Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Brighton BN1 9QG, United Kingdom
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ABSTRACT |
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Jones, Nick,
György Kemenes, and
Paul R. Benjamin.
Selective Expression of Electrical Correlates of Differential
Appetitive Classical Conditioning in a Feeding Network.
J. Neurophysiol. 85: 89-97, 2001.
Electrical correlates of differential appetitive classical conditioning
were recorded in the neural network that underlies feeding in the snail
Lymnaea stagnalis. In spaced training (15 trials over 3 days), the lips and the tentacle were used as CS+ (reinforced
conditioned stimulus) or CS (nonreinforced conditioned stimulus)
sites for behavioral tactile conditioning. In one group of experimental
animals, touch to the lips (the CS+ site) was followed by sucrose (the
unconditioned stimulus, US), but touch to the tentacle (the CS
site)
was not reinforced. In a second experimental group the CS+/CS
sites
were reversed. Semi-intact lip-tentacle-CNS preparations were made from
both experimental groups and a naive control group. Intracellular
recordings were made from the B3 motor neuron of the feeding network,
which allowed the monitoring of activity in the feeding central pattern
generator (CPG) interneurons as well as early synaptic inputs evoked by the touch stimulus. Following successful behavioral conditioning, the
touch stimulus evoked CPG-driven fictive feeding activity at the CS+
but not the CS
sites in both experimental groups. Naive
snails/preparations showed no touch responses. A weak asymmetrical stimulus generalization of conditioned feeding was not retained at the
electrophysiological level. An early excitatory postsynaptic potential
(EPSP) response to touch was only enhanced following conditioning in
the Lip CS+/tentacle CS
group but not in the Tentacle CS+/lip CS
group. The results show that the main features of differential
appetitive classical conditioning can be recorded at the
electrophysiological level, but some characteristics of the conditioned
response are selectively expressed in the reduced preparation.
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INTRODUCTION |
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Invertebrates provide excellent
model systems for studies on the cellular and molecular mechanisms of
learning and memory (Krasne and Glanzman 1995) and have
provided important information on the nature of changes resulting from
associative conditioning. Gastropod mollusks demonstrate both
nonassociative and associative learning (Byrne 1987
;
Carew and Sahley 1986
). They also have relatively simple
nervous systems and large neurons, which are accessible to
intracellular analysis, and this has made them particularly valuable in
the search for the cellular mechanisms of behavioral plasticity. One
such model gastropod is the pond snail Lymnaea stagnalis.
This animal previously was shown to demonstrate a variety of distinct
types of classical and operant conditioning (Audesirk et al.
1982
; Kemenes and Benjamin 1989a
,b
;
Kojima et al. 1996
; Lukowiak et al.
1996
). Appetitive classical conditioning was one particular
learning paradigm that was extensively studied in Lymnaea at
both the behavioral and cellular level (Kemenes et al.
1997
; Staras et al. 1998
, 1999a
;
Whelan and McCrohan 1996
). Following classical
conditioning of the rhythmic feeding behavior using a lip-touch
training paradigm, electrical correlates of nondifferential appetitive
conditioning could be recorded in a semi-intact preparation. Changes
occurred at several different sites in the feeding network, at the
level of the central pattern generator (CPG) interneuronal network, the
motor neurons, and the conditioned stimulus (CS) pathway (Staras
et al. 1999a
).
Kemenes and Benjamin (1989a) also showed that the touch
learning paradigm in Lymnaea could be developed further to
demonstrate a more complex form of appetitive conditioning,
differential classical conditioning, a type of discriminative learning
behavior. In differential conditioning, two CS sites, the lips and the
tentacles, were used in the same animal. At one site (the CS+) touch
was paired with sucrose, and at the other one (the CS
) touch was
specifically unpaired. After 15 spaced trials discriminative learning
was demonstrated. The aim of the present experiments was to determine
whether neural correlates of this behavioral differential appetitive
conditioning could be obtained. This was successful at the level of the
sustained CPG-driven differentially conditioned fictive feeding pattern in both experimental groups. Another aspect of behavioral differential conditioning, weak asymmetrical stimulus generalization, was not retained in the reduced preparation. Moreover, a known
electrophysiological consequence of appetitive conditioning, an
increase of the amplitude of an early excitatory postsynaptic potential
(EPSP) response to touch (Staras et al. 1999a
) was only
seen in the Lip CS+/tentacle CS
group but not in the Tentacle CS+/lip
CS
group, indicating the selective expression of the neural
correlates of behavioral differential conditioning.
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METHODS |
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Animals
Wild-type specimens of adult Lymnaea stagnalis were obtained from animal suppliers (Blades Biological, Kent, UK). The snails were kept in groups in large holding tanks, containing copper-free water at 18-20°C on a 12 h:12 h light, dark regime and fed lettuce three times a week.
Behavioral experiments
Before the experiment, animals were moved into three tanks in the laboratory. During the training procedure the animals were given a little lettuce at the end of each day's training so that they were in a semi-satiated state. All lettuce was removed from the home tanks 45 min before the first trial and the water was replaced daily.
DIFFERENTIAL CONDITIONING TRAINING.
The simpler nondifferential appetitive conditioning paradigm uses lip
touch as the CS followed by sucrose as the unconditioned stimulus (US)
in a spaced training schedule (Kemenes and Benjamin 1989a; Staras et al. 1998
,
1999a
). Differential conditioning involved two sites on
the body, the lips and tentacles, both of which could be used as CS
sites (Kemenes and Benjamin 1989a
). In a balanced design, both lips and tentacles act as reinforced conditioned stimulus
(CS+) or nonreinforced conditioned stimulus (CS
) sites for touch.
Thus touch at the CS+ site is followed by sucrose (the US) dissolved in
water, but touch at the CS
site is only followed by water to equalize
for the displacement caused by the addition of sucrose solution to the
dish after touching the CS+ site (Fig. 1). As well as these two experimental
groups, a third, naive group, was used where spontaneous feeding rates
were measured and the lack of basic feeding responses to touch was
ascertained.
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BEHAVIORAL TESTING OF THE DIFFERENTIALLY CONDITIONED FEEDING RESPONSE. Testing was performed using a blind procedure, by a person who had no knowledge of the training history of the animals. During testing, animals were removed from the home tanks and placed in shallow test dishes containing 100 ml copper-free water. The test dishes were placed on a Perspex box with a mirror angled at 45° inside that allowed the animals to be observed while they were moving around on the bottom of the dish. In Lymnaea, feeding movements (rasps) consist of cycles of mouth opening and closing accompanied by extrusion of the toothed radula of the buccal mass. Each cycle of feeding was counted by visual observation using a hand-held counter.
After emergence of the snail from its shell, any spontaneous feeding activity was recorded for 2 min, and then a touch stimulus was applied to the lips or the tip of the left tentacle. For a balanced test protocol, we alternated the order in which these two different test sites were used from one animal to another. After applying the touch stimulus to the test sites, feeding activity was recorded for a further 2 min before the animals were replaced into the home tanks. After all the animals had been tested on touch at one of the test sites, they were tested again after 90 min for a feeding response to touch to the other site. This testing regime ensured that approximately half of the animals in all three groups were first tested for a response to lip touch followed by a tentacle touch, and the remaining animals within the group were tested in the reverse order.Electrophysiological experiments
The main aim of these experiments was to record electrical changes in semi-intact preparations resulting from differential conditioning of the intact snails. Following training on three consecutive days snails were kept overnight in a sub-satiated state, and electrophysiological experiments were carried out on the following 2 days.
GENERAL PROCEDURES.
Individual snails from experimental and control groups of animals were
dissected under a light microscope in a silicone elastomer (Sylgard)-lined dish containing
N-2-hydroxyethylpiperazine-N-ethanesulfonic acid
(HEPES)-buffered snail saline previously described by Benjamin and Winlow (1981). The preparations were then transferred to
the electrophysiology chamber. Before recording, the outer ganglionic sheath surrounding the CNS was removed from around the buccal ganglia
using a pair of fine forceps, and the inner ganglionic sheath was
softened using a nonspecific protease (Sigma, XIV, Sigma Chemicals,
Poole, UK) to aid intracellular recording. An experimenter who had no
prior knowledge of the training history of the individual animals then
performed the rest of the electrophysiological procedures.
PREPARATIONS.
"Whole lip-tentacle-CNS" semi-intact preparations were developed
that allowed the lips and tentacles to be stimulated by touch while
simultaneously recording from neurons of the feeding system. The CNS
was accessed by a dorsal body incision and all the peripheral nerves
cut except for the paired superior and median lip nerves and the
tentacle nerves. The buccal mass was excised, but a short length of
esophagus was left attached to the brain to allow pinning out. The lips
and the surrounding structures including the tentacles were arranged in
the electrophysiological chamber facing upward so that the CS+ and CS
sites were accessible to tactile stimulation. The tactile stimulus was
applied to either the lips or tentacle using an electromagnetic
coil-driven mechanical probe that was designed to closely mimic the
handheld probe used in the behavioral experiments (see Staras et
al. 1999a
). By pulling the intact CNS down below the peripheral
head structures, the buccal ganglia could be stretched clear of the
head structures over a raised Sylgard block so that the dorsal side was
exposed for intracellular recording, as previously described by
Staras et al. (1998)
.
INTRACELLULAR RECORDING TECHNIQUES.
Glass microelectrodes, with tip resistances ranging from 10 to 60 M
when filled with 4 M potassium acetate solution, were used in the
electrophysiological experiments. These were pulled on a Narashigi
vertical electrode puller. The tips were dipped in black etching ink to
improve visualization. The electrophysiology chamber in which the
preparations were pinned was illuminated with a cold light source, and
the preparations were viewed under a stereo microscope. To allow
intracellular recording, micromanipulators with attached headstage
preamplifiers (Neurolog, Digitimer, Welwyn Garden City, UK) were
arranged around the electrophysiological chamber. Signals were fed into
amplifiers (NL102G, Digitimer) incorporating a bridge-balance circuit
for current injection and then outputted to a storage oscilloscope
(GOULD 1604, Gould Instrument Systems, Hainault, UK) and a chart
recorder (GOULD TA240S). All signals were recorded digitally using a
DAT recorder (BIOLOGIC DTR-1801, Biologic Science Instruments, Claix, France).
INTRACELLULAR RECORDING OF THE B3 MOTOR NEURON.
There were two main goals for the electrophysiological
experiments. The first was to monitor fictive feeding activity in the semi-intact preparations, and the second was to record an early synaptic response to lip touch, previously reported on feeding neurons
(Staras et al. 1999a,b
). Both types of response were
enhanced following training in earlier nondifferential tactile
conditioning experiments (Staras et al. 1998
,
1999a
). The B3 motor neurons were used because food
(sucrose) is known to elicit sequences of burst activity that are
representative of fictive feeding activity throughout the feeding
network. Similar patterns of neural activity were known to underlie
feeding movements in the intact animal (Rose and Benjamin
1979
). Feeding patterns are generated by a CPG consisting of
three main types of interneurons (N1, N2, and N3) and at least three
modulatory and command neurons (Benjamin and Elliott
1989
). The B3 motor neuron receives inhibitory input in the N1,
protraction phase and is excited in the subsequent N2 and N3, rasp and
swallow phases (Rose and Benjamin 1981
). Therefore by
intracellularly recording from the B3 motor neuron, it was possible to
monitor indirectly the activity of all the components of the CPG. We
impaled the B3 with two microelectrodes, one for recording the
electrical signals and one for passing current into the cell. The
current passing electrode was used to maintain the membrane potential
of the cell at
65 mV, the average of previous measurements of B3
resting potential (Staras et al. 1999a
). This current
clamp allowed the early depolarizing synaptic response in B3 due to
touch (input I of Staras et al. 1999a
) to be recorded at
a constant membrane potential. Changes in the amplitude of the early
compound EPSP were recorded as a further monitor of the effect of
differential conditioning.
ELECTROPHYSIOLOGICAL TESTING OF THE DIFFERENTIALLY CONDITIONED FEEDING RESPONSE. After the preparation had been set up by an experimenter who had no knowledge of its training history, spontaneous fictive feeding activity was recorded for 2 min, and then a touch stimulus was applied to one of the CS sites. This was repeated after a 2-min interval. After a further 2-min interval the other CS site was stimulated, and this was repeated again after another 2 min. Randomization of the testing regime was achieved by using preparations provided by a second experimenter that had been randomly selected from the three groups to be tested.
Statistical analysis of behavioral and electrophysiological data
The same types of statistical analysis were used for both
the behavioral and electrophysiological experiments. All analysis was
done blind with the person analyzing the records unaware of the group
from which the snail/preparation had originated. First, the presence of
a normal distribution of both prestimulus (spontaneous) and
poststimulus (touch-evoked) rasp/fictive feeding rate data were
established [Normal Probability Plot, SPSS (Norusus
1995)]. This justified the subsequent use of parametric
analysis techniques to compare data (presented as means ± SE)
both within and between groups. A one-way ANOVA on the prestimulus data
established that all three groups (Lip CS+/tentacle CS
; Tentacle
CS+/lip CS
and Naive) were matched for both spontaneous rasping and
fictive feeding rates before either a lip or a tentacle touch was
applied [F(5,75) = 1.4, P = 0.26, behavior; F(5,58) = 0.64, P = 0.67, electrophysiology].
Responses to touch to the lips or the tentacle were quantified by
awarding a difference score to each animal for each site. This was
calculated by subtracting the number of rasps/fictive feeding cycles in
the minute preceding the tactile stimulus from the number of
rasps/fictive feeding cycles in the minute after the first rasp/fictive
feeding cycle after the tactile stimulus. Poststimulus rasps/cycles
were only counted if the first rasp/cycle occurred within 1 min after
the stimulus; otherwise the poststimulus rate was regarded as zero. For
the analysis of fictive feeding rates, the rates measured before and
after the two stimuli to the same site were averaged. These are the
same methods that were used by Staras et al. (1998,
1999a
) to analyze behavioral and electrophysiological
changes after simple appetitive classical conditioning. Paired
t-tests were then performed on data within each
group to determine whether touch to a site, either lip or tentacle,
induced a significant change in the rasping/fictive feeding rate over
prestimulus levels measured in the same preparations. Differences
between responses to touch applied to the two different sites in the
same group were also analyzed using paired t-tests. To
make comparisons between different groups, first an
ANOVA was performed separately for responses to touch to the lips and
tentacle, respectively. Only when a source of significant difference
was revealed was the data further subjected to post hoc analysis
(Tukey's B test) to determine where the source of significant
difference originated.
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RESULTS |
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Behavioral analysis
LIP CS+/TENTACLE CS GROUP.
In almost all of the Lip CS+/tentacle CS
animals (12 of 13), touch to
the lips at test increased the feeding rate following training. The
mean spontaneous feeding rate before touch was applied to the lips was
1.3 ± 0.8 (SE) rasps/min. After tactile stimulation of the lips,
the mean feeding rate rose to 5.3 ± 1.0 rasps/min (Fig.
2Ai), and statistical analysis
showed that there was a significant difference between the pre- and
poststimulus data (paired t-test, df = 12, t = 6.1; P < 0.001).
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TENTACLE CS+/LIP CS GROUP.
The results with this group of animals were more complex than with the
Lip CS+/tentacle CS
group. As expected, touch to the CS+ site after
training increased feeding rates (in 15 of 17 animals) over spontaneous
levels. The mean feeding rate before touch was 1.2 ± 0.4 rasps/min; this increased to 5.3 ± 0.9 rasps/min after touch, and
this was statistically significant (paired t-test, df = 16, t = 2.6; P < 0.001), indicating
successful conditioning of the CS+ tentacle site. However, increases in
feeding rate were also observed at the CS
lip site (Fig.
2Bii). This occurred in a smaller proportion of animals (10 of 17, ~60% for the lips, compared with 90% for the tentacles), and
the increase in mean feeding rate was smaller (less than twice,
2.2 ± 0.8 rasps/min before; 4.2 ± 1.0 rasps/min after, for
lips compared with more than 3 times for the tentacle), but it was
still significant (paired t-test, df = 16, t = 2.6; P < 0.02). This indicated
that the effects of food reinforcement on the tentacle were being
transferred to the alternative lip site, despite its CS
status. This
suggests that stimulus generalization was occurring, but only in one
direction, from tentacle to lips (Fig. 2Bii) and not from
lips to tentacle (see Fig. 2Aii). Touching the CS+ tentacle
site after training still produced significantly greater conditioned
feeding responses than the CS
lip site in the same animals (Fig.
2Biii; paired t-test, df = 16, t = 2.1; P < 0.05) showing that there
was still a distinction between the effectiveness of the two sites in
eliciting conditioned feeding and that generalization was only partial.
NAIVE GROUP. The naive control group provided baseline data for comparison with the two experimental groups. It also showed that there were no significant differences in the before and after touch scores at either the lips (Fig. 2Ci) or tentacles (Fig. 2Cii), and it follows from this that simply touching either of these sites does not induce any feeding responses (Fig. 2Ciii).
COMPARISONS BETWEEN LIP CS+/TENTACLE CS, TENTACLE CS+/LIP CS
,
AND NAIVE GROUPS.
The inter-group analysis was useful in that it confirmed the basic
discriminative phenomena revealed by the within group analysis. Thus
the effect of lip touch (Fig. 2, Aiii-Ciii, open bars) was significantly greater in the Lip CS+/tentacle CS
group than in either
the Tentacle CS+/lip CS
or the Naive group (Tukey's B test,
P < 0.05). Similarly, the effect of tentacle touch
(Fig. 2, Aiii-Ciii, solid bars) was found to be
significantly greater in the Tentacle CS+/lip CS
group than in either
of the other two groups (Tukey's B test, P < 0.05).
However, the inter-group analysis was less successful in detecting the
stimulus generalization revealed by the paired test in the Tentacle
CS+/lip CS
group. This was indicated by the lack of significance when
the difference score (Fig. 2Biii, open bar) to lip touch in
the Tentacle CS+/lip CS
group was compared with baseline response to
touch in the naive group (Fig. 2Ciii, open bar; Tukey's B
test, P > 0.05). Inter-group analyses are always less
sensitive than their within group equivalents because of group
differences, and this made it more difficult to confirm the fairly weak
stimulus generalization.
Electrophysiology
ANALYSIS OF TOUCH-EVOKED FICTIVE FEEDING. The same animals that had been behaviorally trained over 3 days were kept overnight in a sub-satiated state and semi-intact preparations made for electrophysiological recordings. The person performing the electrophysiological experiments on the snails was unaware of their behavioral scores, and the animals to be dissected were drawn randomly from the three groups (2 experimental, 1 naive).
LIP CS+/TENTACLE CS GROUP.
In 80% of the preparations (8 of 10), touch to the lips (CS+ site)
increased the fictive feeding rate (Fig.
3Ai). The mean fictive feeding
rate following touch increased to 2.2 ± 0.5 cycles/min from
0.85 ± 0.2 cycles/min before touch. When the data were analyzed, using a paired parametric test, the increase in mean fictive feeding rate was found to be significant (df = 9, t = 4.2;
P < 0.002; Fig. 3Ai). This increase in the
fictive feeding rate is substantially lower than the conditioned
feeding rates in response to touch in the intact animals, but similar
differences between intact snails and semi-intact preparations
following nondifferential appetitive conditioning were reported earlier
by Staras et al. (1998
, 1999a
).
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TENTACLE CS+/LIP CS GROUP.
Touch to the tentacle CS+ site increased fictive feeding rates in the
semi-intact preparation (Fig. 3Bi). This occurred in 11 of
the 12 preparations tested (mean fictive feeding rates before 1.3 ± 0.4 cycles/min and after 2.9 ± 0.9 cycles/min), and the increase was statistically significant (paired t-test,
df = 11, t = 4.8; P < 0.001).
This result was similar to the equivalent behavioral response seen in
the whole animals. However, touch applied to the CS
site produced
different results from those obtained in the same animals in the
behavioral experiments. Instead of stimulus generalization and an
enhanced response to touch on the lips, no increase in fictive feeding
was observed in the semi-intact preparations. There was a small
increase in mean fictive feeding rates (from 1.5 ± 0.5 cycles/min
to 1.6 ± 0.4 cycles/min; Fig. 3Bii), but this was not
statistically significant. Most of the preparations (7 of 12) did not
respond at all, with the remainder showing one or two cycles of
activity in the minute after touch. In the example shown in Fig.
4B, the early EPSP evoked a burst of spikes, but then only
one burst of spikes was seen in the 1 min following touch in the B3
motor neuron. This contrasts with the tentacle CS+ site where a more
sustained pattern of fictive feeding was recorded with five bursts of
spikes following the early EPSP-evoked activity (Fig. 4B, bottom
trace).
NAIVE GROUP. As predicted from the behavioral experiments, the naive group did not show fictive feeding responses (Fig. 3Ciii) at either the tentacle or lip test site, and this is illustrated in an example of the electrophysiological records shown in Fig. 4C.
COMPARISONS BETWEEN LIP CS+/TENTACLE CS, TENTACLE CS+/LIP CS
,
AND NAIVE GROUPS.
The statistical comparison of the electrophysiological data
between groups confirmed what was expected from successful differential conditioning. The main significant differences were between the conditioning shown for lip touch in the Lip CS+/tentacle CS
group (Fig. 3Aiii, open bar) versus the other two groups. The
effect of lip touch in this group was significantly greater (Tukey's B
test, P < 0.05) than that of lip touch in the Tentacle
CS+/lip CS
(Fig. 3Biii, open bar) and Naive groups (Fig.
3Ciii, open bar). For tentacle touch the response in the
Tentacle CS+/lip CS
group was significantly greater (Fig.
3Biii, filled bar) than either the Lip CS+/tentacle CS
group (Fig. 3Aiii, filled bar) or the Naive group (Fig.
3Ciii, filled bar; Tukey's B test, P < 0.05).
Analysis of the amplitude of the touch-evoked EPSP in motor neuron B3
Previous studies of Staras et al. (1999a,b
) showed
that the earliest response to lip touch on the B3 motor neuron was a
compound EPSP. The maximum amplitude of the EPSP but not the duration
was enhanced following nondifferential appetitive conditioning
(Staras et al. 1999a
). A similar early EPSP also
occurred in B3 following tentacle touch (e.g., Fig. 4C), so
we examined whether the size of the responses from the two sites would
change after differential conditioning.
Maximum EPSP amplitude (Fig.
5A) was measured with no
knowledge of the origin of the preparation in the Lip CS+/tentacle CS (n = 10), Tentacle CS+/lip CS
(n = 11), and Naive groups (n = 10; Fig. 5B). The
only significant difference was found within the Lip CS+/tentacle CS
group. As predicted by the successful differential conditioning of the
behavioral response and its electrophysiological correlate, fictive
feeding, the amplitude of the lip touch-induced EPSP was greater than
the tentacle touch response (mean EPSP amplitude 7.3 ± 1.0 mV,
5.0 ± 1.3 mV, respectively; paired t-test, df = 9, t = 2.3; P < 0.05; Fig.
5Bi). This is not due to any inherent difference in the
amplitude of the responses from the two sites because there was no
significant difference between the amplitude of EPSP responses to lip
(mean EPSP amplitude 5.3 ± 1.5 mV) and tentacle touch (mean EPSP
amplitude 3.9 ± 1.1 mV) in the naive group response (paired
t-test, df = 9, t = 1.0;
P < 0.3; Fig. 5Biii). However, there was no
significant difference between the amplitudes of EPSPs from lip (mean
EPSP amplitude 7.5 ± 1.2 mV) and tentacle touch (mean EPSP
amplitude 5.1 ± 1.6 mV) in the Tentacle CS+/lip CS
group
(paired t-test, df = 10, t = 1.5;
P = 0.15; Fig. 5Bii) indicating no effect of
differential conditioning on the EPSP response in this group.
|
An ANOVA showed that the differences within the groups were greater
than between the groups [F(5,56) = 1.15, P = 0.67] so no multiple unpaired comparisons could be
justified between groups. Nevertheless, the within group analysis
suggests that differential conditioning of the early EPSP was only
possible in the Lip CS+/tentacle CS group. This asymmetry of the
effect of differential conditioning on EPSP amplitude was different
from the result of the analysis of fictive feeding where differential
conditioning occurred symmetrically in both experimental groups (Figs.
3 and 4).
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DISCUSSION |
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This paper has shown that neuronal correlates of differential appetitive classical conditioning can be recorded from semi-intact preparations of behaviorally conditioned Lymnaea stagnalis. This appears to be the first electrophysiological study performed on a single identified neuron in a preparation made from differentially conditioned whole animals. The main electrical correlate of the differential conditioning was found at the level of the CPG-driven fictive feeding pattern but only selectively at the level of the early EPSP response recorded on B3. Also the asymmetrical stimulus generalization seen behaviorally was not present as a correlated fictive feeding pattern in the semi-intact preparation.
Discriminative learning and conditioned fictive feeding
The behavioral results showed that after differential conditioning
the animals in the Lip CS+/tentacle CS group were able to distinguish
between touch to the lips and touch to the tentacle, with only touch to
the lips increasing the feeding rate. This clearly showed the presence
of discriminative learning. However, in the case of the Tentacle
CS+/lip CS
group, the data were more complex with evidence for weak
stimulus generalization as well as discriminative learning. Because the
lip touch could evoke a generalized feeding response in the Tentacle
CS+/lip CS
group, we have to assume that there is a site of
convergence between the nonreinforced lip-brain and the reinforced
tentacle-brain pathway, and it is likely to be upstream to the site(s)
of plastic change(s) resulting from tentacle CS+ conditioning. On the
other hand, the lack of a generalized response to tentacle touch in the
Lip CS+/tentacle CS
group indicates that the nonreinforced tentacle-brain tactile pathway has no access to neurons that have undergone plastic changes after lip touch conditioning. This asymmetry could be due to differences between the innervation of the lips and
tentacles, the former being innervated only by the lip nerves, whereas
the latter is innervated by both the lip nerves and the tentacle nerve
(Nakamura et al. 1999
). Therefore lip conditioning may
only affect neurons receiving tactile inputs from the lip, but tentacle
conditioning may affect neurons receiving tactile inputs from both the
lip and tentacle. Recently it was suggested that the expression of
cellular correlates of nondiscriminative appetitive classical
conditioning using the lips as the CS site in Aplysia is
also specific to the stimulation of a particular lip-CNS pathway (the
AT4 nerve) but the effect of stimulating other
peripheral nerves was not investigated (Lechner et al.
2000b
). Behavioral work on appetitive classical conditioning in
Aplysia (Lechner et al. 2000a
) also showed
that for the appetitive training to be successful, the US needs to come
into contact with both external (e.g., lips) and internal epithelia
(e.g., foregut) of the animal and subsequently demonstrated that the
esophageal nerve plays an important role in the mediation of the
internal effect of the US during conditioning. These nondifferential
conditioning experiments provided valuable insights into the
organization of the US-mediating pathways, whereas our differential
conditioning experiments provide new insights into the organization of
the CS pathways that contribute to appetitive associative learning.
The robustness of the basic discriminative learning result was
confirmed in the electrophysiological experiments. Here elevation of
fictive feeding rates only occurred at the CS+ sites in both groups.
However, the weak stimulus generalization seen in the behavioral data
in the Tentacle CS+/lip CS group did not survive as an
electrophysiological response in the semi-intact preparation. Previous
work showed that the neuronal correlates of both unconditioned and
conditioned feeding were always weaker than the corresponding behavioral responses in intact animals (Staras et al.
1998
) and as the behavioral stimulus generalization was weak
both in the present and previous experiments (Kemenes and
Benjamin 1989a
), it appears that only the strongest aspects of
the behavioral response survive.
Asymmetrical differential conditioning of the early EPSP response
Touch to the lips produces an early compound EPSP on the B3 motor
neuron that precedes the onset of the rhythmic conditioned fictive
feeding pattern. Previous work using the nondifferential lip touch
conditioning paradigm showed that the amplitude of this EPSP was
enhanced following reinforcement of lip touch with sugar in behavioral
experiments, and this correlated with an increase in fictive feeding
responses to touch (Staras et al. 1999a). If differential conditioning had been completely successful, then it would
be predicted that touch to the CS+ sites in both the Lip CS+/tentacle
CS
and Tentacle CS+/lip CS
experimental groups would produce larger
EPSP responses than the corresponding CS
sites. In fact, only the lip
CS+ stimulus in the Lip CS+/tentacle CS
group produced a significant
enhancement in the amplitude of the EPSP response compared with the
tentacle CS
stimulus, and there was no significant difference between
the effect of touch at the two test sites in the Tentacle CS+/lip
CS
experimental group, despite the occurrence of conditioned fictive
feeding that normally follows the EPSP response.
Why should it be more difficult to use the tentacle as a CS+ site
compared with the lips to condition this early EPSP response? One
possibility is that the early EPSP response to tentacle touch is a
correlate of an alternate behavior, withdrawal. Short latency EPSP
responses to skin touch can be recorded in many neurons in the
Lymnaea CNS, including motor neurons known to mediate whole body withdrawal responses (Ferguson and Benjamin 1991).
It is generally known from vertebrate studies that stimuli that are a
cue for an alternative behavior are more difficult to condition than
completely novel or indifferent stimuli (Shettleworth
1973
). Touch to the lips is more likely to be a part of the
normal food stimulus (see Staras et al. 1999b
) making
touch responses evoked at this site more susceptible to conditioning by
food reinforcers. The touch pathways from tentacles and lips both
produce a similar amplitude EPSP on the B3 motor neuron so the
differential effect of conditioning suggests independent pathways
mediating the early EPSP, one of which is more susceptible to
appetitive conditioning than the other.
The fact that conditioned fictive feeding patterns can occur to
tentacle touch in the Tentacle CS+/lip CS group, despite a lack of
enhancement of the early EPSP, was not entirely unexpected, as the two
components could be dissociated in previous experiments on
nondifferential conditioning by sating the experimental animals between
the behavioral and electrophysiological tests (Staras et al.
1999a
).
Discriminative learning in other model systems
Discriminative learning has been previously demonstrated in other
invertebrate systems, mainly mollusks. For example, differential conditioning of the defensive gill withdrawal reflex was shown in
intact Aplysia (Carew et al. 1983), and more
recently Hawkins et al. (1998)
have shown that
differential conditioning of the same reflex can be achieved in a
reduced preparation. In vitro analogues of differential classical
conditioning also have been used with great success in
Aplysia (Hawkins et al. 1983
; Murphy and Glanzman 1999
; Walters and Byrne 1983
), but
so far no attempt has been made to analyze cellular traces of
discriminative learning in preparations made from differentially
conditioned whole animals. Discriminative learning was also reported in
Pleurobranchaea californica (Mpitsos and Cohan
1986a
,b
), and using muscle recording techniques it was shown
that it survives dissection, and therefore features of whole-animal
differential conditioning can persist into physiological preparations
(Mpitsos and Cohan 1986c
). The above Aplysia
and Pleurobranchaea examples used aversive paradigms, but
appetitive differential classical conditioning also has been
demonstrated in Aplysia (Colwill et al. 1997
)
and the terrestrial slug Limax maximus (Sahley et al.
1990
). Appetitive differential classical conditioning was also
investigated in an insect, the honeybee (Mauelshagen
1993
), where a neuronal correlate of differential appetitive
conditioning also was obtained after training had been performed in an
isolated head preparation. The main advantage of using differential
conditioning paradigms is that each animal/preparation can serve as its
own control. This is particularly important in studies aiming to find
cellular traces of behavioral classical conditioning where the
variability of the data at both the behavioral and electrophysiological
levels can considerably reduce the chances of finding such traces.
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ACKNOWLEDGMENTS |
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G. Kemenes is a Medical Research Council Senior Fellow.
This work was supported by the U.K. Biotechnology and Biological Sciences Research Council.
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FOOTNOTES |
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Address for reprint requests: P. R. Benjamin (E-mail: P.R.Benjamin{at}sussex.ac.uk).
Received 12 June 2000; accepted in final form 14 September 2000.
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REFERENCES |
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