J Comp Physiol A (2006) 192: 69–83
DOI 10.1007/s00359-005-0052-y
O R I GI N A L P A P E R
Gabriela Hermitte Æ Héctor Maldonado
Cardiovascular component of the context signal memory in the crab
Chasmagnathus
Received: 17 June 2005 / Revised: 5 August 2005 / Accepted: 9 August 2005 / Published online: 23 September 2005
Springer-Verlag 2005
Abstract Research on diverse models of memory in vertebrates demonstrates that behavioral, autonomic and
endocrine responses occur together during fear conditioning. With invertebrates, no similar studies have been
performed despite the extensive study of fear memory
paradigms, as the context signal memory (CSM) of the
crab Chasmagnathus granulatus, usually assessed by a
behavioral parameter. Here, we study the crab’s CSM,
considering both the behavioral response and the concomitant neuroautonomic adjustments resulting in a
heart rate alteration. Results show that upon the first
presentation of the visual danger stimulus, a heart arrest
followed by bradycardia is triggered together with a
conspicuous escape response. The latter declines
throughout training, while heart arrests become sporadic
and bradycardia tends to deepen along the session. At
test, 24 h after training, the outcome clearly contrasts
with that shown at training, namely, stimulus presentation in the same context induces lower escape, no heart
arrests and quick suppression of bradycardia. These results support the view that the same memory process
brings about the changes in both responses. High escape,
heart arrest and bradycardia are considered three
parameters of the unconditioned response while minor
escape, no heart arrests and bradycardia attenuation are
three parameters of the learned response.
Keywords Crustacea Æ Heart rate Æ Neuroautonomic
adjustments Æ Conditioned emotional
response Æ Memory
Abbreviations BA: Basal motor activity Æ BA(P1): Basal
motor activity during P1 of trained and untrained
G. Hermitte Æ H. Maldonado (&)
Laboratorio de Neurobiologı́a de la Memoria IFIBYNE,
Depto. Fisiologı́a, Biologı́a Molecular y Celular, Facultad de
Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Pabellón 2 Ciudad Universitaria (1428), Buenos Aires, Argentina
E-mail: [email protected]
Tel.: +54-1-145763348
Fax: +54-1-145763447
groups Æ BA(P4): Basal motor activity during P4 of
trained and untrained groups Æ bpm: Beats per
minute Æ CS: Conditioned stimulus Æ CSM: Context
signal memory Æ ER: Escape response Æ GABA:
Gamma-amino-butyric acid Æ HR: Heart rate Æ HR(P1):
Mean HR during P1 of trained and untrained
groups Æ HR(P4): Mean heart rate during P4 of trained
and untrained groups Æ P1: Phase 1 Æ P2: Phase 2 Æ P3:
Phase 3 Æ P4: Phase 4 Æ P5: Phase 5 Æ pVDS: Previous
interval before VDS Æ TR: Trained Æ UN:
Untrained Æ US: Unconditioned stimulus Æ VDS: Visual
danger stimulus
Introduction
Research on diverse models of memory in vertebrates
demonstrates that behavioral, autonomic and endocrine
responses occur together during fear conditioning (LeDoux and Muller 1997). Therefore, there is a compelling
need for multiple measures indicative of an emotional
state in animals (Antoniadis and McDonald 1999, 2000;
Lee et al. 2001; Ayers and Powell 2002; Yoshida et al.
2004) and accordingly, diverse studies with rodents have
been performed with the purpose of exploring the concurrent cardiovascular and behavioral changes that occur during fear conditioning (Stiedl et al. 2004; Carrive
2000). Although most of the experiments on this subject
were performed in connection with somatomotor aversive conditioning, results have been reported indicating
that a CS-evoked cardioinhibitory process is engendered
by Pavlovian appetitive conditioning (McLaughlin and
Powell 1999).
With invertebrates, no similar studies have been
hitherto performed, despite the extensive study of fear
memory paradigms, as that of the crab Chasmagnathus
granulatus. The sudden presentation of a rectangular
screen overhead, named visual danger stimulus (VDS), is
immediately recognized by the crab Chasmagnathus as
an impending threat and directional escape is elicited.
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Similar observations have been done in the field where
escape is easily triggered by either natural threats or the
presentation of a simulated menace (the rectangular
screen) introduced in the real environment (M. Fathala,
personal communication). Furthermore, in a comparative study between two closely related grapsid species
that diverge widely in ecology, Tomsic et al. (1993)
proposed that the faster acquisition and long-lasting
nature of the learned response to an iterated object
passing overhead observed in C. granulatus might be
explained by the great ambiguity of the signal, since this
crab is immersed in an environment featuring wind-induced oscillations of the upper portion of the cord
grasses which may elicit the escape response. Upon the
iterative presentation of the VDS, the crab’s response
weakens and is replaced by a strong freezing-to-VDS,
which persists over time (Pereyra et al. 1999, 2000). This
long-term memory, which is termed context signal
memory (CSM), is mediated by an association between
the environmental features of the training site (the
context) and the features of the screen moving overhead,
VDS (the signal; Tomsic et al. 1998).
Therefore, crab’s CSM assessment has been thus far
restricted to behavioral measures, with particular
emphasis on the assessment of escape response and
freezing, regarded as manifestations of fear before the
impending threat. However, fear might not only be revealed on the behavioral level but may also involve
concomitant neuroautonomic adjustments resulting in
alterations of heart rate (HR), blood pressure dynamics,
ventilatory rate and hormones and neuromodulators
levels.
Considerable behavioral, physiological and neuroautonomic data on the cardiac nervous system of decapod crustaceans have already accumulated (Maynard
1960; Cooke 1988; Wilkens 1998; McMahon 2001;
Yazawa and Katsuyama 2001). Specifically, several
studies have demonstrated alterations in HR during
environmental disturbances and social interactions
(Schapker et al. 2002; Listerman et al. 2000) and
intriguingly, dramatic changes in cardiac performance
were detected following presentation of sensory stimuli,
though not evoking behavioral responses directly visible
to an observer (Li et al. 2000). However, though significant alterations in HR were shown to be clearly
elicited by life history memory of a past experience (Li
et al. 2000), no study aimed at the role of cardiac activity
in relation to a memory process in decapod crustaceans
has yet been addressed.
Here, we aim to study CSM in the crab Chasmagnathus, assessing the behavioral response together with
the neuroautonomic adjustments that may be elicited
concurrently. Namely, our propose is to determine
whether the same VDS that triggers an escape response
as well as a robust long-term memory after its iterative
presentation has a measurable effect on HR and to
characterize such effect. This implies analyzing the cardiac response as a component of the unconditioned response during the first presentation of the VDS, and
later, the evolution of the escape response and the HR
through the CSM acquisition and retention test
(Experiment 1). In a second experiment, changes in the
pattern of the cardiac response during the first 2.5 s of
the stimulus presentation were investigated in further
detail. Finally, in a third experiment, the independence
of the physiological function and locomotor activity was
assessed by controlling locomotor activity while monitoring changes in HR.
Materials and methods
Animals
The animals were adult male Chasmagnathus crabs 2.7–
3.0 cm across the carapace, weighing around 17.0 g,
collected from water less than 1 m deep in the narrow
coastal inlets of San Clemente del Tuyú, Argentina, and
transported to the laboratory, where they were lodged in
plastic tanks (35·48·27 cm3) filled to 2 cm depth with
diluted marine water, with a density of 20 crabs per
tank. Water used in the tanks and other containers
during experiments was prepared using hw-Marinex
(Winex-Germany), salinity 10–14&, pH 7.4–7.6, and
maintained within a range of 22–24C. The holding and
experimental rooms were kept on a 12 h light–dark cycle
(light on 07:00–19:00 hours). Experiments were carried
out within the first week of the animals’ arrival,
throughout the year, and between 08:00 and 18:00
hours. Each crab was used in only one experiment.
Prolonged handling stresses the animals and alters the
physiological measurements, so crabs were allowed to
recover for a time duration of 48 h and maintained on a
12 h light–dark cycle. Experimental procedures are in
compliance with the Argentine laws for Care and Use of
Laboratory Animals.
Behavioral record: apparatus and procedure
The apparatus was described in detail elsewhere
(Maldonado 2002). Briefly, the experimental unit was
the actometer: a bowl-shaped opaque container with a
steep concave wall 12 cm high (23 cm top diameter and
9 cm floor diameter) covered to a depth of 0.5 cm with
marine water. The crab was lodged in the container,
which was illuminated with a 10 W lamp placed 30 cm
above the animal. During each trial, an opaque rectangular screen (25.0·7.5 cm2), i.e., the VDS, was moved
horizontally over the animal’s head, cyclically from left
to right and vice versa provoking an escape response
(ER) of the crab and subsequent container vibrations
(Fig. 1). A VDS trial could last 9 or 5 s, depending on
whether it comprised two successive cycles of screen
movement with an interval of 2 s between them or
compacted without gap between them, and 3 min of
intertrial interval (Fig. 2b, c). A microphone was centrally cemented to the bottom of the container, so that
71
to the pins of a jack cemented with instant adhesive to
the dorsal carapace in a more anterior position. All the
recording experiments were conducted 2–3 days after
the initial wiring of the animals in order to allow them to
recover from the stressful handling. It has been shown
that such stress alters HR for a few days (Wilkens et al.
1985; Listerman et al. 2000). Prior to an experiment, a
plug connected to the impedance detector (UFI, model
2991) was slotted in each jack, which allowed HR to be
monitored as a measure of dynamic resistance (Fig. 1).
The output from the impedance leads was sent to the
analog-to-digital converter of a computer data acquisition and analysis system. Heart rate was determined by
direct counts of each beat over 9 s intervals and then
converted to beats per minute (bpm). Locomotor
activity and HR were simultaneously recorded in 20
crabs.
Experimental procedure and design
Fig. 1 Apparatus and recording procedures. Each crab was lodged
in the container (C) of each apparatus (actometer). A motor
operated a screen (VDS), which was moved during a 9 or a 5 s VDS
trial from position 1 to position 2. Screen displacements provoked a
crab running response, and subsequent container vibrations were
converted into electrical signals through a microphone (M). These
signals were translated into arbitrary numerical units and then
processed by a computer. Twenty actometers could work simultaneously. To obtain electrocardiograms, two insulated iridium/
platinum wires were placed under the dorsal carapace directly over
the heart and soldered to pins of a jack (J) cemented to the
carapace. Prior to an experiment, a plug (PL) connected to the
impedance detector was inserted in each jack
container vibrations induced electrical signals proportional to the amplitude and frequency of the signal.
These signals were amplified, integrated during either 9 s
(9 s VDS trial) or 5 s (5 s VDS trial) and translated into
arbitrary numerical units ranging from 0 to 8,000, before
being processed by a computer. The experimental room
had 20 actometers, separated from each other by partitions. A computer was employed to program trial sequences, trial duration and intertrial intervals, as well as
to monitor experimental events.
Heart rate record: apparatus and procedure
Heart rate was measured by inserting two impedance
electrodes in holes previously drilled in the cardiac region of the dorsal carapace, which easily pierced the
hypodermis, and were cemented in place with instant
adhesive. The electrodes were made of insulated iridium/
platinum wires (diameter 0.005 in./with coating
0.008 in.; A-M Systems, Inc., Carlsburg, WA, USA).
These two wires were placed to span the heart in a
rostral–caudal arrangement to ensure an accurate
impedance measure during each contraction; one was
located directly above the middle of the heart and the
other was placed 4–5 mm posterior to the first electrode.
The free end of both wires had been previously soldered
The three experiments of this article included a training
session and a testing session, separated by a 24 h interval. Figure 2a describes the experimental protocols. The
gray rectangles stand for phases without VDS presentation, during which both the HR and basal motor
activity (BA) were recorded. The white rectangles stand
for phases in which VDS was presented and both HR
and ER were recorded. The training session consisted of
30 or 15 VDS trials. The testing session always comprised six VDS trials. Figure 2b shows the structure of a
9 s VDS trial used in Experiment 1. The VDS comprised
two cycles of movement (1 and 2), each cycle with a
displacement from position 1 to position 2 (1.25 s) and
vice versa (1.25 s), thus covering 90 and lasting a total
of 2.5 s. Cycles were separated by a rest interval of 2.0 s
and finally, a second rest interval of 2.0 s completed the
9 s trial. Figure 2c shows the structure of a 5 s VDS
trial, used in Experiments 2 and 3. It comprised two
cycles of movement and displacement similar to those of
the 9 s trial, but without rest interval between them or
second rest interval of 2.0 s at the end of the trial. Unlike
the 9 s trial, the 5 s trial was preceded by an interval of
2.5 s before VDS presentation (pVDS) but during which
both the HR and BA were recorded.
Along P2 (training session) and P5 (testing session),
each 9 s VDS trial of Experiment 1 or each 5 s VDS trial
of Experiments 2 and 3 was divided for its analysis into
six or four periods, respectively, named subtrials. Subtrial length matched the VDS cycles, therefore the 9 s
VDS trial included four subtrials of 1.25 s (first, second,
fourth, and fifth subtrials) during screen movement and
two subtrials of 2.0 s (third and sixth subtrials) during
screen arrest (Fig. 2b), while the 5 s VDS trial comprised
four equal subtrials of 1.25 s each (Fig. 2c).
Experiment 1: Procedure A trained (TR) and an untrained (UN) group of 20 crabs each, all previously
wired (Fig. 1), followed one of the two different protocols described in Fig. 2a. Day 1 included three phases as
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Fig. 2 a Experimental procedure and design. The gray rectangles
stand for phases without VDS presentation, during which both
heart rate (HR) and basal motor activity (BA) were recorded.
Trained groups: P1 and P3 (Day 1) and P4 (Day 2); Untrained
groups: P1, P2 and P3 (Day 1) and P4 (Day 2). The white rectangles
stand for phases in which VDS was presented and both HR and ER
were recorded: P2 (Day 1) of the TR group and P5 (Day 2) of both
groups. The training session consisted of 30 or 15 VDS trials (P2)
and the testing session of 6 (P5). Both phases were separated by
24 h. b The 9 s VDS trial design. Two cycles of 2.5 s of VDS
presentation with a gap in between them (gray rectangles). Six
subtrials during which ER (stripped rectangles) and HR (black
rectangles) were recorded. White rectangles indicate the duration of
each subtrial. c The 5 s VDS trial design. Two cycles of 2.5 s of
VDS presentation without gap between them (gray rectangles).
Four subtrials during which ER (stripped rectangles) and HR
(black rectangles) were recorded. White rectangles indicate the
duration of each subtrial. A stripped and a black rectangle
preceding the first subtrial (pVDS) indicates an interval of 2.5 s
for BA and HR records, respectively.
follows. Phase 1 (P1), common to both groups and
during which both BA and HR were recorded while the
animals remained in the actometers. Phase 2 (P2): the
training session, where the TR received 30 trials and the
UN group remained in the actometers during the entire
session (90 min) but without being trained (i.e., without
VDS presentation). ER or BA and HR were recorded in
both groups. Phase 3 (P3), common to both groups and
during which both BA and HR were recorded while the
animals remained in the actometers. Immediately after
P3, crabs were removed from the actometers, individually housed in the resting containers (i.e., plastic transparent bowls covered to a depth of 0.5 cm with water
and kept inside dimly lit drawers) for 24 h. Day 2 included two phases that are described below. Phase 4
(P4), common to both groups and during which both BA
and HR were recorded while the animals remained in the
actometers. Phase 5 (P5): the test session, where both TR
and UN received six trials. ER as well as HR was recorded in both groups. Throughout this experiment a 9 s
VDS trial was used.
Experiment 2: Procedure As Experiment 1, but 15, instead of 30, VDS trials were given during the training
session (P2). Throughout this experiment, a 5 s, instead
of a 9 s, VDS trial was used in both P2 and P5.
Experiment 3: Procedure Experimental design as in
Experiment 2, but without UN group, i.e., only a TR
group was included. Unlike previous experiments, all
crabs were immobilized prior to training and testing
sessions by being enclosed in close-fitting thick elastic
73
bands, with legs positioned in an anterior position and
slightly under their bodies to restrict movement.
Definitions
Subtrial Throughout this paper a subtrial refers to
each of the periods in which the trial time was divided
(Fig. 2b, c).
Bradycardia
basal HR.
Defined operationally as a decrement in
Data analysis and evaluation of memory retention
Long-term memory was assessed by focusing the data
analysis on testing scores (Experiments 1 and 2). Rescorla (1988) argued convincingly for the use of this sort of
analysis instead of a paired training–testing comparison,
stressing the need to distinguish clearly between the time
of input (training session) and the time of assessment
(testing session). The view is certainly justified as much
as it has been demonstrated that long-term retention is
independent of the escape response level at training
(Tomsic et al. 1991). Notwithstanding, in Experiment 3,
where no UN group was included, a comparison between training and testing is performed. Differences in
testing scores between TR and UN groups (Experiments
1 and 2) and between training and testing in TR group
(Experiment 3) were analyzed by ANOVA of repeated
measures (P<0.05).
Results
Evolution of the escape response and the HR through
the CSM acquisition and retention test
The purpose of the first experiment in the present series
was to assess concomitant escape and cardiac responses
along CSM acquisition and retention test. In keeping
with the classical model of CSM experiment (Hermitte
et al. 1999; Pedreira et al. 2002), two groups of equal
number of crabs (n=39) were formed: the TR group and
the UN group. The TR group received 30 training trials
of 9 s each, separated by 3 min intertrial interval; the
UN group remained during the entire training session in
the actometer without VDS presentations. After 24 h,
both groups received a test session of six trials.
The trial analysis
The trial–response curve (Fig. 3a) represents the typical
pattern of performance during CSM acquisition. At the
first trial, the activity of TR group rises promptly from
its basal level of motor activity (BA), recorded previously during the pre-training phase (P1), to the high
point that entails a full ER. This change in motor
activity is concomitant with a sudden decline in HR
(Fig. 3b) of trained animals, from the value recorded
previously during the pre-training session (P1). Thus,
with the first VDS presentation, the TR group displays a
noticeable bradycardia.
Following the first trial, ER of trained animals
reaches P1 level at trial 11, falling in some trials under
the activity level of the UN group. The decrease in escape response over trials is the manifestation of the
increasing substitution of escaping by freezing in the
course of each 9 s trial (Pereyra et al. 1999, 2000).
Concerning the evolution of HR over trials, a persistent
bradycardia is displayed by trained animals throughout
the 29 remaining trials of the training phase (P2), except
a brief but incomplete recovery between fourth and
seventh trials. Intriguingly, the bradycardia of TR group
outlasts the training session and is upheld during the
post-training session (P3).
Thus, the rise in ER of TR group at the first trial is
simultaneous with a fall in HR. A test of correlation
between these two families of data for animal and trial
barely reaches statistical significance (N=39, r= 0.536,
P=0.03). No significant correlation was found along the
following trials. This result, together with the finding
that bradycardia is steadily displayed during the posttraining session (P3), i.e., a session without VDS and
with no overt response, makes hardly tenable the possibility that bradycardia might be a spin-off of the escape
response. On the other hand, the behavior of the UN
group, showed in parallel with that of the trained one,
exhibits a steady motor activity around P1 during the
entire training session P2 and a steady HR around the
respective value of P1 during the phases P2 and P3
(Fig. 3a, b).
An analysis of the topography of the escape and heart
responses during Phases 1–6 of the trial
Though the previous trial analysis discloses both the ER
and HR evolution, allowing us to compare both variables
over consecutive trials, this data treatment is not perhaps
best suited to obtain a full description of both somatomotor and cardiac responses (ER and HR, respectively).
Actually, it is possible that both values change deeply but
briefly, so that a ‘‘within’’ trial analysis could be more
revealing . In a previous work with Chasmagnathus, a
similar analysis was done when behavioral and neuronal
visually elicited responses were compared in peristimulus
time histograms (Tomsic et al. 2003). Here, an analysis of
the topography of the escape and heart responses during
Phases 1–6 of the trial is performed, dividing each trial
into six periods named subtrials. Figure 4 shows the
kinetics of ER and HR corresponding to TR and UN
groups along the first trial.
74
Fig. 3 Evolution of ER and
HR during P2 for both TR and
UN groups. a Ordinate: mean
escape response. Abscissa:
trials. Black squares stand for
TR group; gray squares for UN
group. BA(P1) basal motor
activity during P1, for TR and
UN groups. b Ordinate: mean
HR. Abscissa: trials. Black
circles stand for TR group; gray
circles for UN group. Solid line
HR(P1) mean HR during P1,
TR group. Dashed line HR(P1)
mean HR during P1, UN group.
Scores corresponding to the
following phase (P3) are also
represented
The escape response (Fig. 4a) increases from a basal
level of motor activity corresponding to P1 to the very
high level shown at subtrials 1 and 2, then falls at subtrial 3, recovers at subtrials 4 and 5 and finally falls
down again to the basal level at subtrial 6. Thus, the
kinetics curve for ER consists of two peaks separated by
a valley, ending with a final depression. Such a curve has
a good resemblance with the pattern of VDS presentations (Fig. 4, top). Namely, the two peaks keep step with
the moments of screen movement in the 1–2 and the 2–1
directions, respectively, while the two valleys match up
with the screen arrest (Fig. 2b). A repeated measures
ANOVA on ER data revealed significant UN–TR differences per group, subtrial and interaction (P<0.0001);
while subtrial comparisons showed significant UN–TR
group differences for subtrials 1, 2, 4 and 5, but not for 3
and 6.
On the other hand, the HR kinetics (Fig. 4b) follows
a clearly different course from that of the ER. The cardiac response falls deeply at subtrial 1 from the basal
level of HR corresponding to P1 but starts to recover at
subtrial 2, reaching a value similar to that of P1 at
subtrial 3, which is roughly held during the remaining
trial. A repeated measures ANOVA on HR data showed
significant UN–TR differences for group, subtrial and
interaction (P<0.05; P<0.001; P<0.001, respectively);
while subtrial comparisons revealed significant betweengroup differences for subtrials 1, 2 and 5 but not for 3, 4
and 6.
Here again, the rise in the activity level of TR at the
first subtrial of the first trial is concurrent with a fall in
HR. A test of correlation between these two families of
data for animal and subtrial barely reaches statistical
significance (r= 0.505, P=0.04). No significant correlation was found along the following subtrials of the first
trial.
In short, the subtrial analysis of the first trial demonstrates that the values of both variables change
acutely, though with opposite signs, in response to the
VDS presentation at the first subtrial, but ever since no
75
Fig. 4 Analysis of the
topography of ER and HR
during the first 9 s VDS trial.
a Ordinate: mean escape
response. Abscissa: subtrials.
Symbols as in Fig. 3a. BA(P1)
BA during P1, for TR group
and UN groups. b Ordinate:
mean HR. Abscissa: subtrials.
Symbols as in Fig. 3b. Solid line
HR(P1) mean HR during P1,
TR group. Dashed line HR(P1)
mean HR during P1, UN group.
VDS presentation is shown on
top. Asterisks stand for P level
difference between TR and UN
for the respective subtrials
(***P £ 0.001; **P £ 0.05)
further association is evinced between ER and HR. The
course of the ER values across subtrials seems to
correspond to the screen motion (VDS), but the course
of the HR across subtrials is at least partially detached
from both the screen motion and the resulting crab’s
activity. This noticeable disparity represents an additional support to the view that the cardiac response is
independent of the escape response.
In order to study the topography of the escape and
HR responses during the entire training session (P2),
data of ER were clustered into blocks of six trials and
those of HR into blocks of three trials, since this
treatment more conveniently resulted to reveal the
major trends in the data (Petrinovich and Widaman
1984). Figure 5a–c describes the subtrial analysis per
block of trials corresponding to the three stages of the
training session, namely, initial, middle and final parts
of training. The curves that correspond to the initial
training (Fig. 5a, ER, HR) are, for both responses,
very similar to those presented in Fig. 4 and showed
significant differences for group, subtrial and interaction (P<0.0001) in ER and also for group (P<0.05),
as well as for subtrial and interaction (P<0.0001) in
HR. At the middle of the session (Fig. 5b, ER), the
pattern of the ER curve looks like that of the initial
stage, but the ER level of the TR group has noticeably
decreased, so that now the level corresponding to two
subtrials is lower than that of the UN group. On the
other hand, the pattern of the HR curve repeats that of
the initial training but with a sustained bradycardia
over a larger number of subtrials. A repeated measures
ANOVA on HR subtrial data showed significant UN–
TR differences for group and interaction (P<0.05).
Subtrial comparisons revealed significant differences
76
Fig. 5 Analysis of the topography of ER and HR across blocks of
trials. Evolution of ER and HR during three stages (a–c) of the
training session and during testing session (d). Data of ER per
subtrial were clustered into blocks of six trials and those of HR into
blocks of three trials (see text). a (initial stage) ER (top panel)
corresponds to trials 1–6. Ordinate: mean escape response.
Abscissa: subtrials. BA(P1) BA during P1, for TR group and UN
groups. HR (bottom panel) corresponds to trials 1–3. Ordinate:
mean HR. Abscissa: subtrials. Solid line HR(P1) mean HR during
P1, TR group. Dashed line HR(P1) mean HR during P1, UN group.
Other symbols as in Fig. 3a and b. b (middle stage) ER (top panel)
corresponds to trials 13–18 and HR (bottom panel) to trials 13–15.
Other symbols as in Fig. 3a and b. c (final stage) ER (top panel)
corresponds to trials 25–30 and HR (bottom panel) to trials 25–27.
Other symbols as in Fig. 3a and b. d (testing phase) ER (top panel)
corresponds to trials 1–6. BA(P4) BA during P4, for TR group and
UN group. HR (bottom panel) corresponds to trials 1–3. HR(P4)
mean HR during P4, for TR and UN groups. Other symbols as in
Fig. 3a and b. Asterisks show P level in those subtrials that showed
significant differences between TR and UN (***P £ 0.001;
**P £ 0.005; *P>0.05)
between groups for subtrials 1, 2 and 4 but not for 3, 5
and 6 (Fig. 5b, HR).
Finally, the previous trends are enhanced during the
last block (Fig. 5c, ER, HR). The ER values of the TR
group corresponding to three subtrials are now significantly lower than those of the UN group; and the values
of HR of the TR group are significantly lower than those
of the UN group in the six subtrials. Results corresponding to intermediate blocks (i.e., between initial and
medium and between medium and final) confirm the
above tendency (data not shown).
The same analysis was performed at testing.
According to the method repeatedly used in the laboratory for assessing crab CSM retention, i.e., a method
that excludes before/after assessment of a treatment
(Rescorla 1988), our TR–UN group’s data comparison
was restricted to the test session. Concerning ER
(Fig. 5d, top panel) the ER level of the TR crabs was
significantly lower than that of UN crabs at subtrials 2,
4 and 5. The TR<UN difference for each subtrial is
well illustrated when the ER level of each group is
compared with the basal motor level of P4. Repeated
measures ANOVA on ER subtrial data showed significant UN–TR differences only for subtrial and
interaction (P<0.001; P<0.05, respectively). With respect to HR (Fig. 5d, bottom panel), bradycardia in
the TR group was rapidly attenuated since subtrial 2
onwards, while a persistent bradycardia is established
in the UN group and the HR level of trained crabs was
higher than that of UN crabs for all the six subtrials.
The TR>UN difference for each subtrial is well illustrated when the HR level of each group is compared
with the HR level of P4. ANOVA on HR subtrial data
showed significant UN–TR differences for group and
subtrial (P<0.0001).
In a nutshell, CSM retention at testing is expressed in
trained animals, on one hand, by a reduction in the escape response level in keeping with consistent results of
previous experiments (Maldonado 2002) and, on the
other hand, by a quick and complete suppression of the
training bradycardia.
Changes in the pattern of the cardiac response during
the first 2.5 s of the VDS presentation: the heart arrest is
an early component of the cardiac response
Several previous studies with decapods have reported
that crabs, lobsters and crayfish produce cardiac arrests
to a variety of optical and tactile stimuli (Larimer 1964;
Larimer and Tindel 1966; McMahon and Wilkens 1972;
Uglow 1973; Florey and Kriebel 1974; Wilkens et al.
1974; Cumberlidge and Uglow 1977; Grober 1990). Such
transient alteration in the cardiac performance was also
77
detected in several cardiac responses to VDS in Experiment 1. However, a reliable identification of such
episode proved difficult since each HR record
commenced simultaneously with the VDS presentation,
thus impeding a comparison with an immediately previous record of basal HR. For this reason in the next
experiment the analysis was limited to the first two
subtrials, namely to the first 2.5 s interval upon VDS
presentation (first cycle) of each trial, which was compared with the previous 2.5 s interval before VDS
(pVDS). The experimental protocol was as above but
included 5 s instead of 9 s VDS trials and 15 instead of
30 trials at training that reliably renders a robust
retention at test. A TR group and a UN group of equal
number (n=20) of crabs were formed.
An instance of cardiac response during the first cycle
of the first training and testing trial is exhibited for the
same animal in Fig. 6a, b. A regular rhythm of heart
activity (bpm) with a regular beat pattern is displayed
during the 2.5 s before VDS, but upon VDS presentation, a change in the response is noticeable. The first
beat corresponding to subtrial 1 becomes rather flattened during nearly 1 s, recovering later a steady but
decelerated pace (subtrial 2).
Such pattern, which generally extends during the first
subtrial, could be considered tantamount to a heart arrest. It is found in nearly all the cardiac records of
trained crabs, during the first subtrial of the first training
trials, but seldom during a later phase of training. At
test, while rarely detected in trained crabs, UN animals
frequently exhibit heart arrests during the first subtrial
of the first testing trials and hardly ever during the later
testing phase.
Figure 7 shows the analysis of both ER and HR
during the first subtrial in either training or testing session. In Fig. 7a, ER scores were clustered into a single
subtrial block that comprised 15 training trials and into
a single subtrial block that included the six testing trials.
ER data were normalized with respect to its BA during
the previous 2.5 s interval (pVDS). At the first training
subtrial (Fig. 7a, left panel), the normalized ER scores
of the TR group increases several times, while those of
the UN group remains near the basal level. Significant
TR–UN differences were disclosed for the first subtrial
(t test, P<0.001). In contrast, at the first testing subtrial
(Fig. 7a, right panel), an opposite relationship between
groups is found. The normalized ER scores of the UN
group show a huge increase, while those of the TR group
remain near the basal level (pVDS). Significant TR–UN
differences were disclosed for the first subtrial (t test,
P<0.0001).
Concerning the cardiac activity (Fig. 7b), HR scores
were clustered into a single subtrial block that comprised the 15 training trials and into a single block that
included the 6 testing trials. HR data were normalized
with respect to their basal HR during the previous
2.5 s interval (pVDS). At the first training subtrial
(Fig. 7b, left panel) the normalized HR scores of the
TR reveals a sharp reduction, while no reduction is
observed in the UN group. Significant TR–UN differences were disclosed for the first subtrial (t test,
P<0.0001). At the first testing subtrial (Fig. 7b, right
panel), the normalized HR score of the UN group
reveals a sharp reduction, while a remnant but attenuated bradycardia is observed in TR group. Significant
TR–UN differences were disclosed for the first subtrial
(t test, P<0.05).
The above HR differences could be attributed to the
occurrence of heart arrests, almost entirely limited to the
first subtrial of the first cycle and thus having a different
weight in each group and session. Therefore, it is expected that the larger the number of heart arrests, the
deeper the HR reduction and the greater the differences
between groups within sessions.
Results of this experiment are coherent with those
of the first experiment, confirming an opposite TR–
UN relationship when the ER and HR responses at
test are compared with those of training. In addition,
the heart arrest emerges as a frequent component of
bradycardia at the first subtrial of the first cycle during
early training in the TR group or at test trials in the
UN group, but not at test trials of TR group, thus
confirming the contrasting nature of the cardiac response to the first VDS presentations and that upon
re-exposure at testing.
Effect of the mechanical immobility on the cardiac response at CSM training and testing
Fig. 6 a Instance of an individual record of the cardiac response
during the first cycle of the first training trial. b Instance of an
individual record of the cardiac response during the first cycle of
the first testing trial. Intervals corresponding to pVDS and to the
two subtrials of the first cycle are shown underneath
Previous work of our laboratory established that crabs
impeded from responding to VDS due to GABA
administration were, however, able to acquire CSM and
show memory retention (UN > TR) at testing (Tomsic
et al. 1991). It is now interesting to investigate whether
crab immobility, determined here by mechanical restraint, affects the cardiac component of CSM, namely,
78
Fig. 7 Analysis of the first training and testing subtrial. ER and
HR scores were clustered into a single subtrial block that
comprised the 15 training trials, and into a single subtrial block
that included the 6 testing trials. Black bars stand for TR group;
gray bars stand for UN group. a ER (left panel) Ordinate: mean
escape response. Scores were normalized respect to the basal
activity during pVDS at training. Abscissa: first training subtrial.
Solid line pVDS at training. ER (right panel) Ordinate: mean escape
response. Scores were normalized respect to the basal activity
during pVDS at testing. Abscissa: first testing subtrial. Dashed line
pVDS at testing. b HR (left panel) Ordinate: reduction of the basal
HR. Scores were normalized with respect to the basal HR during
pVDS at training. Abscissa: first training subtrial. Solid line
indicates no reduction of HR during pVDS level at training. HR
(right panel) Ordinate: reduction of the basal HR. Scores were
normalized with respect to the basal HR during pVDS at testing.
Abscissa: first testing subtrial. Solid line shows no reduction of HR
during pVDS level at testing. Asterisks show P level for significant
differences between TR and UN (***P £ 0.001; **P £ 0.05;
P<0.05)
whether crabs impeded from responding at training exhibit nonetheless heart arrest episodes followed by bradycardia at training and bradycardia suppression without
heart arrests at testing. The experimental design was as
that of Experiment 2 but using a single TR group of 20
crabs, previously immobilized by mechanical restraint.
An instance of cardiac response during first cycle of
the first training and testing trials is exhibited for the
same immobilized animal in Fig. 8a, b. A regular beat
pattern is displayed during the previous 2.5 s before
VDS presentation (pVDS). Upon VDS presentation,
those beats corresponding to the first two training subtrials show an extended and rather flattened trace previous to the following peak. Such pattern deformation,
similar to that described in Experiment 2, is found in
practically all the cardiac records of the first subtrial
79
Fig. 8 a Instance of an individual record of the cardiac response of
immobilized animals during the first cycle of the first training trial.
b Instance of an individual record of the cardiac response of
immobilized animals during the first cycle of the first testing trial.
Intervals corresponding to pVDS and to the two subtrials of the
first cycle are shown underneath
upon VDS presentation early at training, but not at
testing (Fig. 8b).
A subtrial analysis on the HR data of immobilized
animals is shown in Fig. 9. HR data has been normalized
in relation to its respective values during the previous
2.5 s period before VDS presentation (pVDS). HR subtrial scores were clustered, both in training and testing,
into subtrial blocks that comprised six trials in order to
compare both phases. At the first and second subtrials,
the normalized HR reveals a sharp reduction at training
( 45 and 42%) and at testing ( 25 and 22%), with
significant differences between sessions. Repeated measures ANOVA showed significant differences between
training and testing values for group and subtrial
(P<0.003 and P<0.001, respectively). Therefore, neither the cardiac response to VDS early at training nor the
observed bradycardia attenuation exhibited at testing is
hindered by the crab’s immobility. This result strongly
supports the view that cardiac response to VDS is independent of the associated escape response.
Discussion
The unconditioned responses
Results from the present study demonstrate that
C. granulatha, being faced for the first time with the
sudden presentation of a VDS, responds with a clear-cut
bradycardia and heart arrest, together with a conspicuous escape response.
A statistical analysis performed on the data corresponding to the first trial of the first two experiments
showed that the sharp decrease in HR correlates with a
sharp increase in motor activity, suggesting that bradycardia is secondary to the production of the escape response. However, such interpretation seems hardly
tenable, since a deep decrease in HR is unveiled when no
escape response to VDS is displayed, as it happens in
many of the following training trials of Experiments 1
and 2 or in Experiment 3 when crabs were immobilized.
Even more, a deep bradycardia is upheld for at least
15 min after the last VDS presentation when the escape
response is no longer displayed (Experiment 1). In
addition, while increased somatomotor demands have
been generally associated with tachycardia, bradycardia
has been frequently linked to immobility (Aagaard et al.
1995; McLaughlin and Powell 1999; Carrive 2000).
Therefore, the changes in cardiac and motor activities at
first trial appear as two simultaneous but independent
manifestations of fear provoked by a token of potential
damage (the VDS presentation). They may be deemed as
part of a constellation of fear-induced physiological responses, which could include, for instance, changes in
blood pressure, secretion of stress hormone, neuromodulators and changes in ventilatory rate.
The change in the cardiac response at first trial starts,
in nearly all the cases, with a brief flattening of the curve
for nearly 1 s, which could be considered as a cardiac
arrest. After such a brief arrest, the trace recovers its
usual waveform, showing a bradycardia than tends to be
attenuated along the remaining seconds of the first trial.
Namely, bradycardia is a noticeable cardiac response to
the first VDS presentations but tends to disappear during the 9 s presentation. Alterations in the waveform
that could be identifiable with long-lasting cardiac arrest
are often shown in other decapods, as when a crayfish
automizes its claw during a battle with another crayfish
(Schapker et al. 2002), or upon 6 min exposure to heavy
metal pollution (Depledge 1984).
Cardio-inhibition resulting from sensory stimulation
was early described in crustacean by Brandt (1865–1866)
and since then, it was repeatedly reported as response to
a variety of optical and tactile stimuli (Dogiel 1894;
von Brucke and Satake 1912; Larimer 1964; Larimer
and Tindel 1966; Mislin 1966; Uglow 1973; Florey
and Kriebel 1974; Wilkens et al. 1974; Cumberlidge and
Uglow 1977; Grober 1990). Struck by the remarkable
sensitivity of the HR, Florey and Kriebel (1974) called
the attention on the possibility that it could serve as an
excellent bioassay of sensory perception in decapods
even in the absence of observable behavior, an opinion
that has been shared by several other authors (Shuranova and Burmistrov 2002; Schapker et al. 2002). It is
worthwhile to notice, however, that cases of tachycardia
without overt behavioral change as a response to small
disturbances in the environment have also been reported
for crayfish (Listerman et al. 2000; Li et al. 2000).
Several attempts have been done to account for the
possible functional role of bradycardia in response to
external stimulation. According to a hypothesis proposed for mammals, bradycardia would play a causal
role in appropriate response selection and thus would
have adaptive significance in their own right
(McLaughlin and Powell 1999). For decapods, a
hypothesis was posed suggesting that bradycardia could
produce concomitant decrease in electrical output and
80
Fig. 9 HR of immobilized
animals during P2 and P5.
Ordinates: mean HR. Abscissa:
first and second subtrials during
P2 and P5. HR subtrial scores,
normalized with respect to
pVDS, were clustered both in
training and testing, into blocks
that comprised six trials. Gray
circles stand for training scores;
black circles stand for testing
scores. Asterisks show P level
for significant differences
between training and testing
(***P £ 0.001; **P £ 0.05)
water movement around the animal (McMahon and
Wilkens 1972), thus helping to disguise the prey from
predators that utilize electrical or mechanical cues for
catching prey (Wilkens et al. 1974). Consequently, bradycardia has been intended as a component of death
feign behavior in decapod crustaceans (McMahon and
Wilkens 1972; Horridge 1965). However, although
induction of ‘‘death-feigning behavior’’ may be in some
species a plausible functional hypothesis for bradycardia, it is unlikely to be the primary explanation in species
in which the decrease in HR is highly correlated with
active movements away from the stimulus (Grober 1990;
our work). Finally, the possibility that cardiac inhibition
would be associated with attentional phenomena (Lacey
and Lacey 1974) has been presented as other possible
role of bradycardia in crustacean, related to the orienting response (Shuranova and Burmistrov 1996).
The learned responses
The pattern of motor and cardiac responses at the first
VDS presentation undergoes noticeable changes
throughout the following training trials. The escape response declines during all the sessions (Fig. 3a), while
the bradycardia tends to deepen along the session
(Fig. 3b) that is consistent with the course of changes
revealed by the subtrial analysis (Fig. 5). In fact, after
the deep bradycardia demonstrated invariably at the
start of each trial, HR tends to recover within the 9 s
trial (Fig. 4b), but such attenuation becomes gradually
smaller during training. Thus, at the end of the training
session, bradycardia is upheld throughout the entire trial
time and also keeps on during the following post-training session (P3).
Results at test on Day 2, for both responses, clearly
contrast with those shown at the first training trial. The
first presentation of VDS elicits a sudden and powerful
escape and a heart arrest followed by bradycardia, while
VDS presentation in the same context, 24 h later, induces lower escaping and a quick suppression of bradycardia without episodes of heart arrest.
Concerning escape response, preceding studies have
led us to conclude that the behavioral changes involve a
contextual fear conditioning. Namely, during training
an association between context (CS) and VDS (US) is
established, which determines at test, lower escaping to
VDS in TR crabs than in UN ones and coincidently,
freezing display in the former but not in the latter
(Pereyra et al. 1999, 2000). Such robust expression of
CSM is not exhibited if VDS is presented in a context
different from that of the training session. We propose
that changes in the cardiac response, here shown, involve the same contextual fear conditioning, which
determines at test, both suppression of the heart arrests
and attenuation of the bradycardia in trained crabs, but
events of heart arrest followed by a bradycardia in UN
crabs. Rationale for this interpretation is based on the
following evidence. First, both fear responses are elicited
throughout by the iterative presentation of the same
VDS in the same context, separated by 3 min intertrial
interval. Secondly, both fear responses are coupled
throughout when facing each VDS presentation. Thus,
the analysis of the topography of the escape and heart
responses shows abrupt changes in ER and HR at the
beginning of each trial, compared with the level exhibited at the end of the previous interval before VDS
presentation (Figs. 7, 9). Thirdly, despite such strong
coupling, results of Experiment 3 show that the changing course of bradycardia, including its attenuation at
test, can occur without the contingent course of ER.
Fourthly, both conditioned responses at test depend
necessarily on VDS presentation at training, but are
independent of the escape performance at training. In
fact, Experiment 3 showed the expected attenuation of
training bradycardia at test despite mechanical immo-
81
bility at training, and previous results from Tomsic et al.
(1991) showed that pre-training injection of GABA,
though producing complete immobilization, fails to
impair long-term retention.
All in all, these results support the view that changes
at test in motor activity and HR are engendered by a
same CSM process. That is, the high escaping and heart
arrests followed by bradycardia during the first VDS
presentation and context exposure could be considered
the three parameters of the unconditioned response,
while minor escaping (most freezing, Pereyra et al.
2000), heart arrest suppression and bradycardia attenuation, during VDS presentation and context re-exposure at testing, three parameters of the CSM learned
response. In accordance, it is widely recognized that
conditioning procedures lead to the elicitation of a
number of non-specific conditioned responses in addition of the specific somatomotor CR being assessed
(Powell 1999; Ayers and Powell 2002). However, though
it seems justified to assume that the HR conditioned
response is also context dependent, a direct demonstration of context dependency, as that shown for ER conditioned response (Tomsic et al. 1998; Hermitte et al.
1999), is pertinent to be included in further research.
This study demonstrates for the first time in invertebrates, a concurrent fear conditioning of both a cardiac
and a behavioral response.
Similar results have been repeatedly reported for
vertebrates, demonstrating that HR is an indicator of
associative learning (Stiedl and Spiess 1997; Stiedl et al.
1999; Butler and Jones 1982; Richardson et al. 1996).
Furthermore, following fear conditioning to environmental context in the rat, a HR rise but preceded by a
period of HR near to the base line, identified as a decelerative period coupled to freezing immobility, has
been reported upon contextual re-exposure. The better
the conditioning, the longer the freezing and the longer
the decelerative period, i.e., the slower the rise in HR up
to reaching tachycardia (Carrive 2000). Nijsen et al.
(1998) suggest a mechanistic explanation for such temporal course of HR, namely, that the HR response
during conditioned fear to context is the result of coactivation of the parasympathetic and sympathetic/
sympathoadrenal systems. Intriguingly, present results
with crab reveal that HR, coupled also with escaping
suppression (freezing), shows at test a temporal course
similar to that described for vertebrates, i.e., a first brief
bradycardia (corresponding to the decelerative period)
followed by a rise in HR up to reach the basal level
(tantamount to tachycardia).
Prior research on the contextual conditioning of escape response in the crab Chasmagnathus showed the
role of protein and RNA synthesis, gene expression as
well as the involvement of diverse receptors on different
memory phases (e.g., Pedreira et al. 1995, 1996;
Maldonado 2002; Pedreira and Maldonado 2003; Merlo
et al. 2005). Such results and present ones call on to
perform similar research to shed light on the possible
mechanisms involved in the regulation of the learned
HR adjustments.
Fear, fight or flight responses, usually associated with
vertebrates are the primary physiological responses that
mediate adaptive responses in front of imminent threat.
These physiological responses in conjunction with the
autonomic nervous system are probably equally evolved
in complex invertebrates (Schapker et al. 2002). Furthermore, learned cardiac changes may play a direct role
in the elaboration of adaptive behavioral responses to
environmental contingencies (McLaughlin and Powell
1999) or be indicative of an affective component of
learning and memory (LeDoux et al. 1986). Our results
strongly suggest that decapods are altogether capable of
displaying a response in essence similar to that of vertebrates mediated by the autonomic nervous system. There
has been interest in the past to anatomical, neural and
endocrine comparisons of a putative autonomic nervous
system in arthropods to that of higher animals (Zavarzin
1941; Field and Larimer 1975a, b; Cooke 1988; Kuramoto and Yamagishi 1990; Wilkens and McMahon 1992;
McMahon 1995; Burmistrov and Shuranova 1996; Lee
et al. 2000; Li et al. 2000). Therefore it will be intriguing
to further investigate if the associated neural and humoral
control of bodily function by the autonomic nervous
system has likely evolved to regulate basic survival
strategy in invertebrates as well as in vertebrates.
Acknowledgements This work was supported by FONCYT (Grants
PICT-1-0662 and PICT Redes 0349) and Universidad de Buenos
Aires (Grant X-362). The authors are grateful for the fruitful
conversations with Daniel Tomsic. Appreciation is given to Diego
Anfossi and Angel Vidal for their technical assistance.
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