after training: their preference for peppermint did not differ significantly from that before training (WCX, P>0.05). All other groups exhibited no significant decay of retention from 2 h to 4 days after training (WCX, P>0.05).
The results indicate that differential conditioning with peppermint-rewarded and vanilla-punished stimuli leads to the highest level of olfactory retention. The following experiments were performed using this conditioning procedure.
Experiment 3: effects of different numbers of differential
conditioning trials
At 2 h after training, the performance of crickets that had been given
four-trial P+/V- conditioning (group 2 in
Fig. 4) was significantly
greater than that of crickets that had been given the two-trial conditioning
(group 1) (M-W, P<0.001) but did not differ significantly from
that of crickets in the six-trial group (group 3). The level of performance at
2 h after training (PI=56±4 %; mean ± S.E.M.) was among the
highest observed in the present study (see Figs
2,3,4,5,6,7).
These results indicate that four trials were sufficient to attain a saturated
level of olfactory retention 2 h after training.
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At 2 h after training, the performance of the group of crickets that had been subjected to two P+ and two V- conditioning trials in block sequence (group 4) was significantly lower (M-W, P<0.001) than that of the four-trial P+/V- conditioning group, which had been subjected to two P+ and two V- conditioning trials in alternating sequence (group 2), and did not differ significantly (M-W, P>0.05) from that of the two-trial P+/V- conditioning group, which had been subjected to only one P+ and one V- conditioning trial (group 1). This result indicates that the number of alternations of appetitive and aversive conditioning trials, in addition to the number of trials, is an important factor in determining the level of 2 h olfactory retention. In all groups, no significant decay of retention was found from 2 h to 4 days after training (WCX, P>0.05).
Experiment 4: non-associative control and the effects of
interstimulus intervals
No significant change in odour preference was found in the three
non-associative training groups, i.e. CS alone, US alone and CS/US unpaired
groups (Fig. 5). The
preferences (PIs) for peppermint at 2 h after training did not differ
significantly from those before training in these groups (WCX,
P>0.05). The backward 4 s group also exhibited no significant
change in odour preference (WCX, P>0.05). The coincident and
forward 5 s groups exhibited a significantly increased level of conditioning:
the preference for peppermint after training was significantly greater than
that before training in these two groups (WCX, P<0.001). The
performance of the forward 5 s group was significantly lower than that of the
coincident group (M-W, P<0.01). The forward 10 s and 20 s groups
exhibited no significant level of conditioning (WCX, P>0.05). The
CS therefore needs to be presented 0-5 s before the onset of US presentation
to achieve conditioning.
Experiment 5: effects of inter-trial interval
At 2 h after training, a significant level of conditioning (WCX,
P<0.001) was found in groups that had been given four-trial P+/V-
conditioning with ITIs of 30 s (group 1), 1 min (group 2), 2 min (group 3), 5
min (group 4) and 10 min (group 5) (Fig.
6). The performance of the 5 min ITI group was significantly
greater than that of the 30 s, 1 min or 10 min ITI groups (M-W,
P<0.001 compared with group 1; P<0.01 compared with
groups 2 and 5) and did not differ significantly from that of the 2 min ITI
group (M-W, P>0.05). The level of performance 2 h after training
was a non-monotonic function of the ITI: it was highest at an ITI of 2-5 min
and was lower at shorter or longer ITIs.
The 1, 2, 5 and 10 min ITI groups exhibited no significant decay in performance from 2 h to 4 days after training (WCX, P>0.05). In contrast, the 30 s ITI group exhibited a significant decay of retention from 2 h to 4 days after training (WCX, P<0.01). This group exhibited a very low, but significant, level of 4 day olfactory retention (WCX, P<0.05).
Experiment 6: effects of anaesthetic treatment with
CO2
In tests performed 5 h after training, the performance of the group that
had received CO2 treatment 60 min before training was not
significantly different from that of the control group that had received
training but no CO2 treatment (M-W, P>0.05)
(Fig. 7). However, groups that
had been given CO2 treatment immediately or 1 min after training
exhibited no significant level of conditioning: their preference for
peppermint did not differ significantly from that before training (WCX,
P>0.05). Apparently, CO2 treatment induced retrograde
amnesia. With an increase in the interval between training and treatment, the
effects of the anaesthetic treatment decreased, and anaesthetic-resistant
memory developed fully 20 min after training. The level of performance of the
20 min interval group did not differ significantly from that of the control
group (M-W, P>0.05). This result indicates that olfactory
retention after differential conditioning can be divided into two phases, i.e.
an early phase sensitive to anaesthetic treatment and a later
anaesthetic-resistant phase.
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Discussion |
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The differential conditioning procedure in which one odour was associated
with a reward and the other with punishment led to the highest levels of 2 h,
1 day and 4 day olfactory memory retention among the five types of
conditioning procedures employed (Fig.
3). Notably, many previous studies on insect olfactory learning
have been carried out using differential conditioning with one odour stimulus
rewarded and a second unrewarded (honeybees,
Mauelshagen, 1993;
Hammer, 1993
), with one odour
stimulus punished and the other unpunished (fruit flies,
Quinn and Dudai, 1976
;
Tully and Quinn, 1985
) or
using elementary appetitive conditioning (honeybees, Menzel,
1990
,
1999
;
Gerber et al., 1998
). The use
of differential conditioning with one odour rewarded and the other punished is
rare. There is one study using fruit flies to determine the effects of
differential conditioning training in which one odour was associated with a
sucrose reward and the other with an electric shock
(Tempel et al., 1983
), but
olfactory retention was examined only up to 24 h, which represents middle-term
rather than long-term memory (Tully et
al., 1994
).
Four-trial appetitive conditioning resulted in a robust memory with no significant decay from 2 h to 4 days after training, but aversive conditioning resulted in no significant 4 day retention of olfactory memory (Fig. 3). This suggests that different cellular processes occur after appetitive and aversive conditioning trials.
The presentation of appetitive and aversive conditioning procedures in alternating sequence led to higher levels of olfactory memory retention than when presented in block sequence (Fig. 4). Thus, the effect of the differential conditioning procedure in which appetitive and aversive conditioning procedures were presented alternately cannot be explained by simple addition of the effects of elementary appetitive and aversive conditioning procedures. An examination of the cellular processes underlying differential conditioning and a comparison with those underlying elementary conditioning are necessary to reveal the neural mechanisms underlying non-additive summation of the effects of appetitive and aversive conditioning procedures.
Crickets attained a saturated level of conditioning with only four trials
of differential conditioning (Fig.
4). Moreover, they had an extremely robust memory with no
significant decay from 2 h to 4 days after training with only two trials of
differential conditioning or elementary appetitive conditioning
(Fig. 2). This excellent
olfactory learning capability of crickets is comparable, among insects, only
with that of honeybees, in which appetitive conditioning trials performed
three times establish a memory that is maintained with no decline for 7 days
(Menzel, 1968). The evident
ability of crickets to change odour preferences by learning and to retain the
altered preferences for a long time may reflect the flexibility and
persistency of their feeding behaviour; crickets are omnivores and select what
is edible or inedible after testing various organic materials and, in
addition, they feed persistently on similar food items from the time of
emergence to adulthood, the latter behaviour being typical of hemimetabolous
insects. Another example of hemimetabolous omnivores with a high olfactory
learning ability is the cockroach Periplaneta americana, which can
retain olfactory memory for at least 4 weeks
(Sakura and Mizunami,
2001
).
We showed that the CS/US interval is a sensitive variable for establishing
CS/US association: significant conditioning was achieved only when the CS was
presented immediately or 5 s prior to the onset of US presentation
(Fig. 5). Similar results have
been noted in other species of insects. Conditioning was achieved when the
onset of the CS preceded the onset of the US by 1-5 s in the honeybee
(Menzel, 1990) and by 1-3 s in
the moth Spodoptera littoralis
(Fan et al., 1997
). The
non-associative control and the backward conditioning groups exhibited no
significant levels of conditioning, in accordance with findings in honeybees
(Bitterman et al., 1983
;
Menzel, 1990
), fruit flies
(Tully et al., 1994
) and moths
(Fan et al., 1997
). Forward
pairing appears to be a property common among various systems of associative
learning in invertebrates and vertebrates
(Carew and Sahley, 1986
;
Rescorla, 1988
).
The highest levels of conditioning 2 h, 1 day and 4 days after training
were achieved with ITIs of 2-5 min, with reduced levels of conditioning with
shorter (0.5-1 min) or longer (10 min) ITIs
(Fig. 6). The finding in the
present study that the shortest ITI (30s) led to only a reduced level of 4 day
olfactory memory retention is in agreement with findings in other species of
insect. In fruit flies, spaced trials (with rest intervals of 5-15 min), but
not massed trials (with no rest interval between pairing trials), established
4 day retention of olfactory memory that represents
protein-synthesis-dependent long-term memory via the cyclic AMP
cascade involving cyclic-AMP-responsive element-binding proteins
(Tully et al., 1994).
Similarly in honeybees, the level of 4 day olfactory memory retention in the
massed training (ITI of 30s) group was much lower than that in the spaced
training (ITIs of 1-20 min) groups (Gerber
et al., 1998
; Menzel et al.,
2001
). The finding in the present study that the longest ITI (10
min) led to a reduced level of conditioning, however, differs from previous
findings in other insects. In the moth Spodoptera littoralis, the
level of 2 h olfactory memory retention after appetitive conditioning training
with a sucrose reward increased monotonically with an increase in the ITI
(Fan et al., 1997
). In fruit
flies, the level of 4 day olfactory memory retention after differential
conditioning training, in which one odour was associated with an electric
shock and the other was not, also increased monotonically with an increase in
the ITI (Tully et al., 1994
).
In honeybees, the level of 1 day olfactory memory retention after an
appetitive conditioning trial with a sucrose reward increased monotonically
with an increase in the ITI, but that of 4 day memory retention depended
non-monotonically on the ITI; i.e. the level of memory retention was lower
with an ITI of 3 min than with an ITI of 1 or 20 min
(Gerber et al., 1998
). It
remains to be determined whether these differences reflect species-specific
features or differences in conditioning procedures.
The olfactory memory of crickets can be divided into two phases: an early
short-term memory (STM) that is susceptible to anaesthetic treatment with
CO2 and a later anaesthetic-resistant memory (ARM)
(Fig. 7). This is in agreement
with findings in honeybees (Menzel et al.,
1974; Erber, 1975
;
Erber et al., 1980
) and fruit
flies (Quinn and Dudai, 1976
).
The time when the anaesthetic-resistant memory developed fully, however,
differed among species: it was 20 min after training in crickets (present
study), 5 min after training in honeybees
(Erber, 1975
;
Erber et al., 1980
) and 30-90
min after training in fruit flies (Quinn
and Dudai, 1976
; Tempel et
al., 1983
). In honeybees and fruit flies, a third memory
component, i.e. protein-synthesis-dependent long-term memory, was evident
(Tully et al., 1994
;
Wüstenberg et al., 1998
;
Menzel et al., 2001
), and we
recently detected this component in crickets: blockade of translation by
cycloheximide had little effect on the level of olfactory conditioning 2 h
after training, partly blocked 1 day olfactory memory retention and completely
blocked 4 day memory retention (Y. Matsumoto and M. Mizunami, unpublished
results). The performance of crickets 4 days after training is therefore a
good measure of the level of long-term memory.
Our study demonstrates that crickets have a high capacity to form olfactory
long-term memory and that a differential conditioning procedure is
particularly effective for the formation of associative memory. Crickets have
been used as models to study neural mechanisms of behaviour because
electrophysiological studies of the activities of brain neurons during walking
are possible (Huber, 1990;
Böhm and Schildberger,
1992
; Kohstall-Schnell and
Gras, 1994
; Staudacher and
Schildberger, 1998
). We have recently developed a method of
recording the activities of brain neurons during olfactory conditioning
training of unrestrained crickets (Y. Matsumoto and M. Mizunami, unpublished
results) using chronic extracellular recording techniques originally applied
to cockroaches (Mizunami et al.,
1998
; Okada et al.,
1999
). Neural correlates of olfactory memory processing in
normally behaving crickets will be given attention in future studies.
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Acknowledgments |
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References |
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