CONDITIONING WITH COMPOUND STIMULI IN DROSOPHILA MELANOGASTER IN THE FLIGHT SIMULATOR
Lehrstuhl für Genetik und
Neurobiologie, Biozentrum, Am Hubland, 97074
Würzburg, Germany
*
Present address: Department of Neurobiology and Anatomy, University of Texas,
Houston Medical School, 6431 Fannin, Houston, TX 77030, USA
Author for correspondence (e-mail:
heisenberg{at}biozentrum.uni-wuerzburg.de
)
Accepted May 31, 2001
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Summary |
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Key words: classical and operant conditioning, blocking, overshadowing, sensory preconditioning, second-order conditioning, Drosophila melanogaster, memory, learning
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Introduction |
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Blocking implies that temporal CSUS pairing does not lead to a
CSUS association if the CS is presented together with another CS that
already fully predicts the US. In a classical blocking design, the first phase
consists of training to one stimulus (CS1+US) until the subject shows a
maximal learning response. Subsequently, a new stimulus (CS2) is added and the
compound is reinforced (CS1+CS2+US). If CS2 is then tested alone, the subject
shows a learning score below that of a parallel group that received a control
treatment instead of the first training. Thus, the first training of CS1 has
`blocked' learning about CS2 in the second phase (Kamin,
1968). Most current models of
associative learning (Pearce,
1994
; Rescorla and Wagner,
1972
; Sutton and Barto,
1990
; Wagner,
1981
) incorporate blocking as
a critical constituent. Blocking is often explained in terms of
predictability. Only if a US is `surprising' (Kamin,
1968
; Kamin,
1969
) can new stimuli having a
predictive value for the US enter into the association.
In sensory preconditioning, temporal CSUS pairing is not necessary for a CS to accrue associative strength. Sensory preconditioning consists of three parts. In the first, the subject is presented with two stimuli (conditioned stimuli; CS1+CS2) without any reinforcement. Second, one of the stimuli (CS1) is reinforced alone. In the third part, CS2 alone is tested. Provided that the appropriate controls exclude alternative explanations, a significant learning score for CS2 demonstrates that the response-eliciting properties of the US have been transferred to the CS2 with which the US has never been paired. Blocking and sensory preconditioning experiments have received much attention because they falsify the old idea that simple temporal pairing of a CS and a US is both a necessary and sufficient criterion for learning to occur: in blocking, CSUS pairings are shown to be insufficient and in sensory preconditioning they are not even necessary for memory formation.
In the flight simulator used here (Fig.
1), a tethered Drosophila can control, with its yaw
torque, the angular velocity and orientation of a circular arena surrounding
it. The arena wall is decorated with different patterns (visual pattern
learning; Wolf and Heisenberg,
1991), allowing the fly to
choose its flight direction relative to these patterns. The fly can be
conditioned by a beam of infrared light delivering instantaneous heat to avoid
certain flight directions (i.e. angular orientations of the arena) and to
prefer others. In a variant of this paradigm, the fly can identify arena
orientations in a uniformly patterned arena if different orientations are
combined with spectrally different arena illuminations (colour learning; Wolf
and Heisenberg, 1997
).
Learning success (memory) is assessed by recording the fly's choice of flight
direction once the training is over. In this study, we first establish that
both patterns and colours are learned separately and symmetrically if both are
presented as a compound during training. In an attempt to find blocking in
Drosophila, two blocking groups are compared with five different
control groups, four of which concern the amount of CS and US experience in
the first training phase and one controls for confounding effects in the
second training phase. Finally, we investigate the occurrence of sensory
preconditioning in our paradigm.
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Materials and methods |
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Apparatus
The Drosophila flight simulator
(Fig. 1) is a
computer-controlled feedback system in which the fly is allowed to control, by
its yaw torque, the rotations of a panorama surrounding it. The core device of
the experimental arrangement is the torque meter. Originally devised by
Götz (Götz,
1964) and repeatedly improved
by Heisenberg and Wolf (Heisenberg and Wolf,
1984
), it measures a fly's
angular momentum around its vertical body axis. The fly, glued to the hook as
described above, is attached to the torque meter via a clamp and
performs tethered flight in the centre of a cylindrical panorama (arena,
diameter 58 mm) homogeneously illuminated from behind
(Fig. 1). The light source is a
100 W, 12 V tungsteniodine bulb. For green and blue illumination of the
arena, the light is passed through monochromatic broadband Kodak Wratten
gelatine filters (nos 47 and 99, respectively). Filters can be exchanged
magnetically within 0.1 s.
Via the motor control unit (K in Fig. 1), an electric motor rotates the arena, making its angular velocity proportional to, but directed against, the fly's yaw torque (coupling factor K=11 ° s-1 10-10 N m). This enables the fly to stabilize the rotational movements of the panorama and to control its angular orientation. The angular position of an arbitrarily chosen reference point on the arena wall delineates a relative `flight direction' of 0-360°. Flight direction (arena position) is recorded continuously via a circular potentiometer (Novotechnik, A4102a306) and stored in the computer memory together with yaw torque (sampling frequency 20 Hz) for later analysis. Reinforcement is achieved by applying heat provided by a light beam (diameter 4 mm at the position of the fly) generated by a 6 V, 15 W Zeiss microscope lamp, filtered by an infrared filter (Schott RG780, 3 mm thick) and focused from above onto the fly. Heat at the position of the fly is switched on and off by a computer-controlled shutter intercepting the beam (Fig. 1).
If patterns alone are used as visual cues (Wolf and Heisenberg,
1991), four black, T-shaped
patterns of alternating orientation (i.e. two upright and two inverted) are
evenly spaced on the arena wall (pattern width
=40°, height
=40°, width of bars 14°, as seen from the position of the fly).
For colours alone as visual cues (see Wolf and Heisenberg,
1997
), the patterns are
replaced by four identical vertical stripes (width
=14°, height
=40°). A computer program divides the 360° of the arena into
four virtual 90° quadrants, the centres of which are denoted by the
stripes. The colour of the illumination of the whole arena is changed whenever
one of the virtual quadrant borders passes a point in front of the fly. If a
compound of colours and patterns is used as the visual cue, the four vertical
stripes are replaced by the four T-shaped patterns, and colour is changed as
described. During training, heat reinforcement (input voltage 6.0 V) is made
contiguous either with the appearance of one of the pattern orientations in
the frontal visual field or with either green or blue illumination of the
arena or with both. Reinforcement of each pattern/colour is always equalized
within groups. During testing, the heat source is permanently switched
off.
Experimental procedures: blocking
Two blocking experiments were performed. Both were designed as
between-groups experiments, each with one blocking and one control group. Both
again consisted of two half-groups, one of which was presented with colours
alone in the first training phase and the other with patterns alone (CS1+US).
Throughout this study (unless indicated otherwise), with patterns alone, the
light of the arena illumination was passed through a 2 mm BG18 Schott
`daylight' (broad-band blue-green) filter which allows for generalization of
pattern memory when switching from daylight to monochromatic blue or green
light in the compound (Liu et al.,
1999). The two experiments
differed in the amount of compound training (CS1+CS2+US) and in the choice of
control procedures. In the first experiment, flies received equal amounts of
first training and compound training. In the second experiment, only half the
amount of compound training was given.
The Kamin control
Four of the five control procedures concern the first phase of the
experiment prior to the compound training. To test whether the flies learned
colours and patterns well during compound training, 103 flies were trained
omitting the first training phase. Four minutes of unreinforced preference
testing with patterns and colours were followed by two 4 min training periods,
interrupted by a 2 min test period. After these 14 min of compound
presentation, the flies were allowed to choose the flight direction either
with the compound as a visual cue (control) or with colours or patterns alone
(experimental groups). A fourth group (exchange group) was presented with a
new compound in which the combination between patterns and colours was
exchanged (e.g. if, during training, flying towards an upright T led to green
illumination of the arena, it would now, during the `exchanged' test phase,
lead to blue illumination). `Overshadowing' (Pavlov,
1927) of one stimulus by the
other would be indicated by a significant difference between the results of
the two experimental groups (control 1).
Improved controls
Two additional control treatments in phase one provided the flies with the
same amount of CS1 and US experience as in the first blocking experiment.
After these treatments, the control and experimental groups differed only in
the associative strength of CS1 a clear advantage over another
frequently used control that employs a novel third stimulus during the first
training. This is accomplished in two different ways. In the control group
stimulated by colours as CS1 during the first conditioning phase, flies were
trained classically by recording their flight orientations and heating regime
in the corresponding blocking group and playing them back to naive flies
(replay experiment; Wolf and Heisenberg,
1991). This implies that the
control flies received the same sensory stimulation as the flies in the
blocking group. However, it has been shown previously that this training is
not sufficient for conditioning the flies (Wolf and Heisenberg,
1991
). Thus, the flies
received the same treatment as the blocking group, but were nevertheless
`naïve' when entering the second phase (control
2).
For the other half of the control flies trained to patterns as CS1, we took
advantage of an effect that had been discovered independently of this study.
We had observed that pattern memory from operant training in white light (no
daylight filter; see above) is lost if monochromatic colours are added to
generate compound stimuli (CS1+CS2; for details of the effects of colour
changes on pattern memory, see Wiener,
2000). This effect was used
for the second group of control flies trained to patterns as CS1. Training
without the daylight filter in the first phase provided the animals with the
same amount of CS1 and US exposure as the animals in the blocking group, but
rendered the flies `naïve' at the onset of
compound training (control 3).
In the second experiment, only half the amount of compound training was given. In this experiment, the control groups did not receive any reinforcement before the compound phase. Instead, they perceived CS1 (either colours alone or patterns with a daylight filter) without reinforcement. If the control flies had developed a latent inhibition to CS1, reinforcement of the compound would have been even less expected, enhancing a potential blocking effect by increasing the control learning scores for CS2. A significant decrease in learning in the blocking versus any of the control groups for CS2 would be indicative of blocking (control 4).
Second-order conditioning control
The fifth control experiment addressed effects during the compound phase.
Two second-order conditioning experiments were conducted differing in the
amount of second-order training (CS1+CS2). The first was similar to the first
blocking experiment, with the difference that the second phase, using the
compound, was shortened by 2 min and included no reinforcement. For the second
experiment, we shortened the second-order conditioning phase even more to only
two 2 min periods (matching the second blocking experiment). The final test
phase for pattern learning (CS2) was for two 2 min periods. Only colours were
used as the conditioned reinforcer. Significant learning in the final test
phase would indicate successful second-order conditioning that might mask a
potential blocking effect (control 5).
Experimental procedures: sensory preconditioning
Two groups of flies were allowed to fly without reinforcement using a
compound of colours and patterns as orientation cues (CS1+CS2) for 10 and 16
min, respectively. The groups were then further subdivided into two
half-experiments each according to which stimulus (colours or patterns) was
chosen as CS1 and was presented during the subsequent single-stimulus phase.
This phase consisted of two 4 min periods of training (CS1+US), with an
intervening 2 min test (CS1 alone). The final 2 min test was conducted with
the alternative stimulus (CS2) alone. Sensory preconditioning is said to have
occurred if this final test shows a significant learning score.
Note that the control experiments commonly used in similar studies to rule out other effects such as generalization or sensitization are not necessary in our design because none of these effects could help the fly determine which of the two patterns or two colours it should avoid.
Data evaluation
The pattern or colour preference of individual flies was calculated as the
performance index:
PI=(ta-tb)/(ta+tb).
During training, tb indicates the time for which the fly
was exposed to the reinforcer and ta indicates the time
without reinforcement. During testing, ta and
tb refer to the times when the fly chose the formerly (or
subsequently) unpunished or punished flight direction, respectively. Thus,
when ta=tb, PI=0, when
ta>tb, the learning score is
positive and when ta<tb, the
learning score is negative.
Statistical analyses
Tests for a normal distribution of performance indices yielded varying
results. Therefore, where possible, non-parametric tests were used, e.g. a
KruskalWallis analysis of variance (ANOVA) to test the hypothesis that
three or more samples were drawn from the same population, a
MannWhitney U-test to compare two independent samples and a
Wilcoxon matched-pairs test to test single performance indices against zero.
For more complicated two-way designs, data were sufficiently close to being
normally distributed to justify a repeated-measures ANOVA whenever within- and
between-group comparisons needed to be carried out.
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Results |
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Blocking
Symmetrical stimuli
In his original design, Kamin (Kamin,
1968) used a simple control
group that received no first training, but was otherwise identical to the
blocking group. We have also tested a group of flies without a first
conditioning phase (Fig. 2).
This primarily ensures that both stimuli give reasonably high learning scores
after compound training without prior conditioning history and serves as one
of the comparisons with the blocking group. We used four groups of flies that
all received identical compound training during the first 14 min of the
experiment (Fig. 2A). In the
subsequent test phase, the first (control) group was scored for the compound
(Fig. 2B). The second and third
groups were presented with colours alone
(Fig. 2C) and patterns alone
(Fig. 2D), respectively
(experimental groups). The fourth group was presented with a new compound in
which the contiguity between colours and patterns was reversed (exchange
group, Fig. 2E). The learning
scores were defined so as to indicate a dominance of colour over pattern when
the score was positive.
A KruskalWallis ANOVA for all four groups revealed a significant difference between groups (P<0.006), encouraging a more detailed analysis. The control group (Fig. 2B) had a large performance index. The difference between the control and exchange groups was highly significant (Fig. 2B versus Fig. 2E; P<0.003, MannWhitney U-test). Moreover, a Wilcoxon matched-pairs test confirmed that the learning scores for the control group and both experimental groups were significantly different from zero (control, P<0.001; colours alone, P<0.005; patterns alone, P<0.001), whereas the performance index for the reversed colour/pattern contiguity was not significantly different from zero (P=0.23). The two experimental groups did not differ significantly from each other (P=0.47, MannWhitney U-test), but the group that had been presented with colours alone differed significantly from the control group (P<0.006, MannWhitney U-test). The difference between the group presented with patterns alone and the control group just failed to reach statistical significance (P=0.07, MannWhitney U-test). We therefore conclude that presenting the individual stimuli alone after binary compound training of patterns and colours in the Drosophila flight simulator led to intermediate, but nevertheless significant, learning scores that did not differ from each other. This result is important for the interpretation of the experiments described below.
The blocking groups
Two blocking experiments were performed that differed in the amount of
compound training and the choice of control procedures (see Materials and
methods). As the outcome was essentially the same, the results of only one of
the experiments are presented here in detail
(Fig. 3). In this experiment,
the final test during the first training phase and the carry-over (i.e. the
amount of learning from the first phase still present in the subsequent phase)
in the first compound test phase of the blocking group did not differ between
the two half-experiments (first training colours and first training patterns)
(P=0.08; between-groups effect in a repeated-measures ANOVA over both
periods and both half-experiments). Therefore, the results of these two
half-experiments have been pooled (Fig.
3A). The same evaluation yielded a significant within-group effect
(P<0.008), rendering the difference between the last test during
pretraining and the carry-over in the first compound test phase statistically
reliable. We did not pool the corresponding control half-experiments
(Fig. 3B,C) because two
different procedures were used for the first training phase (see legend to
Fig. 3 and Materials and
methods).
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In vertebrates, several criteria have been found to be crucial for
blocking. One is the equivalence of the two stimuli. We have shown this
criterion to be met in the present case
(Fig. 2, Fig. 3B,C). Another essential
criterion is the high predictive value of the stimulus trained first. In
operant conditioning, it is not possible to verify a predictive value of 100 %
for a stimulus because there is no reflex-like relationship between a response
and a stimulus. Rather, the animal exhibits active behaviour and controls its
stimulus situation by trial and error (for a discussion of operant behaviour
and initiating activity, see Heisenberg,
1983; Heisenberg,
1994
). Therefore, the first
training is performed until an asymptotic level of performance is reached.
Prolonged operant pattern learning determined this level to be reached after
four 2 min periods of training (Brembs and Heisenberg,
2000
). Moreover, it has been
shown that the level of performance reached after four 2 min periods of
training was very stable, with little extinction over a period of at least 8
min (Guo et al., 1996
; Liu et
al., 1998
; Wolf and
Heisenberg, 1997
; Xia et al.,
1997b
). Therefore, four 2 min
periods of training should be optimal for producing a robust learning score
during the first training phase for the first blocking experiment presented
here (Fig. 3A-C). This will
become very important when the blocking groups are compared with the various
controls.
The Kamin control
Before we compare the first blocking group
(Fig. 3A) with more rigorous
control groups (Fig. 3B,C), it
is interesting to compare it briefly with the `classical' Kamin control
(control 1), despite its apparent lack of control for the first training phase
in the blocking group (data from the two groups in
Fig. 2C,D are pooled for this
comparison). Not surprisingly, the learning score obtained for the compound
stimulus was significantly higher in the blocking group
(Fig. 3A) than in the control
group (Fig. 2A,B) because the
flies had already learned that one of the elements in the compound could be
used to avoid heat (P<0.0002, MannWhitney U-test
between the results of the tests conducted with the compound stimulus prior to
training the flies to the compound). This was still the case during training:
the intermediate compound test score between the two training blocks was
significantly higher in the blocking than in the Kamin control group
(P<0.0001, MannWhitney U-test). Although it seems
that the compound was better predicted throughout the entire compound phase,
the performance indices for the added stimulus in the blocking group were not
significantly different from the corresponding performance indices in the
Kamin control group (P=0.8, MannWhitney U-test). On
the contrary, the performance indices in the blocking group were just as high
as after prolonged, asymptotic training (Brembs and Heisenberg,
2000).
Many blocking experiments control for the CS and US experience in the blocking group by first training, in the first phase, to a novel third stimulus that differs from both CS1 and CS2 prior to training the flies in the second phase to the compound (CS1+CS2+US). Therefore, we have designed more stringent control groups that not only encompass some of the variables controlled for by training a novel stimulus but also cover additional ones (see Materials and methods). Comparing any of these controls with the blocking groups, one might still find a significant difference.
An additional interesting result is revealed by the comparison of single-stimulus learning scores after compound (Fig. 2C,D) and after single-stimulus (Fig. 3A; `first training') training. The significant difference (P<0.007, MannWhitney U-test) indicates an interaction between the two stimuli because patterns and colours are learned better if trained and tested alone than if trained in a compound and tested separately. In other words, in principal, overshadowing does occur in Drosophila if stimulus intensities are chosen appropriately. In our design, however, either stimulus diminished the learning score of the other to the same extent. Thus, with our choice of stimulus intensities in the blocking experiment, overshadowing did not occur. With the appropriate choice of stimulus intensities/saliences, a non-symmetrical overshadowing effect would, however, be expected.
Improved controls
Even though the `classical' Kamin control experiment might be considered a
sufficient control for the first blocking experiments
(Fig. 3), we have addressed
several possible confounding variables using four additional control
procedures.
Similar to the `classical' Kamin procedure, the relevant difference between the experimental and control groups is the carry-over from the performance index in the last test period of the first training to the first test with the compound stimulus. In the experimental group (Fig. 3A), this carry-over should be large (i.e. the generalization decrement should be small), indicating that the reinforcer is well predicted by the pretrained element contained in the compound. In contrast, there should be no significant carry-over in the control groups (Fig. 3B,C). A Wilcoxon matched-pairs test against zero confirmed that the control animals were naive to the compound (P=0.79), whereas the performance index of the experimental group was highly significantly different from zero (P<0.0002). Thus, the application of heat is better predicted in the blocking group, satisfying the most important criterion for blocking to occur. Moreover, comparing the intermediate test period during the compound training phase between experimental and control groups, the experimental group still showed better avoidance than the control groups (P<0.045, MannWhitney U-test), indicating that the US is better predicted not only at the beginning of the compound training, but also throughout the entire compound phase. Just as in the comparison with the Kamin control, there was again a significant difference between the blocking and the control group, demonstrating that the compound was predicting reinforcement better in the blocking group (controls 2 and 3).
Despite the fact that all requirements for blocking seemed to have been met, the final learning score was again indistinguishable between the experimental and control groups (P=0.77, MannWhitney U-test), giving no indication of blocking. The same held true for the second experiment in which the compound training phase was reduced to 4 min and the control groups were spared the reinforcement in the first phase with the single CS to exclude any possible predictive value of US experience (data not shown; control 4).
The second-order conditioning control
Second-order conditioning is very similar to a blocking experiment. Again,
after training with the single stimulus (CS1+US), the compound is presented.
However, compound presentation is not accompanied by reinforcement (CS1+CS2).
In the training phase, CS1 is expected to acquire the response-eliciting
properties of the US and might therefore be able to serve as a second-order US
for CS2 during the compound presentation. One can consider a second-order
conditioning experiment to be a blocking experiment in which reinforcement is
omitted in the compound phase. Thus, second-order conditioning constitutes an
important control for the blocking experiment (control 5): if blocking does
not occur, then this might be due to second-order conditioning masking a
potential blocking effect (Dickinson et al.,
1983). However, the
presentation of the compound without heat after conditioning may attenuate the
CS1-US association (extinction). In addition, extinction might even be
facilitated by the second stimulus (CS2) signalling non-reinforcement of the
compound (CS1+CS2; conditioned inhibition; see, for example, Gewirtz and
Davis, 2000
). Despite these
considerations, we decided to control for second-order conditioning effects
(Fig. 4). Arena illumination
encompasses the patterns and constitutes a major portion of the fly's visual
field. Therefore, only colours were used as CS1 assuming that colour might be
a better second-order US than pattern orientation. To match both blocking
experiments, the experiment was performed twice, with 10 and 4 min of
second-order training. Both yielded only small second-order learning effects
that were statistically significant only if the performance indices of the two
experiments were pooled (P<0.02; Wilcoxon matched-pairs test;
P=0.08 for the two experiments considered separately).
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A steep extinction curve (or conditioned inhibition) is the most likely explanation for the small second-order learning effect. By the first 2 min period of the second-order training phase, avoidance of the colour previously combined with heat was down to a performance index of approximately 0.2 from 0.6 for colour alone after the initial training. Again, only pooling the data from the two experiments (Fig. 4A,B) yielded a statistically significant difference from zero (P<0.02, Wilcoxon matched-pairs test). For the second 2 min period, even pooling the two experiments failed to produce a statistically reliable positive performance index (P=0.15, Wilcoxon matched-pairs test). Summing up, we could find only a slight second-order conditioning effect that was presumably too small to mask any strong blocking.
Sensory preconditioning
Formally, sensory preconditioning is the temporally reversed analogue of
second-order conditioning. In sensory preconditioning, exposure to the
compound (CS1+CS2) precedes training (CS1+US). Hence, no extinction can occur
between training and testing. Flies were exposed to 16 min of unreinforced
flight in which flight directions were designated by compound stimuli
consisting of colours and patterns (CS1+CS2). If, immediately afterwards, one
of the stimuli is paired with heat (CS1+US), the other one (CS2) is regarded
as a predictor of safe and dangerous flight orientations, respectively, in the
subsequent test (Fig. 5B). No
statistically significant performance index was observed in the final test
with only 10 min of preconditioning (Fig.
5A). The difference between the results of these two experiments
was statistically significant (P<0.01, MannWhitney
U-test). This is in line with what has already been concluded on
logical grounds (see Materials and methods), namely that other,
non-associative effects such as generalization and sensitization cannot
account for the significant sensory preconditioning effect found in
Fig. 5B: presenting the
compound stimulus for only 10 min is not sufficient to produce sensory
preconditioning. In each of the two experiments, the two half-experiments
(using colours or patterns as CS1, respectively) yielded statistically
indistinguishable results, justifying the pooling of the corresponding data
sets.
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Discussion |
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Second, in all our experiments, reinforced compound presentation produced
equally strong associations for both stimuli. We found no blocking and no
overshadowing of colours over patterns or vice versa with our choice
of stimulus intensities. We did, however, find reduced learning scores for
either of the two stimuli after training with the compound stimulus compared
with single-stimulus training. This implies, not unexpectedly, that
overshadowing does exist in Drosophila. Overshadowing is a well-known
phenomenon in classical (e.g. James and Wagner,
1980; Rauhut et al.,
1999
; Rubeling,
1993
; Tennant and Bitterman,
1975
) and operant (e.g.
Farthing and Hearst, 1970
;
Miles and Jenkins, 1973
)
conditioning in vertebrates and invertebrates (e.g. Couvillon et al.,
1996
; Pelz et al.,
1997
; Smith,
1998
). With our choice of
stimuli, the finding that the overshadowing effect was symmetrical might be
taken as a further indication of the absence of a significant difference in
effectiveness between colours and patterns.
If prior conditioning history leads to the pretrained stimulus
overshadowing the added stimulus, the effect is called blocking. Blocking is a
cornerstone of modern learning theories (e.g. Pearce,
1994; Rescorla and Wagner,
1972
; Sutton and Barto,
1990
; Wagner,
1981
). While its discovery by
Kamin (Kamin, 1968
) has had a
large impact on vertebrate research (see Cheatle and Rudy,
1978
; Holland and Gallagher,
1993
; Jones et al.,
1990
; Kimmel and Bevill,
1996
; Mackintosh, 1990;
Pearce, 1997
; Roberts and
Pearce, 1999
; Thompson et al.,
1998
), the ecological
significance (Dukas, 1999
) and
neural mechanisms underlying blocking are little understood (Fanselow,
1998
; Schultz and Dickinson,
2000
). The literature on
blocking in invertebrates is more scarce. Moreover, in the few instances where
blocking has been reported (Couvillon et al.,
1997
; Rogers and Matzel,
1995
; Rogers et al.,
1996
; Sahley et al.,
1981
; Smith,
1997
; Smith,
1998
), confounding effects
have been pointed out and remain to be solved (Farley et al.,
1997
; Gerber and Ullrich,
1999
). In particular, the case
of honeybee proboscis extension learning has been intensely debated. Smith and
co-workers first found blocking (Smith,
1996
; Smith,
1997
; Smith and Cobey,
1994
). Gerber and Ullrich
(Gerber and Ullrich, 1999
)
later identified confounding variables in the work of Smith that produced a
blocking-like effect and showed that eliminating these variables also
eliminated blocking. Most recently, Hosler and Smith (Hosler and Smith,
2000
) have again reported
blocking in the honeybee, but only with chemically similar odours. We could
find no unambiguous or undisputed evidence in the literature that
invertebrates exhibit blocking.
We conducted two blocking experiments that varied in the amount of compound
training. The experimental design used for these experiments was derived from
experiments showing asymptotic (Brembs and Heisenberg,
2000) and robust (Guo et al.,
1996
; Liu et al.,
1998
; Wolf and Heisenberg,
1997
; Xia et al.,
1997b
) single-stimulus
learning. Five different control procedures were carried out. Four of them
qualitatively and quantitatively varied the CS, US and flight simulator
experience of the flies prior to compound conditioning. One of the groups
controlled for a second-order conditioning effect that might mask a potential
blocking effect (Dickinson et al.,
1983
). In all experiments, the
key conditions allowing the detection of a blocking effect have been met,
namely, control and experimental groups differed in the predictive value of
the compound (e.g. Fig. 3).
Specifically, in the blocking group (Fig.
3A), each of the stimuli presented individually in the first phase
of training retained its effectiveness in the second phase, i.e. in the
training to the compound. The first training phase caused neither
overshadowing of one stimulus over the other
(Fig. 2) nor a large
second-order conditioning (Fig.
4). Nevertheless, despite varying the compound training and
control procedures (see Materials and methods), no blocking effect could be
detected. While this is one more piece of evidence that blocking might be
absent from invertebrates, let us consider other potential explanations for
why it was not found in our experiments.
There are two types of reason for blocking not to occur in the flight simulator: (i) some qualitative property of the paradigm may interfere with it; and (ii) blocking might not be obtainable over the experimental time course used here.
First, blocking was initially shown to occur in classical (Pavlovian)
conditioning paradigms (e.g. Holland,
1997; Kamin,
1968
; Kimmel and Bevill,
1996
; Marchant and Moore,
1973
; Miller and Oberling,
1998
; Tennant and Bitterman,
1975
). It was later extended
to instrumental (operant) conditioning, using discriminative stimuli (SDs;
e.g. Feldman, 1971
; Feldman,
1975
). Operant SDs, however,
share a feature with `classical' CSs: they are at most only partially
controlled by the animal. They are very different from stimuli controlled
entirely by the animal, as in our approach. SDs can be perceived as `setting
the occasion' upon which behaviourreinforcer contingency occurs. Their
predictive relationship to the US is therefore only indirect (via the
behaviour). We do not know of any study using our type of operant conditioning
to produce blocking. It could therefore be that the high degree of operant
control of the stimuli prevents blocking. We do not consider this explanation
very likely, however, because we have evidence that visual learning in the
flight simulator is a case of classical learning in which the operant
behaviour facilitates CSUS acquisition (Brembs and Heisenberg,
2000
).
Bitterman (Bitterman, 1996)
argued that blocking in bees can only be shown within and not between sensory
modalities (Couvillon et al.,
1997
). Colours and patterns
might represent two modalities. Moreover, in honeybee proboscis extension
conditioning, odours might have to be sufficiently similar to produce blocking
(Hosler and Smith, 2000
). In
our case, the colours and patterns may be too dissimilar.
Second, and more likely, the failure to obtain blocking could be due to a significant generalization decrement of learning upon the introduction of the second CS in the compound phase (Fig. 3A). In addition, conditioned inhibition of generalized learning was observed in the second-order conditioning experiments (Fig. 4). This quick decay of the memory effect might continue in the presence of the US in the blocking experiment, attenuating the predictive value of the CS1 sufficiently strongly to render the flies almost naive even in the shorter blocking experiment (results not shown). In this case, the compound stimulus (CS1+CS2) might be sufficiently `surprising' for the new stimulus (CS2) to acquire associative strength. The possible occurrence of this `surprise' element may constitute the main difference between the blocking experiments conducted in invertebrates and vertebrates. Whereas, in our experiments, training in the first phase of the experiment lasted for no longer than 8 min, in the experiments on vertebrates it lasted for long periods, sometimes for a whole week.
Vertebrates may use this extensive training to explore the situation and to
generate memory templates with much higher reliability than can ever be
obtained with our design. In the flight simulator, in particular, the fly with
a single degree of behavioural freedom has little opportunity to explore the
situation and to increase its level of `orientedness' (for an explanation of
this term, see Heisenberg and Wolf,
1984). In addition, 8 min in
the life of a fly might well be as long as several days in the life of a rat
or a pigeon. Perhaps blocking occurs only if the initial training has not only
rendered the CS1 a certain or almost certain predictor of the US, but has, in
addition, been stored in the memory reliably enough to render CS1 particularly
difficult to extinguish during further training. However, flies will show
significant avoidance for at least 8 min if no changes are made to the
experimental arrangement after training (Guo et al.,
1996
; Liu et al.,
1998
; Wolf and Heisenberg,
1997
; Xia et al.,
1997b
).
Thus, while some conditioned inhibition was expected to occur, such a rapid
decay of avoidance behaviour upon compounding the colours with the patterns is
surprising. Maybe the standard procedure (Wolf and Heisenberg,
1991), even though it has been
shown to produce asymptotic (Brembs and Heisenberg,
2000
) and robust (Guo et al.,
1996
; Liu et al.,
1998
; Wolf and Heisenberg,
1997
; Xia et al.,
1997b
) learning, is
insufficient to produce the required CS1 processing. However, the
significantly larger learning scores of both the carry-over and the
intermediate compound test provide clear evidence that the compound becomes a
reliable predictor of the US after only 6 min, rendering conditioned
inhibition a less than obvious explanation. A more extensive first training
(CS1+US; Xia et al., 1997a
)
could perhaps decrease the generalization decrement as well as minimize
conditioned inhibition. If it were possible to obtain a clear second-order
conditioning by attenuating the rapid extinction, this would open the
possibility that the reinforcement during compound conditioning in a blocking
experiment might have the same effect. In other words, reinforcement of the
compound might decrease both the generalization decrement and extinction,
resulting in an augmented second-order conditioning that might, in turn, mask
blocking that would otherwise be visible. Such effects remain to be
discovered. However, Cheatle and Rudy (Cheatle and Rudy,
1978
) showed that, in their
study, reinforcing the compound blocked second-order conditioning. While it
seems reassuring that the second-order conditioning effect in this study is
too small to mask any substantial blocking, conditioned inhibition still needs
to be completely excluded as an explanation for our failure to find blocking.
For the reasons given above, however, one would expect at least partial
blocking in the present experiments, since the compound is, indeed, better
predicted in the blocking than in the control groups throughout the entire
compound phase, despite conditioned inhibition.
While there are a number of reasons why blocking might exist in Drosophila but was not detected in the present study, the interesting possibility remains that flies, if not invertebrates in general, might lack blocking altogether in their learning performance. Why should blocking be a speciality of vertebrates? Clearly, whether to add further stimuli to the already existing CSs or not is a cost/benefit trade-off. There is no reason not to remember a stimulus, even if it is only vaguely predictive for the US, if this improvement of the memory template can be obtained at low cost. Apparently, for vertebrates, this cost exceeds the benefit: they show blocking. Since one would not assume the costs for a more elaborate memory template in invertebrates to be lower than those in vertebrates, one could argue that, for invertebrates, the benefit of improving memory templates is comparatively large because their reliability is generally low. In other words, blocking may not be implemented in invertebrates because their memory templates never convey the same high degree of reliability as those of vertebrates.
The experiments with reinforcement of the compound provide some evidence
for complex stimulus processing in Drosophila. More clear-cut results
than in the blocking experiment were obtained when the compound was
experienced without reinforcement: we revealed a direct interaction between
the two components in the compound stimulus because they formed a reciprocal
association in our second-order conditioning and sensory preconditioning
experiments. This is obvious in second-order conditioning, in which the CS1
assumes the role of the US. In sensory preconditioning, the preference of
CS2+ and avoidance of CS2 (respectively) in the final test revealed
that CS1+ and CS2+ as well as CS1 and CS2 have formed
specific associations during the preconditioning phase. There are some earlier
reports of sensory preconditioning in invertebrates (Couvillon and Bitterman,
1982; Kojima et al.,
1998
;
Müller et al.,
2000
; Suzuki et al.,
1994
). Sensory preconditioning
can most readily be perceived as a form of `incidental learning' in which two
equally salient stimuli are associated in a symmetrical manner (as opposed to
the asymmetric relationship between the CS and US in normal associative
learning). There is ample evidence for the symmetry in this association.
Simultaneous pairings show stronger effects than sequential ones in honeybees
(Müller et al.,
2000
) and in rats (Lyn and
Capaldi, 1994
; Rescorla,
1980
). Also, in zebrafish
(Brachydanio rerio), Hall and Suboski (Hall and Suboski,
1995
) successfully used
simultaneous stimulus presentations. In mammals, backward pairing with respect
to the stimulus in the final test leads to excitatory, rather than inhibitory,
sensory preconditioning associations (Hall,
1996
; Ward-Robinson and Hall,
1996
; Ward-Robinson and Hall,
1998
). In the flight
simulator, the colour of the arena illumination was changed exactly between
two patterns, providing neither a forward nor a backward relationship between
colours and patterns. This difference between incidental learning (for a
review, see Hall, 1996
) and
normal conditioning is no surprise because the asymmetric dependence on the
temporal arrangement of the CS and US in normal conditioning is reflected by
the difference in biological significance between the CS and US (for a review
on this timing dependence, see Sutton and Barto,
1990
).
Dill and Heisenberg (Dill and Heisenberg,
1995) have reported one case of
incidental learning in the flight simulator termed `novelty choice'. Flies
without heat reinforcement remember patterns and compare them with other
patterns later. Novelty choice learning seems to be considerably faster than
the preconditioning effect observed in the present study. In the novelty
choice paradigm, even a 1 min exposure biased the subsequent pattern
preference (Dill and Heisenberg,
1995
), while in the present
experiment a 10 min preconditioning phase was not enough for a significant
association to be formed (Fig.
5A). Hence, establishing a memory template for a visual pattern is
a fast process, whereas associating different types of sensory stimuli takes
more time. The fly links patterns and colours during preconditioning because
the sudden changes in the colour of the illumination are firmly coupled to
certain changes in angular pattern position. To detect such coincidences, the
fly must compare the temporal structure of the various sensory channels. The
same mechanism has recently been postulated for normal associative
conditioning because, to separate the CS from the context, the animal needs to
compare the temporal structure of the various sensory stimuli present during
training (Liu et al., 1999
).
In both instances, normal conditioning and sensory preconditioning, transient
storage of the incoming sensory data is probably a prerequisite. In novelty
choice learning, pattern storage might be a single step. In summary, one can
propose that incoming sensory data are briefly stored to allow a search for
temporal coincidences. Memory templates with a similar temporal structure are
bound together and kept in storage for an additional period.
To conclude, reinforcing compounds of our choice of colours and patterns always produced symmetrical conditioning to the two CSs, regardless of previous conditioning history (no blocking). It would be premature, however, to conclude that simple temporal CS-US pairing is always sufficient to produce CS-US associations in Drosophila. We have demonstrated, however, that unreinforced presentation of the compound can lead to memory formation, proving that CS-US pairings are not necessary for a CS to accrue associative strength (sensory preconditioning). As in vertebrates, associative learning in invertebrates requires complex processing of sensory stimuli during memory acquisition. Further research is needed to determine the extent to which these processes are shared across phyla.
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References |
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Bitterman, M. E. (1996). Comparative analysis of learning in honeybees. Anim. Learn. Behav. 24,123 -141.
Brembs, B. and Heisenberg, M. (2000). The
operant and the classical in conditioned orientation in Drosophila
melanogaster at the flight simulator. Learn. Mem.
7, 104-115.
Brogden, J. W. (1939). Unconditional stimulus-substitution in the conditioning process. Am. J. Psychol. 52,46 -55.
Cheatle, M. D. and Rudy, J. W. (1978). Analysis of second-order odoraversion conditioning in neonatal rats: implications for Kamin's blocking effect. J. Exp. Psychol. Anim. Behav. Process. 4,237 -249.[Medline]
Couvillon, P. A., Arakaki, L. and Bitterman, M. E. (1997). Intramodal blocking in honeybees. Anim. Learn. Behav. 25,277 -282.
Couvillon, P. A. and Bitterman, M. E. (1982). Compound conditioning in honey bees. J. Comp. Physiol. Psychol. 96,192 -199.
Couvillon, P. A., Mateo, E. T. and Bitterman, M. E. (1996). Reward and learning in honeybees: Analysis of an overshadowing effect. Anim. Learn. Behav. 24, 19-27.
Dickinson, A., Nicholas, D. J. and Mackintosh, N. J. (1983). A reexamination of 1 trial blocking in conditioned suppression. Q. J. Exp. Psychol. B 35, 67-80.
Dill, M. and Heisenberg, M. (1995). Visual pattern memory without shape recognition. Phil. Trans. R. Soc. Lond. B 349,143 -152.[Medline]
Dill, M., Wolf, R. and Heisenberg, M. (1995). Behavioral analysis of Drosophila landmark learning in the flight simulator. Learn. Mem. 2, 152-160.[Medline]
Dukas, R. (1999). Costs of memory: Ideas and predictions. J. Theor. Biol. 197, 41-50.[Medline]
Fanselow, M. S. (1998). Pavlovian conditioning, negative feedback and blocking: mechanisms that regulate association formation. Neuron 20,625 -627.[Medline]
Farley, J., Reasoner, H. and Janssen, M. (1997). Potentiation of phototactic suppression in Hermissenda by a chemosensory stimulus during compound conditioning. Behav. Neurosci. 111,320 -341.[Medline]
Farthing, G. W. and Hearst, E. (1970). Attention in pigeon testing with compounds or elements. Learn. Motiv. 1,65 -78.
Feldman, J. M. (1971). Added cue control as a function of reinforcement predictability. J. Exp. Psychol. 91,318 -325.
Feldman, J. M. (1975). Blocking as a function of added cue intensity. Anim. Learn. Behav. 3, 98-102.
Gerber, B. and Ullrich, J. (1999). No evidence
for olfactory blocking in honeybee classical conditioning. J. Exp.
Biol. 202,1839
-1854.
Gewirtz, J. C. and Davis, M. (2000). Using
Pavlovian higher-order conditioning paradigms to investigate the neural
substrates of emotional learning and memory. Learn.
Mem. 7,257
-266.
Götz, K. G. (1964). Optomotorische Untersuchung des visuellen Systems einiger Augenmutanten der Fruchtfliege Drosophila. Kybernetik. 2, 77-92.[Medline]
Guo, A., Liu, L., Xia, S.-Z., Feng, C.-H., Wolf, R. and Heisenberg, M. (1996). Conditioned visual flight orientation in Drosophila; dependence on age, practice and diet. Learn. Mem. 3,49 -59.[Abstract]
Hall, D. and Suboski, M. D. (1995). Sensory preconditioning and second-order conditioning of alarm reactions in zebra Danio fish (Brachydanio rerio). J. Comp. Psychol. 109,76 -84.
Hall, G. (1996). Learning about associatively activated stimulus representations: Implications for acquired equivalence and perceptual learning. Anim. Learn. Behav. 24,233 -255.
Heisenberg, M. (1983). Initiale Aktivität und Willkürverhalten bei Tieren. Naturwissenschaften 70,70 -78.[Medline]
Heisenberg, M. (1994). Voluntariness (Willkürfähigkeit) and the general organization of behavior. Life Sci. Res. Rep. 55,147 -156.
Heisenberg, M. and Wolf, R. (1984). Vision in Drosophila. In Genetics of Microbehavior (ed. V. Braitenberg), pp. 1-250. Berlin, Heidelberg, New York, Tokyo: Springer.
Holland, P. C. (1997). Brain mechanisms for changes in processing of conditioned stimuli in Pavlovian conditioning: Implications for behavior theory. Anim. Learn. Behav. 25,373 -399.
Holland, P. C. and Gallagher, M. (1993). Effects of amygdala central nucleus lesions on blocking and unblocking. Behav. Neurosci. 107,235 -245.[Medline]
Hosler, J. S. and Smith, B. H. (2000). Blocking
and the detection of odor components in blends. J. Exp.
Biol. 203,2797
-2806.
James, J. H. and Wagner, A. R. (1980). One-trial overshadowing evidence of distributive processing. J. Exp. Psychol. Anim. Behav. Process. 6, 188-205.[Medline]
Jones, S. H., Gray, J. A. and Hemsley, D. R. (1990). The Kamin blocking effect, incidental learning and psychoticism. Br. J. Psychol. 81, 95-109.[Medline]
Kamin, L. J. (1968). Attention-like processes in classical conditioning. In Miami Symposium on Predictability, Behavior and Aversive Stimulation (ed. M. R. Jones), pp.9 -32. Miami: Miami University Press.
Kamin, L. J. (1969). Predictability, surprise, attention and conditioning. In Punishment and Aversive Behavior (ed. R. M. Church), pp.279 -296. New York: Appleton-Century-Crofts.
Kimmel, H. D. (1977). Notes from `Pavlov's Wednesdays': sensory preconditioning. Am. J. Psychol. 90,319 -321.[Medline]
Kimmel, H. D. and Bevill, M. J. (1996). Blocking and unconditioned response diminution in human classical autonomic conditioning. Integr. Physiol. Behav. Sci. 31, 18-43.[Medline]
Kojima, S., Kobayashi, S., Yamanaka, M., Sadamoto, H., Nakamura, H., Fujito, Y., Kawai, R., Sakakibara, M. and Ito, E. (1998). Sensory preconditioning for feeding response in the pond snail, Lymnaea stagnalis. Brain Res. 808,113 -115.[Medline]
Lattal, K. M. and Nakajima, S. (1998). Overexpectation in appetitive Pavlovian and instrumental conditioning. Anim. Learn. Behav. 26,351 -360.
Liu, L., Wang, X., Xia, S. Z., Feng, C. H. and Guo, A. (1998). Conditioned visual flight orientation in Drosophila melanogaster abolished by benzaldehyde. Pharmac. Biochem. Behav. 61,349 -355.[Medline]
Liu, L., Wolf, R., Ernst, R. and Heisenberg, M. (1999). Context generalization in Drosophila visual learning requires the mushroom bodies. Nature 400,753 -756.[Medline]
Lyn, S. A. and Capaldi, E. D. (1994). Robust conditioned flavor preferences with a sensory preconditioning procedure. Psych. Bull. Rev. 1,491 -493.
Mackintosh, N. J. (1975). A theory of attention: Variations in the associability of stimuli with reinforcement. Psychol. Rev. 82,276 -298.
Mackintosh, N. J. (1983). Conditioning and Associative Learning. Oxford: Clarendon Press.
Marchant, H. G. and Moore, J. W. (1973). Blocking of the rabbit's conditioned nictitating membrane response in Kamin's two-stage paradigm. J. Exp. Psychol. 101,155 -158.[Medline]
McHose, J. H. and Moore, J. N. (1976). Expectancy, salience and habit: an noncontextual interpretation of the effects of changes in the conditions of reinforcement on simple instrumental responses. Psychol. Rev. 83,292 -307.
Miles, C. G. and Jenkins, H. M. (1973). Overshadowing in operant conditioning as a function of discriminability. Learn. Motiv. 4,11 -27.
Miller, R. R. and Oberling, P. (1998). Analogies between occasion setting and Pavlovian conditioning. In Occasion Setting: Associative Learning and Cognition in Animals, vol. XXI (ed. N. A. Schmajuk and P. C. Holland), pp. 3-35. Washington, DC: American Psychological Association.
Müller, D., Gerber, B., Hellstern,
F., Hammer, M. and Menzel, R. (2000). Sensory preconditioning
in honeybees. J. Exp. Biol.
203,1351
-1364.
Pavlov, I. P. (1927). Conditioned Reflexes. Oxford: Oxford University Press.
Pearce, J. M. (1994). Similarity and discrimination: a selective review and a connectionist model. Psychol. Rev. 101,587 -607.[Medline]
Pearce, J. M. (1997). Animal Learning and Cognition. Hove, UK: Psychology Press.333 pp.
Pelz, C., Gerber, B. and Menzel, R. (1997).
Odorant intensity as a determinant for olfactory conditioning in honeybees:
roles in discrimination, overshadowing and memory consolidation. J.
Exp. Biol. 200,837
-847.
Rauhut, A. S., McPhee, J. E. and Ayres, J. J. (1999). Blocked and overshadowed stimuli are weakened in their ability to serve as blockers and second-order reinforcers in Pavlovian fear conditioning. J. Exp. Psychol. Anim. Behav. Process. 25, 45-67.[Medline]
Rescorla, R. A. (1980). Simultaneous and successive associations in sensory preconditioning. J. Exp. Psychol. Anim. Behav. Process. 6,207 -216.[Medline]
Rescorla, R. A. and Wagner, A. R. (1972). A theory of Pavlovian conditioning: Variations in the effectiveness of reinforcement and nonreinforcement. In Classical Conditioning II: Current Research and Theory (ed. A. Black and W. F. Prokasy), pp.64 -99. New York: Appleton-Century-Crofts.
Roberts, A. D. L. and Pearce, J. M. (1999). Blocking in the Morris swimming pool. J. Exp. Psychol. Anim. Behav. Process. 25,225 -235.[Medline]
Rogers, R. F. and Matzel, L. D. (1995). Higher-order associative processing in Hermissenda suggests multiple sites of neuronal modulation. Learn. Mem. 2, 279-298.[Abstract]
Rogers, R. F., Schiller, K. M. and Matzel, L. D. (1996). Chemosensory-based contextual conditioning in Hermissenda crassicornis. Anim. Learn. Behav. 24, 28-37.
Rubeling, H. (1993). Pavlovian conditioning in human skilled motor behavior. Integr. Physiol. Behav. Sci. 28,29 -45.[Medline]
Sahley, C., Rudy, J. W. and Gelperin, A. (1981). An analysis of associative learning in a terrestrial mollusc. J. Comp. Physiol. A 144, 1-8.
Schultz, W. and Dickinson, A. (2000). Neuronal coding of prediction errors. Annu. Rev. Neurosci. 23,473 -500.[Medline]
Skinner, B. F. (1938). The Behavior of Organisms. New York: Appleton.
Smith, B. H. (1996). The role of attention in
learning about odorants. Biol. Bull.
191, 76-83.
Smith, B. H. (1997). An analysis of blocking in odorant mixtures: An increase but not a decrease in intensity of reinforcement produces unblocking. Behav. Neurosci. 111, 57-69.[Medline]
Smith, B. H. (1998). Analysis of interaction in binary odorant mixtures. Physiol. Behav. 65,397 -407.[Medline]
Smith, B. H. and Cobey, S. (1994). The
olfactory memory of the honeybee Apis mellifera. II. Blocking between
odorants in binary mixtures. J. Exp. Biol.
195,91
-108.
Sutton, R. S. and Barto, A. G. (1990). Time-derivative models of Pavlovian reinforcement. In Learning and Computational Neuroscience: Foundations of Adaptive Networks (ed. M. Gabriel and J. Moore), pp. 497-537. Boston, MA: MIT Press.
Suzuki, H., Sekiguchi, T., Yamada, A. and Mizukami, A. (1994). Sensory preconditioning in the terrestrial mollusk, Limax flavus. Zool. Sci. 11,121 -125.
Tennant, W. A. and Bitterman, M. E. (1975). Blocking and overshadowing in two species of fish. J. Exp. Psychol. Anim. Behav. Process. 1,22 -29.[Medline]
Thompson, R. F., Thompson, J. K., Kim, J. J., Krupa, D. J. and Shinkman, P. G. (1998). The nature of reinforcement in cerebellar learning. Neurobiol. Learn. Mem. 70,150 -176.[Medline]
Wagner, A. R. (1981). SOP: A model of automatic memory processing in animal behavior. In Information Processing in Animals: Memory Mechanisms (ed. N. E. Spear and R. P. Miller), pp. 5-47. Hillsdale: Erlbaum.
Ward-Robinson, J. and Hall, G. (1996). Backward sensory preconditioning. J. Exp. Psychol. Anim. Behav. Process. 22,395 -404.
Ward-Robinson, J. and Hall, G. (1998). Backward sensory preconditioning when reinforcement is delayed. Q. J. Exp. Psychol. B 51,349 -362.
Wiener, J. (2000). Kontext-Generalisierung in Drosophila melanogaster. Department of Genetics, Würzburg: Julius-Maximilians Universität Würzburg.
Williams, B. A. (1994). Blocking despite changes in reinforcer identity. Anim. Learn. Behav. 22,442 -457.
Wolf, R. and Heisenberg, M. (1991). Basic organization of operant behavior as revealed in Drosophila flight orientation. J. Comp. Physiol. A 169,699 -705.[Medline]
Wolf, R. and Heisenberg, M. (1997). Visual space from visual motion: Turn integration in tethered flying Drosophila.Learn. Mem. 4,318 -327.[Abstract]
Wolf, R., Wittig, T., Liu, L., Wustmann, G., Eyding, D. and
Heisenberg, M. (1998). Drosophila mushroom bodies
are dispensable for visual, tactile and motor learning. Learn.
Mem. 5,166
-178.
Xia, S. Z., Liu, L., Feng, C. H. and Guo, A. (1997a). Memory consolidation in Drosophila operant visual learning. Learn. Mem. 4, 205-218.[Abstract]
Xia, S. Z., Liu, L., Feng, C. H. and Guo, A. K. (1997b). Nutritional effects on operant visual learning in Drosophila melanogaster. Physiol. Behav. 62,263 -271.[Medline]