CS–US delay does not impair appetitive conditioning in Chasmagnathus

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

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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).


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


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.


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.


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.


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.




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.


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).


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.


0 are shown in Fig. 8. No significant difference

between groups during testing was disclosed.

Moreover no trend of increase in scores of the





sent the mean scores of the block of trials 2�/5. (0) Zeros denote

significant differences between first day of training and testing

day.





sent the mean scores of the block of trials 2�/5.


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


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


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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CS–US delay does not impair appetitive conditioning in Chasmagnathus

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