Post on 11-Apr-2023
CS�/US delay does not impair appetitive conditioning inChasmagnathus
Beatriz Dimant, Ariana Rossen, Gabriela Hermitte *
Laboratorio de Neurobiologıa de la Memoria, Departamento de Biologıa, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Pab II, 1428 Buenos Aires, Argentina
Received 10 January 2001; received in revised form 1 May 2002; accepted 1 May 2002
Abstract
Habituation and appetitive conditioning have been already described in the crab Chasmagnathus . The purpose of this
work is to study whether associative learning can be obtained despite a long conditioned stimulus�/unconditioned
stimulus interval. Results of the first experiment show that the weakening of temporal contiguity does not prevent
appetitive conditioning to occur while after a long 4-h delay, conditioning wanes completely. A second experiment was
conducted, after one and three days of training respectively, confirming the above results. Though initially neutral the
context trace may be still available immediately after training and for the period of two but not after 4:00 h,
demonstrating a forward limit for the conditioning window. After 3 days of training, a further decrease in the
exploratory activity suggested that a longer training could increase the relative weight of habituation. Conditioning and
habituation seem to work as opponent processes in the crab Chasmagnathus granulatus : if habituation training in the
box is followed by the administration of reinforcement after a short period of time, appetitive conditioning will take
place. However, as this interval is increased, habituation prevails. A persistent effect of the exposure to a given
environment that may underlie trace conditioning in this crab is discussed in adaptive terms.
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Appetitive conditioning; Chasmagnathus granulatus ; CS�/US interval; Crab; Habituation; Trace conditioning
1. Introduction
A long-term memory process has been found in
the crab Chasmagnathus granulatus , which is
triggered by the iterative presentation of a danger
visual stimulus and assessed by changes in the
escape response (Lozada et al., 1990; Maldonado
et al., 1997; Pedreira et al., 1998; Tomsic et al.,
1998; Hermitte et al., 1999). Aiming at widening
the study of different types of learning and
memory paradigms, other studies were oriented
towards exploratory and feeding behavior in
Chasmagnathus .* Corresponding author
Behavioural Processes 60 (2002) 1�/14
www.elsevier.com/locate/behavproc
0376-6357/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 6 - 6 3 5 7 ( 0 2 ) 0 0 0 6 4 - 5
When the crab Chasmagnathus explores a newenvironment in the wild or wanders in an experi-
mental box for the first time, it exhibits a great
display of activity. However, when the animal
voluntarily enters the experimental box after
successive intertrial intervals, the exploratory ac-
tivity fades away. This decrement of the activity
persists 24:00 h later and has been considered a
case of habituation (Dimant, 1991; Dimant andMaldonado, 1992; Hermitte, 1995). On the con-
trary, if the crab finds food in the box during each
training trial, context reveals as predictive of the
presence of reinforcement. In this condition, an
increase of the exploratory activity is observed
which is also present 24:00 h later. This type of
learning has been named appetitive conditioning ,
where the experimental context is the conditionedstimulus (CS) and the food the unconditioned
stimulus (US) (Dimant and Maldonado, 1992;
Hermitte, 1995).
Some preliminary results have shown that if
Chasmagnathus is appetitively reinforced up to an
hour after the habituation phase had finished it
nonetheless shows an increase in the exploratory
activity. This paper is aimed at trying to determinewhether associative learning can be obtained in
spite of such a long CS�/US interval.
Early views of Pavlov (1927) and Locke (1974)
believed that the occurrence of two events in close
temporal proximity (contiguity hypothesis ) was
critical determinant of learning. Consistent with
this hypothesis is the finding that contiguity
detection (as measured by the optimal intervalthat allows the animal to learn) is surprisingly
constrained across species and learning paradigms.
For example, for defensive conditioning with an
aversive US, the optimal interval CS and US onset
is typically between 500 ms to 2 s (Abrams and
Kandel, 1988). However, in other forms of learn-
ing as in the conditioned taste aversions the
temporal parameters are known to differ markedlyand acquisition occurs despite lengthy delays as
long as hours (Barker and Smith, 1974).
The informational hypothesis (Egger and Miller,
1963; Kamin, 1969; Rescorla, 1972; Rescorla and
Wagner, 1972; Cantor, 1981) emerged as perhaps
the most-widely accepted alternative to the princi-
ple of contiguity. According to the informational
hypothesis the mere contiguous occurrence of two
events is not sufficient for the formation of an
association between their representations. Instead,
learning is presumed to occur only when one event
predicts the occurrence of the other. Thus, a CS
must provide anticipatory information about the
US for associative learning to occur.
Despite the considerable importance of tem-
poral variables in Pavlovian conditioning, time has
played a surprisingly limited role within the
traditional theories of associative learning men-
tioned above. The prevailing view has been that
animals do not learn about the temporal proper-
ties of the stimulus events in Pavlovian condition-
ing experiments. Thus, temporal factors serve only
as a facilitative role in the formation of associa-
tions, and the closer the two events are in time
during training, the more robust the resulting
association is presumed to be. The organism
acquires no representational knowledge about
the temporal relationship of the paired stimulus
events.
However, the temporal coding hypothesis has
questioned this assumption (Matzel et al., 1988;
Barnet et al., 1991; Miller and Barnet, 1993).
According to this point of view, an association
consists of more than a mental link between the
representations of two paired events. Rather, the
temporal relationship between the events that
prevailed during training is encoded as part of
the association. Thus, the temporal conditions
(e.g. the CS�/US interstimulus interval) are not
merely catalysts in the formation of associations,
but are also part of the content of learning
(Savastano and Miller, 1998). In accordance, the
capacity of responding on the basis of temporal
information is shared by very different animal
species (Pedreira et al., 1998).
In keeping with the previous view we propose
that temporal relationships from different phases
of training might be encoded as memory repre-
sentations and integrated into a single one that
determines the nature and strength of the condi-
tioned response. Thus the following predictions
are going to be tested: if habituation training in the
box is followed by the administration of reinforce-
ment after a short period of time, appetitive
B. Dimant et al. / Behavioural Processes 60 (2002) 1�/142
conditioning will take place. However, as thisinterval is increased, habituation prevails.
2. Materials and methods
2.1. Subjects
The crab Chasmagnathus granulatus is foundalong the coast of Southern Brazil, Uruguay and
Argentina, occupying mud flats of the mesolitoral
and supralitoral zones of estuaries, namely, zones
of fresh water transition (Boschi, 1964). This semi-
terrestrial and euryhaline crab makes up crowded
communities dwelling in individual borrows
placed very near one another or sometimes sharing
the same refuge. This species inhabits an upperintertidal zone densely vegetated by cord grass
Spartina alterniflora or Spartina densiflora .
For this study, the animals were adult males
Chasmagnathus crabs 2.8�/3.0 cm across the car-
apace, collected from water less than 1 m deep in
the rias (narrow coastal inlets) of San Clemente
del Tuyu, Argentina. Later transported to the
laboratory, where they were lodged in plastictanks (35�/48�/27 cm3) filled to a 2 cm depth
with water without aeration, at a density of 20
crabs per tank. Water used in tanks and other
containers during experiments was prepared with
hW-Marinex salt (Winex-Germany) whose salinity
was 12 � and pH varied between 7.4 and 7.6. The
holding room was maintained on a 12-h light�/
dark cycle (lights on 07:00�/19:00 h). Animalswere fed rabbit pellets (Nutrientes S.A.) every 3
day and after feeding the water was changed.
Temperature of both the holding and experimental
rooms as well as the alley between them was
maintained within a range of 19�/24 8C. Experi-
ments were conducted during daylight between the
2nd and 10th day after arrival. Each crab was used
in only one experiment.Chasmagnathus can be captured during the
whole year except for the coldest winter days
(water temperature below 10 8C). Experiments
of the present study were performed between
October and May (i.e. late spring, summer and
fall).
2.2. Apparatus and procedure
2.2.1. Short-box
Each experimental unit, the box in Fig. 1a,
consisted of a plastic container (25�/25�/15 cm3)
divided in two compartments of equal size (25�/
12.5�/15 cm3) by a central partition: the dark
compartment (DC) and the light compartment
(LC). DC had its walls painted black and a
removable roof that prevented light from entering.A 10 W lamp illuminated LC which had its walls
painted white. A sliding door in the central
partition, which could be raised or lowered by a
motor, allowed the crab to pass from DC to LC or
viceversa. An infrared emitter-receptor (ER) sys-
tem was located 10 cm from the partition in LC.
When reinforcement was available in the training
session it was placed in a small platform located inthe distal end of LC. A computer monitored the
sliding door and recorded the interval of time
between the moment when the door was raised and
when the crab first interrupted the infrared beam.
Throughout the first experiment this time interval
is called: latency value .
Twenty-four hours before starting an experi-
ment, the animals were fed. Each crab was movedfrom the holding to the experimental room and
placed in DC. After 10 min of adaptation the door
was raised. If a crab remained in DC for another
10 min, the door was lowered again and a latency
value equal to 600 s was computed (cutoff score).
On the contrary, if the crab entered LC and
interrupted the infrared beam, the actual latency
value was recorded and the first 5-min trial started.During this time, the animal could both find the
food pellet located in the small platform in LC or
cross freely from one side of the box to the other.
At the end of the 5-min trial those animals found
in LC were gently pushed to DC and the doors
were closed. After a 10 min resting period a new
trial began. Each session consisted of 5 trials after
which the animals were moved back to theirrespective home tanks were they remained for
24:00 h. A training day comprised a single training
session. After this period all the animals under-
went a testing session of only 2 trials, similar in
every other respect with the only exception that
reinforcement was not administered.
B. Dimant et al. / Behavioural Processes 60 (2002) 1�/14 3
When a conditioning procedure was conducted,a food pellet was offered as reinforcement each
time the animal reached the distal part of the box.
When a habituation procedure was followed,
reinforcement was never administered in any trial.
When a delay of reinforcement procedure was
chosen, food was not administered during training
in the box but later in a novel environment. Four
different delay intervals were assessed: 0 h (im-mediate reinforcement); 1:00; 2:30 and 4:00 h. The
reinforcement containers were plastic circular
containers (20�/15 cm2) where 5 pellets of food
were placed. These represent the same amount
administered to those animals reinforced in the
experimental box during conditioning. The time
available for the animals to feed was of 30 min.
2.2.2. Long-box
In the short-box, changes in Chasmagnathus
behavior were only estimated by measuring the
time employed by the animal to reach the distal
position of the target compartment (latency)(Dimant and Maldonado, 1992; Hermitte, 1995).
In order to improve measurement and obtain more
information about the crab’s behavior some
changes in the apparatus were introduced in the
second experiment. Each unit of this apparatus
(Fig. 1b) consisted of a plastic box of 15�/52.5�/
25 cm3, so that DC was of 12.5 cm and LC of 40
cm long. Two infrared ER systems were located at
two positions of LC: proximal position, close to
the central partition permitting the instant the crab
entered LC to be recorded and a distal position, at
the end of LC, allowing the moment the animal
reached the reinforcement to be recorded. As in
the short-box, food (F) was placed in a small
square platform of 1 cm located in the distal end of
LC.
Whenever the long-box was used some modifi-
cations in training procedure were introduced, as
follows. Each crab was individually lodged in the
DC. After 10 min of adaptation the door was
raised. If the crab entered LC within the following
10 min, the time interval between the moment the
door was opened and that when the animal
interrupted the proximal infrared beam was com-
puted as the initial latency, thus starting the first
10-min trial. On the contrary if a crab remained in
DC for another 10 min the door was lowered again
and a latency value equal to 600 s was computed
(cutoff score). During the trial time, the animal
that entered LC could continue its walk and
interrupt the distal beam. The time elapsed be-
tween the moments the animal interrupted the
initial beam and that when it reached the distal one
was computed as travel time . When the animals
Fig. 1. (a) Short-box: Sliding door (D), raised and lowered by motor (M); DC with removable roof (RR); LC; infrared ER system;
food location (X). (b) Long-box: Sliding door (D), raised and lowered by motor (M); DC with removable roof (RR); LC; (1) infrared
ER system in proximal position and (2) distal position; food location (X).
B. Dimant et al. / Behavioural Processes 60 (2002) 1�/144
remained in LC for another 10 min withoutinterrupting the distal beam, a travel time equal
to 600 s was computed (cutoff score). In all other
respects, the new experimental procedure was like
the former.
2.3. Statistical analysis
Retention of learning was assessed during thetesting session comparing latency values that were
analyzed by means of an ANOVA of a priori
planned comparisons (Rosenthal and Rosnow,
1985). In the first experiment 2 trials were admi-
nistered during the testing session. A noteworthy
point is that first latency values of multitrial
trained crabs often failed to be significantly
shorter than those of controls. Thus, more thana single testing trial seems to be necessary to
disclose the appetitive behavior acquired during
training, an outcome consistent with a line of
evidence stemming from the classic reinstatement
paradigm (Campbell and Jaynes, 1966).
In the second experiment 5 trials were adminis-
tered during the testing session in order to obtain
more information about the course of retention.Here, the block of trials 2�/5 was considered for
statistical analysis. A t-test was performed on the
mean scores obtained for the block of trials 2�/5.
3. Experiment 1
3.1. Effect of the immediate reinforcement after
training
As stated above, some preliminary results had
shown that if Chasmagnathus is appetitively re-
inforced up to an hour after the habituation phase
was finished, it nonetheless shows an increase in
the exploratory activity. We were then interested
in determining if such increase can be considered
appetitive conditioned responding in spite of thefood being delivered in a novel context and after
training in the box had already finished.
Twenty-four hours before the experiment, 120
crabs were selected and fed ad libitum with rabbit
pellets. The animals were distributed into three
groups of 40 crabs each, as follows: the condi-
tioned group (CON) was trained with the con-ditioning procedure; the habituated group (HAB)
with the habituation procedure and the delayed-0
group (DEL-0) with the delay of reinforcement
procedure (Section 2). All the animals were then
individually lodged in plastic containers where
they remained for 24:00 h and immediately after-
wards exposed to a 2-trial test session.
3.1.1. Results and discussion
Results of training session are shown in Fig. 2a.
From the analysis of latency both HAB and DEL-
0 groups showed a decrease in the exploratory
activity. This reduction was revealed by the
increasing scores of latency compared to those
found in the first trial. On the contrary, CON
showed the same low scores throughout the wholetraining session.
Results at testing are shown in Fig. 2b. Planned
comparisons of the data of second trial revealed
significant differences both for HAB vs. CON
(F�/12.02; P B/0.005) as for HAB vs. DEL-0 (F�/
9.03; P B/0.005) but not between CON and DEL-
0. Namely, the exploratory activity in the habitu-
ated crabs is shown to decrease compared with theconditioned ones. In contrast, the exploratory
activity of the animals not explicitly reinforced
for such activity, recovered its pre-training latency
values during testing. Results point to the conclu-
sion that the weakening of temporal contiguity
does not prevent appetitive conditioning to occur.
Nevertheless, the possibility that the reduction in
latency shown by DEL-0 could at least be partiallyexplained by a metabolic effect of food cannot be
completely ruled out. The performance of this
group was compared during testing with a group
that had not been fed (HAB). Food could induce a
general metabolic increase that may enhance
exploratory activity reducing the latency values
(Wallace, 1973; Cervino et al., 1995). In the
following experiments this alternative explanationwill be evaluated.
3.2. Effect of delayed reinforcement after training
As was previously proposed, after long intervals
between training context and reinforcement, con-
ditioning may be weakened simultaneously with
B. Dimant et al. / Behavioural Processes 60 (2002) 1�/14 5
the strengthening of the habituation process. On
the contrary, if a metabolic or stimulating effect of
food accounts for the increase in the exploratory
activity, no decrease will ever be observed in
delayed groups, no matter how long the interval
between CS and US. Thus the following experi-ment was aimed at studying the effect of a long
CS�/US time interval on the conditioning process.
The general procedure was similar to that used
in the first experiment except for that the delayed
group was fed 4:00 h after box training. Three
groups were formed of 40 crabs each: CON, HAB
and delayed-4 group (DEL-4).
3.2.1. Results and discussion
Results of training session are shown in Fig. 3a.
From the analysis of latency both HAB and DEL-
4 groups showed a decrease in the exploratory
activity. This reduction was revealed by the
increasing scores of latency compared to those
found in the first trial. On the contrary, CONshowed the same low scores throughout the whole
training session.
Results at testing are shown in Fig. 3b. Planned
comparisons on the data of the second testing trial
revealed significant differences both for CON vs.
HAB (a: F�/8.03; P B/0.025) as for CON vs.
DEL-4 (b: F�/5.3; P B/0.05) but not between
HAB and DEL-4.
The exposure to a given environment must have
some persistent effect if delayed reinforcement is to
produce conditioning. Though initially neutral
after the habituation phase, the context trace
may be still available immediately after training
but not after 4:00 h, demonstrating a forward limit
for the conditioning window. Moreover, the ob-
served changes in latency scores rule out the
possibility of an explanation in terms of the
metabolic effect of food. An additional evidence
of an associative phenomenon versus a more
general motivational/energizing effect of the re-
inforcement is to test for stimulus specificity. This
could be done by exposing trained crabs to test in
different environments. When this was done and
animals that had only received habituation train-
ing encountered a different environment during
testing, latencies decreased compared with the
scores obtained from animals trained and tested
in the same environment (data not shown). Ap-
parently, novelty by itself was responsible for the
changes found in performance. In accordance with
these results it was considered that this kind of
context shift test was not suited for our present
experiments.
Fig. 2. Effect of immediate reinforcement. (a) Training session: the CON group is fed inside the box; the DEL-0 was not fed during
training in the box but immediately afterwards in a novel environment and the HAB group is never fed. (b) Testing session: comparison
among CON, DEL-0 and HAB groups. Histograms represent mean testing latency of the second trial. (*) Asterisks denote significant
differences between treatments.
B. Dimant et al. / Behavioural Processes 60 (2002) 1�/146
4. Experiment 2
4.1. Time course of habituation and appetitive
conditioning after 1 day of training. The study of
latency and travel time
The latency value used in the short-box de-
scribed both the drive to cross to the LC as well as
the tendency to arrive at the target, by a single
parameter. Thus, exploratory activity and goal-
tracking behavior may have been easily con-
founded. A better discrimination between themcould be possible if behavior is analyzed in a
longer box. The modifications introduced in the
apparatus being used in the present experiment
(Fig. 1b) were aimed at analyzing the crab’s
exploratory activity more accurately with the
help of a new testing parameter, the travel time.
The animals were distributed into six groups of
40 crabs each and the performance of a group ofanimals that had been conditioned (CON) was
compared with each of the other three groups
(HAB, DEL-0 and DEL-4).
4.1.1. Results
Testing results corresponding to the three com-
parisons are shown in Fig. 4. Both the initial
latency and travel time scores for the CON group
showed significantly lower values than that of the
HAB group (Fig. 4a). Statistical analysis over
testing data, corresponding to block of trials 2�/
5, revealed differences between groups for latency
(t (1, 78)�/2.73; P B/0.05) and for travel time (t(1,
78)�/2.47; P B/0.05), confirming for both vari-
ables what had been previously been shown for a
single one.Comparison between CON and DEL-4 is shown
in Fig. 4b. Results are similar to those obtained
before but only regarding travel time. Statistical
analysis of data showed a significant difference
between groups for this variable (t (1, 78)�/2.18;
P B/0.05) but not for latency.
No significant differences were disclosed for
CON vs. DEL-0 (Fig. 4c), neither for latency or
travel time.The following conclusions can be drawn from
these results:
The basic finding of the first experiment was
confirmed using the long-box. Namely, during
testing the HAB group showed a decrease in
exploratory activity expressed by higher values of
latency and travel time as compared with those of
the CON group. On the other hand, the explora-
tory activity in the DEL-4 group decreased, as
compared with that of the CON group, only for
the variable travel time. Similarity between DEL-0
group and CON group during testing confirms
above results (short-box): a short delay between
Fig. 3. Effect of delayed reinforcement. (a) Training session: the CON is fed inside the box; the DEL-4 was not fed during training in
the box but later in a novel environment and the HAB group is never fed. (b) Testing session: comparison among CON, DEL-4 and
HAB groups. Histograms represent mean testing latency of the second trial. (*) Asterisk denotes significant differences between
treatments.
B. Dimant et al. / Behavioural Processes 60 (2002) 1�/14 7
context and reinforcement allows appetitive con-
ditioning to occur even when food is offered after
box training and in a novel environment.
4.2. Time course of habituation and appetitive
conditioning after 3 days of training
It has been shown that animals could establish
an association between context and reinforcement
even though the latter was administered shortly
after box training and in a novel context. On the
contrary, when an interstimulus interval of 4:00 h
was used, the association was weakened and could
no longer be established, as was determined by the
use of at least one parameter, travel time. How-
ever, the length of time over which the association
between context (CS) and reinforcement (US) can
be formed has not been established. Consequently
Fig. 4. Time course of habituation and appetitive conditioning after 1 day of training. Analysis of the latency (top panel) and travel
time (bottom panel) during testing. (a) comparison between CON and HAB groups; (b) comparison between CON and DEL-4 groups;
(c) comparison between CON and DEL-0 groups. Histograms represent the mean testing scores of the block of trials 2�/5. (*) Asterisks
denote significant differences between treatments.
B. Dimant et al. / Behavioural Processes 60 (2002) 1�/148
the experiments that follow were aimed at defining
more accurately this time window as well as
studying the time course of habituation and
appetitive conditioning after three consecutive
days of training.
Each experimental group of 40 animals was
trained during three consecutive days with a
testing session in the fourth day. The performance
of a group of animals that had been conditioned
(CON) was compared with each of the other four
groups: DEL-4; delayed-2:30 (DEL-2:30); delayed-
1 (DEL-1) and DEL-0.
4.2.1. Results
Statistical analysis over all training and testing
data, corresponds to block of trials 2�/5.
Results of the comparison between CON and
DEL-4 are shown in Fig. 5. Throughout the three
training days, DEL-4 showed a marked tendency
to increase latency and travel time scores.
During testing, both latency and travel time
scores shown by CON were significantly lower
than those shown by DEL-4 (t(1, 78)�/2.38; P B/
0.05, for travel time; t(1, 78)�/1.98; P B/0.05, for
latency). A comparison between the first training
Fig. 5. Time course of habituation and appetitive conditioning
after 3 days of training. Analysis of the latency (top panel) and
travel time (bottom panel) during training and testing. Com-
parison between CON and DEL-4 groups. Histograms repre-
sent the mean scores of the block of trials 2�/5. (*) Asterisks
denote significant differences between treatments and (0) zeros
denote significant differences between first day of training and
testing day.
Fig. 6. Time course of habituation and appetitive conditioning
after 3 days of training. Analysis of the latency (top panel) and
travel time (bottom panel) during training and testing. Com-
parison between CON and DEL-2:30 groups. Histograms
represent the mean scores of the block of trials 2�/5. (0) Zeros
denote significant differences between first day of training and
testing day.
B. Dimant et al. / Behavioural Processes 60 (2002) 1�/14 9
day and testing day in DEL-4 showed a significant
difference both for latency and travel time.
Results of comparison between CON and DEL-
2:30 are shown in Fig. 6. No clear-cut differences
were observed between groups during testing
session. However, DEL-2:30 did show a significant
increase when both variables scores were com-
pared between the first training day and testing
day (travel time, t(1, 78)�/2.49; P B/0.025; latency
t(1, 78)�/2.22; P B/0.05).
Results of comparison between CON and DEL-
1 shown in Fig. 7 are similar to Fig. 6. No
significant difference between both groups could
be observed during testing either for latency or
travel time. However, DEL-1 did show a signifi-
cant increase both in travel time (t(1, 78)�/2.1;
P B/0.01) and in latency (t(1, 78)�/2.02; P B/0.05)
when the first training day was compared with the
testing day.
Results of comparison between CON and DEL-
0 are shown in Fig. 8. No significant difference
between groups during testing was disclosed.
Moreover no trend of increase in scores of the
Fig. 7. Time course of habituation and appetitive conditioning
after 3 days of training. Analysis of the latency (top panel) and
travel time (bottom panel) during training and testing. Com-
parison between CON and DEL-1 groups. Histograms repre-
sent the mean scores of the block of trials 2�/5. (0) Zeros denote
significant differences between first day of training and testing
day.
Fig. 8. Time course of habituation and appetitive conditioning
after 3 days of training. Analysis of the latency (top panel) and
travel time (bottom panel) during training and testing. Com-
parison between CON and DEL-0 groups. Histograms repre-
sent the mean scores of the block of trials 2�/5.
B. Dimant et al. / Behavioural Processes 60 (2002) 1�/1410
DEL-0 group was observed when the first trainingday was compared with the testing day.
4.2.2. Discussion
After 3 days of training, the exploratory activity
of DEL-4, DEL-2:30 and DEL-1 as well as its
tendency to enter the LC, decreased significantly
as revealed by both latency and travel time,
suggesting that a longer training could increase
the relative weight of habituation training.Regarding CON groups involved in the four
experiments both parameters do not show a
decrease after 3 days of training, implying that
the performance of these animals could not be
further improved between sessions, although pre-
vious results from our laboratory strongly suggest
that this is not a floor effect. Moreover an
extinction effect is suggested by the strong ten-dency exhibited by these groups to increase both
parameters during testing. However this compar-
ison is not analyzed due to the fact that the
conditions during training and testing are not the
same.
During testing, the time course of both latency
and travel time corresponding to DEL-0 (Fig. 8) is
indistinguishable from a CON. Again, the weak-ening of the contiguity between context and
reinforcement does not prevent appetitive condi-
tioning.
Reinforcement 1:00 or 2:30 h after training still
allows association to take place, so that the
performance of the animals in both DEL-1 and
DEL-2:30 during testing results indistinguishable
from that of animals which were reinforced in thebox (CON group). The extension of CS�/US
interval allows the appetitive conditioning to occur
between limits. However, when crab performance
of the first training day is compared with that of
testing day, significant differences appear, suggest-
ing an increasing effect of the habituation training.
With a further extension of the CS�/US interval
(DEL-4 group) conditioning waned completely,disregarding an associative process.
The introduction of the new variable (travel
time) for the measurement of learned changes in
exploratory behavior proved useful. Not only the
above results (short-box) were confirmed, but also
these parameters showed differential response to
training. Travel time showed a rapid response totraining (Fig. 4b) while latency could be tailored
only after 3 days (Fig. 5).
Conditioning and habituation seem to work as
opponent processes in this learning situation,
being both present in the same context. However,
at this juncture we cannot be sure whether one
imposes over the other, being either present
together at the same time or perhaps the acquisi-tion of habituation precludes conditioning from
taking place.
5. General discussion
Only the association between food and context
and not a metabolic effect of food can account for
the observed changes in performance. Thus as waspreviously anticipated appetitive conditioning can
arise when habituation training in the box is
followed by the administration of reinforcement
after a short period of time. However, as this
interval is increased, habituation becomes evident.
We can conclude that though initially neutral after
the habituation phase, the context trace may be
still available immediately after training for itsassociation with the positive reinforcement but not
after 4:00 h. So in keeping with the temporal
coding hypothesis the different episodes encoun-
tered by the animal along its itinerary might be
encoded in memory and the presentation of one
event should activate the representation of the
other event as well as its temporal location.
Results with Chasmagnathus are a furtherexception to the contiguity principle; a delay of
almost 2:30 h allows an associative process to take
place. However, the inadequacy of the contiguity
principle at the empirical level need not mean that
the theoretical principle that forms the basis of
conditioning should be abandoned. Therefore, it
can be assumed that activity might persist for some
time after the eliciting stimulus has terminated,thus ensuring some overlap in the times of activa-
tion in its representation. It is to be expected then,
that some conditioning might be possible even
when there is an interval between offset of the CS
and onset of the US. Increasing this interval by
allowing time for the activity in the CS to wane
B. Dimant et al. / Behavioural Processes 60 (2002) 1�/14 11
completely will reduce the size of any effect (Hall,1994).
But what remains a problem for the principle of
contiguity is that conditioning should occur at
lengthy delays of the CS�/US interval of hours and
what is most intriguing about trace conditioning is
that the internal representation of the conditioned
stimuli is probably encoded in a ‘hold-on ’ position
for hours suggesting that the CS and US areprocessed as independent events.
Several lines of evidence have shown that CS
and US events can be dissociated in time. The
formation of the gustatory trace or gustatory
engram which mediates conditioned taste aver-
sions can be disrupted by treatment with anesthe-
sia or electroconvulsive shock (Garcia and
Forthman, 1984; Shaw, 1988; Bures and Buresova,1989). Furthermore, the latent inhibition para-
digm has shown that the acquisition of a taste
memory can be isolated from the association of
this taste with a salient external reinforcement by
pre-exposure to the sensory stimulus (Lubow,
1989; Misanin and Hinderliter, 1989; De la Casa
and Lubow, 1995). Also in the memory of a
delayed aversion learning in chicks, an associationcould be established between pecking at a green
light-emitting diode (LED) and the onset of
sickness (Barber et al., 1989). The intracranial
administration of 2-D-Gal prior to the chick
pecking the LED has been shown to disrupt
memory of an event that did not have any great
significance until half an hour later, when the
chicks became ill. The LED representation, despitebeing an apparently neutral and tasteless stimulus,
must have been stored in the brain very soon after
the chick pecked at it.
In the trace conditioning found in Chasmag-
nathus, it is the internal representation of the
context what may be encoded in a ‘hold-on ’
position for hours. It may well be the case in this
crab that context is readily associated with adelayed reinforcement event. Since behavior is
adapted to the conditions that occur with high
probability during lifetime, learning about con-
textual features in the environment in order to
prepare for a later encounter with food can be
another case of selective association . Animals in
the wild probably keep capturing contextual
representations that encode in hold-on positionand are later used as predictive stimuli or aban-
doned for new contextual landmarks.
Trace conditioning can be regarded as an
ethological learning, opposed to ‘unnatural ’ para-
digms often employed in research (Seligman and
Hager 1972; Domjam, 1982). In this case, it has
been accepted that acquisition of a selective
association not only depends on the levels ofconcurrent activation of the CS and US represen-
tation but also on the effects of a modulatory
process, which is determined by the past history of
the CS as a predictor of USs of that type, acting on
the link between them (Garcia and Koelling,
1966).
The latency value used in the short-box (Section
3) described both the drive to cross to the LC aswell as the tendency to arrive at the target, in a
single parameter. Thus, exploratory activity and
goal-tracking behavior may have been easily con-
founded. The long-box used in the Section 4
permitted a better description of the crab’s beha-
vior. The main finding was that the parameter
travel time was more easily modified by the
habituation procedure (Section 4.1). Probablythis parameter is measuring the tendency to
continue with the exploratory activity in an
already known environment. On the contrary, in
order to slow down the drive to cross to the LC
(latency increase) more training was required
(Section 4.2). These differences between both
parameters could be showing different behavioral
tendencies related with the natural environment ofChasmagnathus. Crossing to the LC could be
compared with an animal that is coming out
from the burrow and could reflect a natural drive
to explore a potentially new environment. This
tendency could be difficult to be reduced by
training (habituation) since conditions in the
natural environment show great variability and
the failure of an excursion may have no influenceover later ones. Another possible explanation
suggests that it could obey to an endogenous
rhythm regulated by environmental factors such
as cycles of light�/darkness and tides (circadian
rhythms and tidal rhythms). De la Iglesia et al.
(1994) have described something similar when they
referred to the opening and subsequent walking
B. Dimant et al. / Behavioural Processes 60 (2002) 1�/1412
out from the burrows of Uca uruguayensis . Thisanimal, which shares with Chasmagnathus the
same ecological environment, shows a behavior
that is clearly adjusted to the periods of light and
tide.
Acknowledgements
We would especially like to thank Dr Hector
Maldonado for his invaluable and decisive sup-
port. We are also grateful to Dr Arturo Romano
for helpful discussion and suggestions and Angel
Vidal for technical assistance. This work wassupported by grants from the Universidad de
Buenos Aires (Grant TY04), Consejo Nacional
de Investigaciones Cientıficas y Tecnicas (PID
4537/96) and ANPCYT (PICT 01863).
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