Antennal movements reveal associative learning in the American cockroach Periplaneta americana
1 Arizona Research Laboratories, Division of Neurobiology, 611 Gould-Simpson
Building, PO Box 210077, The University of Arizona, Tucson, AZ 85721,
USA
2 Dept of Biological Sciences, 6270 Medical Research Building III,
Vanderbilt University, 465 21st Ave. South, Nashville, TN 37235,
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: behavior, insect, cockroach, Periplaneta americana, memory, multimodal integration, antennal movement
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Introduction |
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While behavioral studies on cockroaches have demonstrated their suitability
for learning and memory studies
(Balderrama, 1980;
Gadd and Raubenheimer, 2000
), a
valid argument against using this taxon is that the behavioral paradigms have
been designed for free-moving animals and are thus unacceptable for studies at
the cellular level. In this and the succeeding paper
(Kwon et al., 2004
), we
describe learning paradigms that have been developed for use on restrained
animals so that, as in the case of the honey bee's proboscis extension reflex,
these can be employed for intracellular and biochemical studies.
Experiments described here rely on a stereotyped foraging behavior. This is
the antennal projection response (APR), which is reminiscent of sniffing in
mammals (Gray and Skinner,
1988) or antennular flicking in crayfish and spiny lobsters
(Mellon, 1997
;
Derby, 2000
). Such actions are
used to assess a continuously changing olfactory milieu and provide the brain
with data for locating smells. In lobsters, the frequency and directional
control of antennular flicking behaviors increase as mixtures of odor
components increase (Mellon,
1997
). Other modalities can also trigger antennal projection
responses. For example, in honey bees, antennal scanning can be elicited by
visual, olfactory and mechanical cues
(Erber et al., 1993
), and
antennal movements can be operantly conditioned
(Kisch and Erber, 1999
). When
crickets track moving objects, their antennae move in the same direction as
the object (Honegger,
1981
).
Here, we describe experiments that demonstrate a plastic behavior that can be driven in immobilized cockroaches. The behavior, which is expressed by APRs towards an olfactory stimulus source, can be classically conditioned and can be used for studying spatial context in learning and memory. We describe classical conditioning of APRs towards a neutral stimulus [a green light cue (conditioned stimulus, CS)] coupled with an odor source (unconditioned stimulus, US). The classical conditioning results in an APR towards the green light cue (CS), mimicking the response towards an odor source (US). The study explores whether an APR is indicative of recognition by the visual system of a stimulus location. The paradigm used here demonstrates a simple form of association between visual and olfactory information and shows that APRs can be used to test learning performance in immobilized cockroaches.
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Materials and methods |
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Arena and stimuli
All behavioral experiments were conducted inside a 1.5 mx1 mx1
m chamber surrounded uniformly by black curtains. An infrared heat lamp
(Supreme Co., Mullins, SC, USA) was positioned above the behavioral chamber to
provide warmth and red light illumination, a non-visible wavelength for
cockroaches, for video recording. The 30 cm-diameter arena was made of
polyethylene with 10 cm-high walls. Green light-emitting diodes (LEDs; peak
wavelength 565 nm; diameter 3 mm; E166; Gilway Technical Lamp Co., Woburn, MA,
USA) were positioned at regular intervals on the wall of the arena, to the
right of the cockroach's midline. These provided stationary light flashes.
Green light was presented during the pre-training, training (conditioning) and
test trials. A single red LED (625 nm, E100; Gilway Technical Lamp Co.), a
wavelength not visible to the cockroach, was positioned alongside the green
LED for spatial continuity and was used in a control test to determine if
sounds from the light switches were being detected.
Food odors (peanut butter; Skippy; Bestfoods Co., Eaglewood Cliffs, NJ, USA) were presented through an odor delivery system consisting of a syringe needle and a polyethylene tube (1 mm inner diameter) that were connected to odor sources. Pure air puffs (charcoal filtered; air pressure 10 Pa; stimulus duration 1 s) were blown through a cartridge containing the odor and controlled by a solenoid valve (General Valve Co., Fairfield, NJ, USA). Permanent air flow was provided by an exhaust fan system placed above and behind the arena to remove odors from the inside of the arena between trials. Peanut butter odor was used as the unconditioned stimulus (US). Odor was delivered from immediately above the green LED used for conditioning trials.
Stimuli and their sequences were controlled by a Grass S88 stimulator (Grass Instrument Co., Quincy, MA, USA). Light and odor cues used for training trials were 15 cm from the cockroach head and at an angle of 5° with respect to the midline of the head (Fig. 1A).
Monitoring and video recording of antennal movements
Antennal movements were video recorded with either an 8 mm Camcorder (Sony)
or a digital video camera (Panasonic). Video sequences of test cockroaches
were digitized every 167 ms for 20 s using Motus software (Peak Performance
Technologies, Inc., Englewood, CO, USA), which produced 120 images per
trial. From the digitized images, antennal angles were measured from the tip
and base of the right antenna and the green light position
(Fig. 1A).
Responses to sensory stimuli
Antennal responses to odor, light, mechanosensory and auditory cues were
tested to evaluate the unconditioned arousal responses of cockroaches. This
was done to control for arousal due to sensitization. Odor cues were given in
the form of peanut butter; light cues were in the form of a green LED;
mechanosensory cues were in the form of high-current air puffs; auditory cues
were given at a frequency of 1.8 kHz
(Shaw, 1994). In the absence
of any other stimuli, a cue was presented for 5x1 s, with a 1 min
interval.
Training
Cockroaches were first trained to project their right antenna towards a
green light as the CS, coupled with a food odor as the US. Procedures were
forward, simultaneous and backward conditioning
(Fig. 1B). In all three
procedures, the duration of visual and odor stimuli was 2 s and 1 s,
respectively. In forward conditioning, the CS was presented for 2 s and, after
a 2 s interval, the US was presented for 1 s
(Fig. 1Bi). In simultaneous
conditioning, the CS was presented for 2 s and, 1 s into the presentation, the
US was presented for 1 s (Fig.
1Bii). In backward conditioning, the US was presented for 1 s
followed by a 2 s interval and then 2 s CS
(Fig. 1Biii). Experimental
procedures consisted of three pre-training trials, in which only the CS was
presented, followed by five training trials (which was determined to be the
optimal number of training trials; data not shown) in which CS and US were
presented. Five minutes after the last training trial, either (1) three test
trials in which the CS was presented followed by three control trials in which
red light was presented or (2) three control trials of red light followed by
three test trials of the CS (Fig.
1C) were performed. The inter-trial intervals of all trials were 1
min.
Memory retention
Initial experiments showed that simultaneous conditioning was most
effective (Fig. 4). Using
simultaneous conditioning, short-term memory retention was measured at 5 min,
10 min, 20 min and 30 min after training. Long-term memory retention was
measured at 1 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h after training.
Throughout the tests, cockroaches remained restrained and were provided with
water to prevent dehydration.
|
APR as a measure of learning
An APR by an exploring cockroach is defined as a directed movement towards
the location of a specific stimulus, such as an odor, that is then followed by
local sampling movements of the antenna in the vicinity of the location to
which the antenna extended. Baseline movements of the antenna are small
deflections of the antenna that do not involve a defined movement towards a
stimulus. The conditioned response is the induced APR towards the position of
the CS. Baseline movement and antennal position are determined by analyzing
three 10-s time frames before beginning the training protocol. The APRs during
pre-training, training, testing and control are compared to this baseline.
Responses are scored as a `1' if an APR is elicited and is significantly
different from the baseline and `0' if there was no APR 10 s after
stimulation. Percentages of APRs were calculated by summation of all scores
during a given trial, as assessed by video observation.
Statistics
The Friedman test was used to compare APRs within subjects. Once a
significant difference was established, the Wilcoxon signed-rank test was
applied to compare each value of the repeated measurements. The Mann-Whitney
U test was used to test differences between two groups. Values shown
depict the responses `0' or `1' in percentages. Statistics were 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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Arousal versus conditioning: antennal responses to sensory
stimuli
Are antennal projection responses during the test phase due to arousal or
conditioning? To determine this, different sensory stimuli were presented
alone and the APRs analyzed (Fig.
3). The only significant APR to any stimulus was to odor
(Mann-Whitney U test, P<0.00001); there was no
significant antennal response to visual, mechanical or auditory stimulation
alone (Mann-Whitney U test, P>0.5). Thus, the pairing of
the olfactory stimulus, which induces an antennal projection, to the
non-arousing visual stimulus indeed provides a classical conditioning
paradigm.
|
Learning performance during different learning conditioning
sequences
Antennal projection responses elicited by forward (N=13),
simultaneous (N=21) and backward (N=11) conditioning showed
significant differences during pre-training, training and testing (Friedman
test, P<0.003; Fig.
4). APRs to green LEDs were below 10-20% during the pre-training
trials, during which cockroaches responded spontaneously, if at all, to the
light cues. During the five training trials, animals showed clear evidence of
learning to associate light cues with food odors and were not different from
testing trials (Wilcoxon signed-rank test, P>0.19). APRs were
significantly higher in simultaneous conditioning than those in forward and
backward conditioning (Mann-Whitney U test, P<0.001) and
showed no significant difference between forward and backward conditioning
(Mann-Whitney U test, P>0.2). Five minutes after
training, APRs of cockroaches to green LEDS were significantly increased
compared with pre-training levels (Friedman test, P<0.001).
Learned APRs after training
To determine memory retention, APRs were tested to green LEDs presented at
5 min, 10 min, 20 min and 30 min after training trials (N=18). APRs
to the visual cue were retained for at least 30 min after training
(Fig. 5A). APRs before and
after training were significantly different (Friedman test,
P<0.0001). A high percentage of APRs (80-90%) to the visual cue
were retained at 5 min, 10 min, 20 min and 30 min following training and
showed no difference in these intervals (Wilcoxon signed-rank test,
P>0.3). A red LED was presented with the same duration as that of
the green LED either before testing at 5 min or after testing at 5 min (Figs
1C,
5A). Cockroaches are
insensitive to red light (Seelinger and
Tobin, 1981), so that control tests with the LED should reveal
whether or not the insect has learned to associate the odor with sensory
modalities, other than the visual cue, that may have been present and not
evident to the experimenters or whether the response was due to an increased
antennal movement due to sensory arousal. Pre-training and control tests were
not significantly different (Wilcoxon signed-rank test, P>0.09).
Only spontaneous antennal movements were observed in response to the red LED
(Fig. 5A and
Fig. 2 D1-D3), indicating that
cockroaches learned to associate visual cues with the odor but not other
concurrent sensory stimuli that may have been present and there was no
increase in antennal movement due to arousal or sensitization.
|
Decay of the learned response over time
Antennal projection responses to the CS were tested three times at 5 min,
10 min, 20 min and 30 min after training and thereafter at 1 h, 3 h, 6 h, 12
h, 24 h, 48 h and 72 h. Antennal projection responses decrease after 30 min
from 90% to 60%, but even after 72 h were still significantly higher in four
of nine animals than at pre-training (Friedman test, P>0.7,
N=9). The persistence of this learned response to a visual cue may
suggest the establishment of long-term memory
(Fig. 5B).
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Discussion |
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Motor learning without a reward is not without precedent. After scanning an
object within the range of their antennae, honey bees will continue to make
antennal movements towards the position of the object even after it is removed
and even without receiving a reward (Erber
and Pribbenow, 1997).
Effects of stimulation intervals on learning performance
The inter-stimulus interval (ISI) and sequence of the unconditioned and
conditioning stimuli strongly influence retention. In honey bees, olfactory
learning, as assayed from the proboscis extension reflex, demonstrates that an
optimal learning performance is achieved when the ISI between the presentation
of the CS (odor) and the US (sucrose) is between 0 s and 5 s. If there is a
longer ISI, inhibitory learning results upon the presentation of the two
stimuli. This is shown by backward conditioning, where the ISI of the US and
CS exceed 1 s and result in inhibitory learning
(Menzel et al., 1993).
Backward conditioning in honey bees showed that an ISI of 15 s between the US
and CS induced maximum inhibitory learning
(Hellstern et al., 1998
),
suggesting that contiguity between the CS and US is critical in reward-based
learning performance. A reinforcer must be temporally connected to a stimulus.
The acquisition of a gill-withdrawal reflex after using electric shock as a
negative reinforcer in Aplysia showed that an ISI of 0.5 s between
the backward pairing of the CS and US induces no learning
(Hawkins et al., 1986
).
In the present experiments, ISIs of 2 s between the CS and US in forward and backward conditioning result in a weak learning performance compared with simultaneous conditioning (Fig. 4). That these responses are learned responses rather than sensitization is demonstrated by the observation that animals do not show significant responses during control testing, indicating that APRs induced by the CS after conditioning are due to associative learning rather than non-associative effects. Interestingly, short ISIs between the odor and visual cue in backward conditioning elicited moderate learning performance, suggesting that temporally close stimuli can be learned and that concurrent stimuli are not a prerequisite. In nature, foraging animals detect salient cues before the reward, implying that the ISI is a critical factor in reinforcement-based conditioning. Visual learning with food odors used here suggests that cockroaches can learn to associate visual cues with food odors if the ISI is less than 2 s.
Effect of inter-trial interval on learning
Intervals between training trials (ITIs) have an important influence on
learning and memory retention. Gerber et al.
(1998) examined the effect of
different ITIs (30 s, 1 min, 3 min or 20 min) on intermediate (1 day) or
long-term (4 days) memory. These authors demonstrated that proboscis extension
reflexes evoked during training using ITIs of 20 min and 1 min showed stable
intermediate and long-term retention, while 3 min and 30 s showed stable
intermediate but not long-term retention. The impairment of long-term memory
during the 30 s intervals may reflect massed training results and habituation.
Impairment using 3 min ITIs may be due to the disruption of consolidation of
each training trial. These results suggest that there is an ITI dependence of
the molecular mechanism involved. At the level of gene expression, spaced
training of Aplysia results in the expression of new protein
synthesis, which is essential for long-term memory formation, whereas massed
training did not (Alberini,
1999
).
In the present study, the interval between training trials was 1 min,
creating a `spaced training' protocol. Cockroaches showed a significant
learning performance after five training trials (Figs
4,
5A). Although learning
behaviors to varying ITIs were not tested, our results suggest that an ITI of
1 min with repeated presentation of mutimodal information in the absence of
rewards is sufficient to support long-term retention
(Fig. 5B) similar to that shown
in the honeybee (Gerber et al.,
1998).
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Acknowledgments |
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