Spatial learning in the restrained American cockroach Periplaneta americana
1 Dept of Biological Sciences, 6270 Medical Research Building III,
Vanderbilt University, 465 21st Ave. South, Nashville, TN 37235,
USA
2 Arizona Research Laboratories, Division of Neurobiology, 611 Gould-Simpson
Building, PO Box 210077, The University of Arizona, Tucson, AZ 85721,
USA
Author for correspondence (e-mail:
dlent{at}u.arizona.edu)
Accepted 29 September 2003
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Summary |
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Key words: place memory, cockroach, Periplaneta americana, antennal movement, behavior
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Introduction |
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The antennal motor system of insects can be used to develop novel
behavioral paradigms for studying associative memory (Lent and Kwon, 2004)
and, by extension, spatial memory. Antennal motor actions can be elicited by
different modalities, including olfactory, tactile and visual stimuli
(Erber et al., 1993;
Erber and Pribbenow, 1997
).
Antennal movements elicited by visual stimuli demonstrate active sensory
exploration, such as by restrained honey bees that move their antennae towards
the direction of a moving grating (Erber
and Pribbenow, 1997
). Visual inputs have also been shown to elicit
antennal movements in crickets (Honegger,
1981
). Such behaviors have been utlized by experiments in which
animals were operantly conditioned to extend their antennae towards a target
in order to receive a reward (Erber et al.,
1993
; Kisch and Erber,
1999
). In nature, directed antennal movements that are elicited by
a sensory stimulus (here termed antennal projection responses or APRs) may be
employed to locate an olfactory stimulant, such as the odor of food, a
conspecific or a predator (Bell,
1981
). As shown previously, APRs can be conditioned to point to a
visual cue after its learned association with a food odor (Lent and Kwon,
2004).
The present study describes a novel visual association paradigm to demonstrate spatial learning on restrained cockroaches, again exploiting antennal movements as an indicator of learning. The present results show that restrained insects can learn to recognize spatial relationships between distant cues. Because this can be demonstrated on an immobilized animal, the results provide a crucial step towards investigating place memory at the level of defined circuitry.
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Materials and methods |
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Arena and stimuli
As described previously, experiments were conducted in an arena enclosed
within a visually uniform chamber illuminated with an infrared lamp
(Fig. 1A). A restrained
cockroach was positioned in the middle of the arena and aligned with respect
to five green LEDs on the arena wall positioned at 15° intervals to the
right of the insect (Fig. 1A).
The distance from the insect's head to the position of these cues was 15 cm.
Each diode was given a number from 1 to 5. Four white LEDs (E1000; Gilway
Technical Lamp Co., Woburn, MA, USA) were positioned on the wall of the arena
to the left of the insect. These contralateral reference stimuli (ConRS) were
also spaced at 15° intervals with respect to the cockroach and named
A-D.
Food odors controlled by a solenoid valve were presented through an odor delivery system positioned at green diode 1. The duration of the odor stimulation was 1 s. A ventilation system was placed above the arena to remove odor after each trial (see Lent and Kwon, 2004 for details).
Monitoring and video recording of antennal movements
Antennal movements were captured with either an 8 mm Camcorder (Sony) or a
digital video camera (Panasonic) and recorded on tape. Digitized images
provided the raw data used to analyze antennal movements in each trial.
Antennal movements recorded for 10 s after stimulation were digitized by the
Motus program (Peak Performance Technologies, Inc., Englewood, CO, USA), which
captured images every 167 ms, producing 60 images per trial. From these
digitized images, the tip and base of the right antenna of each test cockroach
and the position of the green light cue were recorded to obtain angle data
with respect to the midline of the head. These antennal angles thus quantified
antennal movements with respect to different positions of the green LED.
Training procedures
The white diode at position A was switched on during the pre-training and
training trials. One pre-training trial was followed by five training trials.
These were succeeded by 3-8 test trials, depending on the experiment. These
protocols are summarized in Fig.
1B.
During the training trials, peanut butter odor was emitted under solenoid control at a position coincident with green LED 1 to provide the unconditioned stimulus (US). The green LED at position 1 served as the conditioned stimulus (CS). A Grass S88 stimulator (Grass Instrument Co., Quincy, MA, USA) controlled the sequence of the US and CS. As described by Lent and Kwon (2004), the US was given 1 s after CS onset, thus providing a simultaneous conditioning protocol. In all experiments, except test 1 (see below), pre-training consisted of a 2 s presentation of the green LED at position 1, without an odor cue, and during continuous illumination by the white diode (ConRS) at contralateral position A (referred to as A+1). Pre-training measured a cockroach's innate response in the presence of the A+1 configuration. In the training trials, the green LED at position 1 was coupled with the food odor. The CS and US were presented in the context of continuous contralateral reference illumination by the white LED at position A (A+1+odor) for five trials, with an inter-trial interval (ITI) of 1 min. Post-training tests began 5 min after the last training trial and each lasted 1 min, with a 3 min ITI between each test. For the duration of each test, one of the white contralateral LEDs at one of positions A-D was illuminated. Then, one of the green LEDs at one of positions 1-5 was presented for 2 s. Between each test, the animal was covered with a black box (15 cmx15 cmx20 cm) while a white LED was switched on at a new position (A-D), after which the box was removed. After 40 s, the animal's APR was tested by illuminating the ipsilateral eye with a green LED at a new position for 2 s. Because of the time required to change the positions of the contralateral visual cues (Fig. 1B) 3 min ITIs were maintained during test trials.
Testing procedures
Testing procedures determine whether an APR can be elicited when the CS
alone is displaced from its original position (test 1), when the CS is
displayed with a ConRS (test 2), when the CS is displaced but the ConRS is not
(test 3), when the ConRS is displaced but the CS is not (test 4) or when both
CS and ConRS are displaced in various combinations (test 5).
Scoring APRs and statistics
An APR for the trial was scored as `1' if there were antennal movements
towards the green LED (±2.5°) during the 10 s that followed
stimulation. A score of `0' was assigned if there were no antennal projection
movements or if there was only antennal tremor but no projections towards the
green LED. APRs are shown as percentages of `1' responses during a given
trial, as assessed by video observation. Non-parametric analytical tests were
performed to compare APRs during pre-training and testing. The Friedman test
was used to compare APRs within subjects. Once a significant difference was
shown, a Wilcoxon signed-rank test (Z statistic) was performed in
parallel to compare each value of every trial. The Kruskal-Wallis test
(H statistic) was performed to compare the antennal responses between
groups. Mann-Whitney U tests (U statistic) were used to test
the differences between two groups. Values shown depict the responses `0' or
`1' in percentages. Statistical analysis was carried out using Statistica 5.5
for Windows and results were regarded as `not significant' if
P>0.05.
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Results |
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Test 2: CS and ConRS fixed
This experiment determines whether a cockroach can project its right
antenna towards the unchanged position of the green light cues during the
simultaneous presentation of ConRS at a fixed position `A'. The positions of
the ConRS and the CS at position 1 (A+1) were maintained throughout this
experiment. APRs to the CS at position 1 were scored
(Fig. 3). APRs of the right
antenna towards the green light position after training were significantly
increased compared with pre-training (Fig.
3; Wilcoxon signed-rank test, Z=2.37, N=7,
P<0.02), showing that cockroaches learn the CS in the presence of
additional visual information. These results do not show how or if the animal
is using this additional sensory information.
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Test 3: CS displaced and ConRS fixed
If APRs can be elicited by the changed position of the CS (as described in
test 1), does this still occur when the CS is displaced but ConRS is
maintained at A throughout? During pre-training, APRs to the green LED at
position 1 were tested in the absence of odor cues but in the presence of the
ConRS at position A. Training of APRs was to the CS+US in the presence of the
ConRS. Insects were then tested with altered positions of the CS while
maintaining the position of the ConRS. The spatial configurations to which the
insects were tested are thus A+1, A+2, A+3 and A+4
(Fig. 4).
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Throughout tests, APRs to different green light positions showed
significant differences (Fig.
4; Friedman test, N=18, 2=33.82, d.f.=5,
P<0.0001). The first test in response to A+1 revealed a
significant increase in APRs compared with pre-training
(Fig. 4; Wilcoxon signed-rank
test, N=18, Z=2.95, P<0.005). APRs to A+2, A+3
and A+4 were not significantly different from those during pre-training
(Wilcoxon signed-rank test, Z=1.83, 1.46, 0.534, respectively,
N=18, P>0.1). To control for sensitization or arousal,
APRs to A+1 were tested again ('A+1 again'); the tests showed a significant
difference compared with tests using A+2, A+3 and A+4 (Wilcoxon signed-rank
test, N=18, Z=2.04, 2.04, 2.69, respectively,
P<0.05). These results are evidence that the ConRS plays a role in
place recognition of the CS and may be used in relation to the CS during
learning. In contrast to test 1 above, the animal no longer points to the
green LED if the LED position is shifted from that learned during
training.
Test 4: CS fixed and ConRS displaced
After training, tests were made with the ConRS positions changed from A to
B, C or D, with the final test made with the ConRS position returned to A
again. Throughout these tests, the position of the CS remained at position 1.
The spatial configurations that tested APRs were thus A+1, B+1, C+1, D+1 and
`A+1 again'. The sequence of changed positions of the ConRS was randomized,
but the first and last trials were always A+1
(Fig. 5).
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APRs to the CS at position 1 coupled with the altered positions of the
ConRS were significantly different throughout the tests (Friedman test,
N=17, 2=38.2, d.f.=5, P<0.0001). APRs
towards A+1 and `A+1 again' were significantly increased compared with those
of pre-training (Wilcoxon signed-rank test, N=17, Z=2.69,
3.06, respectively, P<0.01). However, APRs elicited by B+1, C+1
and D+1 were not significantly different from APRs in pre-training (Wilcoxon
signed-rank test, N=17, Z=0.535, 0.535, 0.535, respectively,
P>0.5). This experiment provides further support that the animal
is using the relationship between the CS and ConRS during learning.
Test 5: CS and ConRS displaced
Finally, we investigated whether APRs could be elicited to a changed
position of the CS if the ConRS position was correspondingly changed. We
compared tests in which the angular relationships between the CS and ConRS
were the same as the original trained angular relationship of the ConRS at
position A and the CS at position 1 (see
Fig. 1A). In these tests, the
original angular relationships were preserved when A was shown with 1 (A+1), B
with 2 (B+2), C with 3 (C+3) and D with 4 (D+4). Random sequences were tested,
after which APRs were tested using different angular relationships, such as
B+3, C+4 and D+5. The final test was for APRs towards `A+1 again'
(Fig. 6).
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APRs towards A+1, B+2, C+3 and D+4, which all had the same angular relationships, showed significant differences from pre-training APRs (Fig. 6A; Wilcoxon signed-ranked test, N=17, Z=3.18, 3.18, 2.93, 2.37, respectively, P<0.02). However, APRs towards B+3 (N=14), C+4 (N=16) and D+5 (N=14), all of which had angular relationships that differed from the combination of the training stimulus A+1, were not significantly different from APRs during pre-training (Fig. 6A; Wilcoxon signed-rank test, Z=1.47, 0.53, 0.91, respectively, P>0.14). Within APRs to the same angular relationships, responses to D+4 were significantly different from those towards A+1 and B+2 (Wilcoxon signed-rank test, N=17, Z=2.20, 2.20, respectively, P<0.03) but not C+3 (Wilcoxon signed-rank test, N=17, Z=1.47, P>0.14). APRs towards B+3 (angle mismatch) showed no difference from C+3 (Wilcoxon signed-rank test, N=14, Z=1.82, P>0.06) and D+4 (Wilcoxon signed-rank test, N=14, Z=0.73, P>0.4). APRs towards `A+1 again' increased significantly in the last trial of the tests and were significantly different from APRs during pre-training (Wilcoxon signed-rank test, N=7, Z=2.36, P<0.03).
APRs towards the same and discrepant angular relationships of the green
light and ConRS were pooled to compare overall behaviors.
Fig. 6B elaborates on the
patterns of APRs towards the same and discrepant angular relationships
compared with during pre-training. APRs to the green light were significantly
influenced by the ConRS, being generally maintained when the angles between
the green light and ConRS were maintained (Kruskal-Wallis test, N=53,
68 and 44 for pre-training, same and different angular relationships,
respectively, H=40.95, P<0.0001). The average APRs to
green light positions when the angular relationships between the ConRS and
green light positions were maintained were 70%
(Fig. 6B). This was
significantly different from APRs in response to the green light positions
when the angle between these and ConRS were altered (Mann-Whitney U
test, U=746, P<0.0001). Also, compared with APRs towards
A+1 in the tests (N=21) in which positions of visual cues were not
changed throughout (CS and ConRS fixed;
Fig. 3), APRs towards green
light positions having the same angular relationships (N=68) showed
no significant difference (Mann-Whitney U test, U=640,
P=0.47). Pre-training APRs showed no difference from APRs towards
green light positions having angular relationships to ConRS that were
different from A+1 (Mann-Whitney U test, U=1162.5,
P>0.9).
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Discussion |
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If the recognition of the learned CS is a function of point-to-point retinotopic matching, does this mean that retinotopic matching itself requires two discrete retinal stimuli? Experiments in which the position of the CS and ConRS are changed suggest that retinotopic matching also involves the retintopic subtense of the two stimuli. When the learned spatial configuration [the arc distances (angle)] of the two stimuli is maintained, the animal projects its antenna to the new location of the CS (Fig. 6A). But if the arc distances are altered from that provided during training (e.g. B+3, C+4 and D+5 in Fig. 6A), the antenna is not projected to the new position of the CS. Therefore, APRs to the CS in the context of a second visual cue rely on the recognition of angular matching rather than retinotopic matching.
Does this occur in nature? Rust et al.
(1976) showed that cockroaches
turn their heads towards a pheromone source to facilitate antennal scanning in
the direction of the odor plume. This behavior indicates that head movements
follow antennal movements and that by realigning their antennae insects
achieve greater precision of information about an odor source
(Murlis, 1992
). However, as
far as we are aware, the role of visual cues in such olfactory-driven
behaviors has not been investigated. Here, we have provided evidence that
animals are able to localize food sources in the absence of information
provided by an odor plume given that they have previously learned to associate
the food source with visual cues. We suggest that external reference cues
provide increased precision in localizing odor sources.
Behavioral and neural correlates of spatial learning
Where and how memory templates of sensory scenes are formed, stored and
compared in the insect brain is still unknown. A requirement for investigating
underlying mechanisms, using electrophysiological methods, is to have
behavioral paradigms for spatial learning. This and our previous study (Lent
and Kwon, 2004) demonstrate spatial learning abilities in a restrained insect
using antennal movement patterns to provide reliable behavioral indicators.
Evidence from lesion studies suggests that the mushroom bodies play a crucial
role in visual associative and spatial learning
(Mizunami et al., 1998). When
adapted for intracellular recordings, the present behavioral paradigm should
provide new insight into mechanisms of spatial learning in insects.
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Acknowledgments |
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Footnotes |
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References |
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