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 Previous Article
The Journal of Neuroscience, December 1, 1999, 19(23):10595-10602
Cellular Analog of Differential Classical Conditioning in
Aplysia: Disruption by the NMDA Receptor Antagonist
DL-2-Amino-5-Phosphonovalerate
Geoffrey G.
Murphy1 and
David L.
Glanzman2
1 Interdepartmental Graduate Program in Neuroscience,
School of Medicine, and 2 Departments of Physiological
Science and Neurobiology and the Brain Research Institute, University
of California, Los Angeles, California 90095-1761
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ABSTRACT |
We previously showed that the associative enhancement of
Aplysia siphon sensorimotor synapses in a cellular analog of
classical conditioning is disrupted by infusing the
Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid
into the postsynaptic motor neuron before training or by training in
the presence of the NMDA receptor antagonist
DL-2-amino-5-phosphonovalerate (APV). Our earlier
experiments with APV used a nondifferential training protocol, in which
different preparations were used for associative and nonassociative
training. In the present experiments we extended our investigation of
the role of NMDA receptor type potentiation in learning in
Aplysia to differential conditioning. A cellular analog of
differential conditioning was performed with a reduced preparation that
consisted of the CNS plus two pedal nerves. A siphon motor
neuron and two siphon sensory neurons, both of which were
presynaptically connected to the motor neuron, were impaled with sharp
microelectrodes. One sensorimotor synapse received paired stimulation
with a conditioned stimulus (brief activation of a single sensory
neuron) and an unconditioned stimulus (pedal nerve shock), whereas the
other sensorimotor synapse received unpaired stimulation. Training in
normal artificial seawater (ASW) resulted in significant differential
enhancement of synapses that received the paired stimulation. Training
in APV blocked this differential synaptic enhancement. A comparison of
the present data with the data from earlier experiments that used
nondifferential training is consistent with the possibility that
differential training comprises competition between the presynaptic
sensory neurons. Synaptic competition may contribute significantly to the associative effect of paired stimulation in the differential training paradigm.
Key words:
Aplysia californica; classical conditioning; learning and memory; long-term potentiation (LTP); NMDA; synaptic
plasticity; synaptic competition
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INTRODUCTION |
The siphon-withdrawal reflex of the
marine snail Aplysia can express a form of classical
conditioning. The conditioned stimulus (CS) used for this form of
associative learning is weak stimulation of the siphon; the
unconditioned stimulus (US) is strong electrical shock of the tail
(Carew et al., 1981 ). The original demonstration of classical
conditioning of the withdrawal reflex used a nondifferential training
protocol, in which separate groups of animals received either
associative training paired presentation of the CS and USor
nonassociative trainingpresentation of the US alone or unpaired
presentation of the CS and US. A more rigorous demonstration of the
dependence of classical conditioning of the withdrawal reflex on the
paired presentation of the CS and US was provided by the report of
differential classical conditioning by Carew et al. (1983). In their
study each animal received two conditioned stimuli, which consisted of
weak stimulation of two separate sites on the skin of the animal. The
delivery of one of the stimuli (CS+) was paired with
the US, whereas the delivery of the other stimulus (CS ) was explicitly unpaired with the US. After
differential training, animals exhibited a longer withdrawal response
to the CS+ than to the CS. An
advantage of differential conditioning is that each animal serves as
its own control. This not only permits a more convincing separation of
the associative and nonassociative effects of training but also
provides an advantageous experimental paradigm for a cellular analysis
of classical conditioning in Aplysia. In 1983 two groups,
using somewhat different experimental paradigms, demonstrated a
cellular analog of differential conditioning in Aplysia
(Hawkins et al., 1983; Walters and Byrne, 1983). In this cellular
analog of differential conditioning, synapses made by two different
sensory neurons, each of which was connected to same motor neuron, were differentially trained. Activation of the CS+
sensory neuron was paired with the US (tail or tail nerve shock), whereas activation of the CS sensory neuron was
unpaired with the US. Both Hawkins et al. (1983) and Walters and Byrne
(1983) found that the synapses made by the CS+
sensory neurons were significantly more enhanced after training than
were the synapses made by the CS sensory neurons.
Based on these results, the two groups jointly proposed a cellular
mechanism to explain classical conditioning in Aplysia. This
mechanism, known as "activity-dependent presynaptic facilitation
(ADPF)" is hypothesized to be an elaboration of the mechanism of
presynaptic facilitation of sensorimotor synapses (Byrne and Kandel,
1996). As originally conceived, ADPF is a non-Hebbian mechanism,
involving strictly presynaptic processes.
Evidence that classical conditioning in Aplysia might be
mediated by a different mechanism came from the discovery by Lin and
Glanzman (1994a,b, 1997) that sensorimotor synapses possess the
capacity for Hebbian long-term potentiation (LTP). This discovery led
to the hypothesis that classical conditioning of the withdrawal reflex
is mediated, in part, by Hebbian potentiation (Glanzman, 1994; Lin and
Glanzman, 1994b; Glanzman, 1995). In support of this hypothesis we
previously showed that the cellular analog of nondifferential classical
conditioning of the withdrawal reflex is blocked by infusing the rapid
Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid
into the postsynaptic motor neuron (Murphy and Glanzman, 1996) and by
the NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (APV) (Murphy and Glanzman, 1997). In the present experiments we tested
whether the cellular analog of differential conditioning (Hawkins et
al., 1983; Walters and Byrne, 1983), like the cellular analog of
nondifferential conditioning, depends on activation of NMDA-type
receptors (Collingridge et al., 1983; Dale and Kandel, 1993).
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MATERIALS AND METHODS |
Preparation. A reduced preparation was used (Hawkins
et al., 1983; Murphy and Glanzman, 1996, 1997). Aplysia
californica (100-200 gm, obtained from local suppliers) were
anesthetized by injecting isotonic MgCl2 (equal to
approximately one-half body weight) into the hemocoel. The CNS, except
for the buccal ganglia, was removed from the animal, together with two
posterior pedal nerves (P9) that innervate the animal's tail. The
tail, together with the rest of the animal's body, was discarded.
After the abdominal ganglion was lightly fixed with glutaraldehyde
[0.5% in 50% MgCl2 and 50% normal artificial seawater
(ASW; 460 mM CaCl2, 11 mM
CaCl2, 10 mM KCl, 55 mM
MgCl2, and 10 mM HEPES buffer, pH
7.6)], the preparation was pinned to the Sylgard-lined bottom of a
recording chamber containing 50% MgCl2 and 50% normal ASW
(Fig. 1A). The abdominal ganglion was then partially desheathed, and the two P9 nerves
were drawn into suction electrodes. Afterward the
MgCl2-containing solution was washed out by perfusing the
recording chamber with normal ASW for at least 30 min before the start
of the experiment.
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Figure 1.
Experimental preparation and pattern of
stimulation used for the cellular analog of differential classical
conditioning. A, Illustration of the reduced preparation
(see Methods and Materials for details). Note that the abdominal
ganglion is shown artificially enlarged relative to the other ganglia.
Sharp microelectrode recordings were made from a single small siphon
(LFS) motor neuron and two siphon sensory (LE) neurons, both of which
were presynaptically connected to the motor neuron. B,
Differential conditioning was performed by pairing activation of one of
the sensory neurons, the CS+ sensory neuron, with
the US (tail nerve shock), whereas the activation of the other sensory
neuron, the CS sensory neuron, was specifically
unpaired with the US. The US was delivered once per 5 min; the
CS stimulation occurred 2.5 min after the US. All
stimuli were presented five times during training (see Fig. 2).
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Electrophysiology. A single small siphon (LFS) motor
neuron (Frost et al., 1988; Hickie and Walters, 1995) and two siphon sensory (LE) neurons (Byrne et al., 1974), each of which was
presynaptically connected to the motor neuron, were impaled with sharp
micropipettes (20-30 M ); the micropipettes were filled with a
solution of 2 M K-acetate, 0.5 M KCl, and 10 mM HEPES, pH 7.2. Stimulation and recording were performed
using standard methods, and the electrophysiological data were
digitized with a Maclab 4e system (ADInstruments, Castle Hill,
Australia). Before the start of an experiment the motor neuron was
hyperpolarized to 50 mV below its resting potential by passing negative
current through the bridge circuit of the amplifier. This
hyperpolarizing current was maintained throughout the experiment to
prevent both spontaneous firing by the motor neuron and evoked firing
in response to test stimulation of the sensory neurons. Action
potentials were elicited in LE neurons by brief (20 msec) injections of
positive current.
Experimental protocol. The differential training protocol
was similar to that of Hawkins et al. (1983). The CS was brief
activation of the sensory neuron (12 action potentials at 25 Hz) (Figs.
1B, 2). The level of
current used to stimulate the sensory neuron during the CS was twice
the threshold level for eliciting an action potential. The specific
levels of current used for the CS were 0.4-1.8 nA. The US was a 1 sec
shock (3 msec pulses at 25 Hz) delivered to the P9 nerves via the
suction electrodes. The intensity of the US was six times the threshold
level for evoking an EPSP in the motor neuron. This shock intensity was
7-9 V, measured from the front panel of the stimulator (S88; Grass
Medical Instruments, Quincy MA). During training only the activity of
one sensory neuronthe CS+ sensory neuronwas
paired with the US. Activity in the other sensory neuron (the
CS sensory neuron) was specifically unpaired with
the US. Assignment of a sensory neuron to the CS+ or
CS condition was based on the size of its EPSP
evoked on pretest 2; the assignment was made to minimize the difference
between the CS+ and CS groups
(and between the CS+/APV and
CS/APV groups) with respect to the mean group
value of the pretest 2 EPSP. In each experiment the US was presented
five times once per 5 min. Sensorimotor synapses in the
CS+ condition received five paired presentations of
the CS and US. During each bout of paired (CS+)
stimulation, the onset of the CS preceded that of the US by 500 msec.
The unpaired CS (CS) occurred 2.5 min after the
US. Before training there were four pretests with a 15 min interval
between the tests. During each pretest the two sensory neurons were
each fired one time, and the resulting EPSPs were recorded from the
motor neuron. Training was begun 5 min after the fourth pretest. Two
post-tests were performed on each synapse, one at 15 min and the other
at 60 min, after the delivery of the final CS in each experimental
condition. For example, the 15 min post-test for the
CS synapse occurred 15 min after the last unpaired
CS and 2.5 min after the 15 min post-test for the
CS+ synapse (Fig. 2). One of the pretests (pretest
2) and both of the post-tests were performed in the presence of a
modified ASW that contained elevated levels of Mg2+
and Ca2+. This so-called "2:1" ASW (NaCl, 368 mM; CaCl2, 13.8 mM; KCl, 8 mM; MgCl2, 101 mM;
MgSO4, 20 mM; and HEPES buffer, 10 mM, pH 7.6) reduces the interneuronal contribution to the
sensorimotor EPSP (Trudeau and Castellucci, 1992). The 2:1 ASW was
perfused into the recording chamber after pretest 1 and was washed out immediately after pretest 2. The 2:1 ASW was reintroduced after the
final bout of paired stimulation and remained in the recording chamber
for both post-tests. The training was performed with normal ASW in the
recording chamber. To test the potential contribution of NMDA-type
receptors to associative plasticity, in some experiments APV
(Sigma, St. Louis, MO) was dissolved in normal ASW (100 µM) and perfused into the recording chamber immediately
after the pretest 2; it was washed out immediately after the fifth
delivery of the CS.
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Figure 2.
Experimental protocol used for differential
conditioning. Four pretests were performed on each of the two
sensorimotor synapses at an intertest interval of 15 min. The first was
performed in normal ASW. After the first pretest, 2:1 ASW (see
Materials and Methods), indicated by the black bars above
the time line, was perfused into the recording chamber. The
second pretest was then performed in 2:1 ASW. After the second pretest
the 2:1 ASW was replaced with normal ASW, and the third and fourth
pretests were performed. Training began 5 min after pretest 4 and was
performed either in normal ASW or in ASW that contained 100 µM APV (indicated by the hatched bar above the
time line). Each of the stimuli (CS+, US,
and CS) was presented 5 times. The filled
arrows indicate paired presentations of the CS+
and US; the open arrows indicate presentations of the
CS. The paired stimuli were delivered at a rate of
one per 5 min, and the unpaired stimuli were separated from the paired
stimuli by 2.5 min. Immediately after training 2:1 ASW was again
perfused into the recording chamber, and two posttests were performed;
the post-tests occurred 15 and 60 min after the final delivery of the
CS for each synapse (see Materials and Methods). Representative traces
are from the motor neuron and the two presynaptic sensory neurons
recorded during the sequential presentation of the stimuli
(CS+, US and CS) in an
experiment in which APV was present during training. Notice that the US
produced strongdepolarization and firing of the motor neuron.
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Statistics. The summary statistics are presented as
means ± SEM. In the figures and text n refers to the
number of preparations. A nonparametric paired t test
(Wilcoxon signed rank test) was used to assess the statistical
significance of differences between two conditions within the same
preparations. Intragroup comparisons (between pre- and post-tests) were
also made with Wilcoxon signed rank tests. Planned comparisons between
two experimental groups were made with unpaired nonparametric
Mann-Whitney tests, and planned multiple comparisons were made with a
nonparametric ANOVA (Kruskal-Wallis). All reported levels of
statistical significance represent two-tailed values.
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RESULTS |
Effect of APV on differential conditioning
There were no differences among the mean raw EPSP amplitudes
recorded on pretest 2 in the four different experimental groups (CS+ EPSP = 5.0 ± 0.6 mV;
CS EPSP = 6.3 ± 1.6 mV;
CS+/APV EPSP = 5.0 ± 1.5 mV;
CS/APV EPSP = 5.4 ± 1.3 mV;
Kruskal-Wallis = 2.6; p > 0.05). Synapses that
received paired stimulation (CS+ group; Fig.
3) showed significant enhancement for
both the 15 min post-test (normalized EPSP = 194 ± 17%) and
60 min post-test (normalized EPSP = 163 ± 8%), based on the
comparison with the pretest 2 EPSP (p < 0.01 for each comparison). Unpaired presentations of the CS and US
(CS group; Fig. 3) did not produce a significant
enhancement of the EPSP for either the 15 min post-test (normalized
EPSP = 127 ± 16%) or 60 min post-test (normalized EPSP = 80 ± 9%), compared with the pretest 2 EPSP
(p > 0.05 for both comparisons). The training protocol produced significant differential enhancement of synapses in
the CS+ group, as indicated by the significant
difference between the CS+ EPSP and
CS EPSP on both post-tests
(p < 0.01 for each comparison).
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Figure 3.
Training in normal ASW produces differential
enhancement of the synapse made by the CS+ sensory
neuron. A, Representative records of the responses of the
two presynaptic sensory neurons (bottom traces) and the
postsynaptic motor neuron (top traces) during the second
pretest and the 60 min post-test. Both tests were performed in 2:1 ASW
(see Materials and Methods). B, Group data for experiments
in which training was performed in normal ASW. The amplitude of the
EPSP on each of the posttests was normalized to the amplitude of the
EPSP on pretest 2 (see Fig. 2). The data are presented as means ± SEM. The EPSP in synapses that received paired CS-US stimulation (the
CS+ group) was significantly enhanced compared with
the normalized pretest EPSP (indicated by the dashed line).
Furthermore, the CS+ EPSP was significantly more
enhanced after training than was the EPSP in synapses that received
unpaired training (the CS group; see
Results).
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Paired stimulation in the presence of APV did not produce significant
enhancement of the EPSP (CS+/APV group; Fig.
4). Neither the mean normalized EPSP for
the 15 min post-test (101 ± 12%) nor that for the 60 min
post-test (105 ± 10%) was significantly different from the
pretest 2 EPSP (p > 0.7 for each comparison).
Furthermore, the presence of APV during the training significantly
blocked differential synaptic enhancement. The mean EPSP in the
CS+/APV group was not significantly different from
the mean EPSP in the CS/APV group for either the
15 min post-test (CS/APV EPSP = 131 ± 17%) or the 60 min post-test (CS/APV EPSP = 129 ± 14%) (p > 0.05 for each
comparison). Unpaired stimulation with the CS and US in the presence of
APV produced a small increase in mean EPSP amplitude on the post-tests
compared with the pretest 2 EPSP. This increase was statistically
significant for the 15 min post-test (p < 0.05), although not quite for the 60 min post-test
(p > 0.07). Therefore, in contrast to its
disruption of associative synaptic enhancement, APV did not reduce the
effect of unpaired stimulation on the EPSP. The mean EPSP in the
CS/APV group was not significantly different from
the mean EPSP amplitude in the CS group trained
without APV (Fig. 3) for the 15 min posttest (p > 0.05) and was actually significantly greater for the 60 min post-test (p < 0.01).
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Figure 4.
Training in APV blocks differential synaptic
enhancement. A, Representative records of the responses of
the two presynaptic sensory neurons (bottom traces) and the
postsynaptic motor neuron (top traces) during the second
pretest and the 60 min post-test. Both tests were performed in 2:1 ASW
(see Materials and Methods). B, Group data for experiments
in which training was performed in ASW containing APV (100 µM). The amplitude of the EPSP on each of the post-tests
was normalized to the amplitude of the EPSP on pretest 2 (see Fig. 2).
The data are presented as means ± SEM. The EPSP in synapses that
received paired stimulation in APV (the CS+/APV
group) did not exhibit significant enhancement compared with the
normalized pretest EPSP (indicated by the dashed line) on
either post-test. Furthermore, the CS+/APV EPSP was
not significantly different from the CS/APV EPSP
on either post-test.
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Effect of APV on the excitatory drive of the motor neuron
during tail nerve shock
A potential explanation for the disruption of associative synaptic
plasticity by APV is that the drug might have reduced the efficacy of
excitatory or modulatory interneurons that are activated by tail shock
(Frost and Kandel, 1995). The effect of the excitatory interneurons is
to strongly depolarize the siphon motor neurons during the US. A
decrease in postsynaptic depolarization during the US would be expected
to interfere with Hebbian potentiation of the sensorimotor synapses. To
assess the possibility that APV might have interfered with US-induced
excitatory drive on the motor neuron during paired training, we
quantified the amount of postsynaptic depolarization produced by the US
in our experiments. This was done by mathematically integrating the
area under the postsynaptic membrane during the US (Fig. 5A,
shaded area). This integration was
made for each bout of paired stimulation for both the
CS+ and CS+/APV groups. (The
integration was performed off-line with software options provided by
our data acquisition system. See Materials and Methods.) The presence
of APV during training did not decrease the amount of depolarization
produced during the US, as indicated by the results from integrating
the area under the postsynaptic membrane potential
(p > 0.05; Fig. 5B). As a second
method of quantifying the US-induced drive on the motor neuron in our
experiments, we also counted the number of action potentials (spikes)
produced in the motor neuron during the US. The drug did not decrease
the number of postsynaptic spikes during the US
(p > 0.05; Fig. 5B). Therefore, the
block of associative plasticity produced by APV cannot be attributed to
a decrease in the activity, or efficacy, of excitatory interneurons
during training.
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Figure 5.
APV does not alter the effect of the US on the
postsynaptic motor neuron. A, Examples of the responses of a
motor neuron and the two presynaptic sensory neurons during a bout of
paired stimulation (CS+ and US). The excitatory
drive on the motor neuron produced by the US (tail nerve shock) was
approximated by mathematically integrating the area under the
postsynaptic membrane potential during the 500 msec of the US
(shaded area). B, Group data (means ± SEM)
for the experiments performed in normal ASW and the experiments
performed in ASW containing APV. There were no significant differences
between the two groups in the amount of postsynaptic depolarization or
in the number of postsynaptic action potentials (spikes) produced by
the US.
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These data, together our previous demonstration that APV does not alter
the nonassociative enhancement of the sensorimotor EPSP resulting from
unpaired stimulation (Murphy and Glanzman, 1997), permit us to rule out
a significant effect of the drug on the actions of excitatory and
facilitatory interneurons. We cannot, however, completely exclude an
effect of APV on interneuronal pathways. Notice that sensorimotor EPSPs
were relatively more enhanced on the 60 min posttest after unpaired
training in APV than after unpaired training in normal ASW (compare
Figs. 3B, 4B). This effect may be
attributable to a disruptive effect of APV on the influence of
presynaptic inhibitory interneurons activated by the US (Mackey et al.,
1987; Marcus et al., 1988).
Possible recruitment of synaptic competition during
differential conditioning
Our present results differed in one major respect from the results
of our previous conditioning experiments, which used a nondifferential
training protocol wherein preparations received only paired or unpaired
stimulation (Murphy and Glanzman, 1997). In the earlier nondifferential
experiments we observed significant synaptic enhancement 15 min after
training in preparations that received unpaired training in normal ASW
(Murphy and Glanzman, 1997, their Fig. 2D; the
CS EPSP was significantly greater than its pretest
value in both the 15 and 60 min post-tests). But we did not observe
significant synaptic enhancement attributable to unpaired training in
the present experiments for the 15 min post-test (Fig. 3; the
CS EPSP in the 15 min post-test was not
significantly different from its pretest value). Figure
6 shows the post-test data from Murphy
and Glanzman (1997), together with the post-test data from the present
study, for those experiments in which training was performed in normal
ASW. The data from the two studies have been replotted as the mean
difference in millivolts between the EPSPs for pretest 2 and the
post-tests. The data are plotted in this manner to permit examination
of the absolute results from the two independent studies. In the study
by Murphy and Glanzman (1997) the mean synaptic enhancement in the
CS+ group (2.5 ± 0.8 mV) was not significantly
different from that in the CS group (1.5 ± 0.7 mV) for the 15 min post-test (Fig. 6A;
p > 0.05, Mann-Whitney test). However, the difference
between the two groups was significant for the 60 min post-test. Paired
training produced a mean synaptic enhancement of 2.7 ± 1.2 mV
(CS+ group), whereas unpaired training produced
almost no enhancement (0.1 ± 0.6 mV, (CS
group; p < 0.05, Mann-Whitney test). In contrast to
the nondifferential training protocol, the differential training
protocol used in the present experiment resulted in significant
differences between the CS+ and
CS groups for both posttests (Fig.
6B). The mean synaptic enhancement for the 15 min
post-test was 4.4 ± 1.0 mV in the CS+ group
but only 0.1 ± 1.2 mV in the CS group
(p < 0.01, Mann-Whitney test). The mean
synaptic enhancement for the 60 min post-test was 3.0 ± 0.5 mV in
the CS+ group; in the CS group
there was a synaptic decrease of 1.7 ± 0.9 mV
(p < 0.0001, Mann-Whitney test).
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Figure 6.
Comparison of the synaptic effects of
nondifferential and differential training. A, Replotted data
from Murphy and Glanzman (1997). The data are expressed as the
mean ± SEM difference (in millivolts) between the non-normalized
EPSP on each of the post-tests and the non-normalized EPSP on pretest 2 (see Fig. 2). Paired and unpaired training in these experiments was
performed on different preparations. B, Data from the
present experiments, in which paired and unpaired training was
performed on the same preparations. The data are presented as
difference scores as in A. Statistical comparisons between
groups (CS+ vs CS) in
A and B were made using Wilcoxon signed rank
tests. Asterisks indicate statistical significance for the
between-group comparison; *p < 0.05;
**p < 0.01.
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What might account for the discrepancy between our nondifferential and
differential data on the 15 min post-test? One possibility, suggested
by related experiments on sensorimotor synapses in cell culture
(Glanzman et al., 1991; Schacher et al., 1997; Wright et al., 1999), is
that the presynaptic sensory neurons compete with each other for
synaptic interaction with the motor neuron. According to this idea, the
significant effect of paired stimulation at 15 min after training in
the differential experiments (Figs. 3, 6B)
predominately reflects a competitive interaction between the two
presynaptic cells. In the differential experiments both the
CS+ and CS sensory neurons
synapsed onto the same target motor neuron. By contrast, only one
presynaptic sensory neuron per preparation was studied in the
nondifferential experiments; no effect of synaptic competition would
therefore be expected in the nondifferential results. Consequently, the
significant synaptic enhancement for the 60 min post-test in the
CS+ group in the nondifferential experiments is
probably attributable to a purely associative mechanism. The lack of a
difference between the paired and unpaired results at 15 min after
training in the nondifferential experiments suggests that this
associative synaptic mechanism has a relatively delayed onset, being
absent at 15 min but present at 60 min after training. We believe that
the significant difference between the CS+ and
CS groups at 60 min after training in the
differential experiments is also attributable to a delayed associative
synaptic enhancement. However, it is possible that synaptic competition
contributes to the 60 min post-test results in the differential
experiments as well (see Discussion).
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DISCUSSION |
Comparison with earlier results
The present results demonstrate that the strengthening of the
monosynaptic sensorimotor synapses that occurs during the cellular analog of differential classical conditioning in Aplysia
requires activation of NMDA, or NMDA-related, receptors (Dale and
Kandel, 1993). These results, together with our previous findings
(Murphy and Glanzman, 1996, 1997), support the hypothesis that Hebbian potentiation of sensorimotor synapses (Lin and Glanzman, 1994a,b, 1997)
plays a role in classical conditioning of Aplysia's
siphon-withdrawal reflex. In our previous study examining the potential
contribution of NMDA-type LTP of sensorimotor synapses to classical
conditioning (Murphy and Glanzman, 1997), we used a nondifferential
protocol. Experiments involving paired stimulation (both in normal ASW
and in APV) were performed separately from those involving unpaired stimulation using different preparations. It has therefore been suggested that the apparent disruption of associative plasticity by APV
we observed in the earlier study might have been caused, instead, by
fluctuation in the magnitude of the nonassociative effects of training
over time (Hawkins, 1998). The present data, in which associative and
nonassociative training were performed within the same preparation at
the same time, obviate this interpretation of the effect of APV. Our
new data therefore confirm our previous conclusion that APV blocks
associative, conditioning-related synaptic enhancement of sensorimotor
synapses (Murphy and Glanzman, 1997, 1998).
Potential contribution from synaptic competition in
differential conditioning
An unexpected result from our earlier study was that there was no
associative effect of paired training in normal ASW on the 15 min
post-test (Fig. 6A; also see Murphy and Glanzman,
1997, their Fig. 2D). This result contrasts with
those of Hawkins et al. (1983), who found significant associative
synaptic enhancement in the CS+ group as early as 5 min after five bouts of paired stimulation. The study by Hawkins et al.
(1983), unlike our earlier study, used a differential paradigm. In the
present study, which also used a differential paradigm, we observed an
associative effect of paired training on the 15 min post-test for the
CS+ versus CS protocol (Figs.
3, 6B). This suggests that the early (15 min) associative effect of paired training is peculiar to the differential paradigm. We hypothesize that the differential conditioning paradigm, unlike the nondifferential paradigm, recruits synaptic competition between the CS+ and CS sensory
neurons, and that this competition accounts for the associative effect
we observed at 15 min after training in the present study.
Evidence from studies of sensorimotor cocultures supports the idea that
the strength of different sensory inputs to a common target motor
neuron is regulated by a competitive interaction, during both
development and learning-related plasticity. Glanzman and colleagues
(1991) found that the mean size of the sensorimotor EPSP of
"mature" (5-d-old) sensorimotor cocultures with two presynaptic neurons was half the size of the EPSP of cocultures with one
presynaptic neuron. Furthermore, Schacher and colleagues (1997) found
that long-term associative enhancement of a connection made by one sensory neuron [attributable to paired stimulation with serotonin (5-HT) and tetanus] interferes with long-term nonassociative
enhancement (attributable to 5-HT alone) of the connection made by a
second sensory neuron with the same target motor neuron. Schacher et al. (1997) refer to this phenomenon as "pathway-specific
facilitation." These in vitro results parallel the
disparity we observed between our nondifferential and differential
conditioning experiments with respect to the CS
data for the 15 min post-test (Fig. 6). Interestingly, Schacher et al.
(1997) found that both postsynaptic hyperpolarization and APV disrupt
pathway-specific facilitation. The results of Schacher et al. (1997)
indicate that an associative increase in strength of the connection
made by one sensory neuron with a motor neuron comes at the expense of
the potential nonassociative enhancement of the connection made by a
second sensory neuron with the same motor neuron. The results further
indicate that postsynaptic NMDA-type receptors may play a role in the
apparent competition among presynaptic inputs.
The hypothesis that synaptic competition is recruited during
differential conditioning in Aplysia is supported by a
negative correlation we observed in our data between a large initial
size of the non-normalized CS+ EPSP relative to that
of the non-normalized CS EPSP [pretest 2 CS+ (mV)  pretest 2 CS
(mV)], and the absolute amount of differential conditioning produced by the training [( CS+ EPSP amplitude) ( CS EPSP amplitude), where CS× EPSP = posttest CS×
EPSP pretest 2 CS× EPSP; Murphy and
Glanzman, unpublished data]. We have considered and rejected several
alternative hypotheses that might explain this negative correlation.
For example, one possible explanation for the negative correlation is
the existence of a ceiling effect for EPSP size. According to a ceiling
effect hypothesis, initially large CS+ EPSPs undergo
less positive differential conditioning because of an intrinsic upper
limit for sensorimotor EPSP size. But we found no significant
correlation, either negative or positive, between the non-normalized
size of the pretest 2 CS+ EPSP (in millivolts) and
the absolute change in the size of the CS+ because
of training [posttest CS+ EPSP (mV) pretest
2 CS+ EPSP (mV)] for either the 15 or 60 min
post-test, contrary to what a ceiling effect hypothesis would predict
(G. G. Murphy and D. L. Glanzman, unpublished data).
A mechanism that could account for our conditioning data, as well as
for the data from in vitro studies of the sensorimotor synapse (Glanzman et al., 1991; Schacher et al., 1997), is homeostatic regulation of the sizes of multiple sensory inputs to a common motor
neuron. According to a current theory (Miller, 1996), the total
strength of excitatory inputs to a postsynaptic cell is relatively
fixed. Consequently, if some inputs increase in strength, the strength
of others must correspondingly decrease. Such a homeostatic mechanism
has been referred to as "postsynaptic normalization" (Buonomano and
Merzenich, 1998). A model incorporating postsynaptic normalization can
explain the pattern of synaptic alterations we observed during the
cellular analog of differential classical conditioning. According to
this model, conditioning is a zero-sum situation: a large increase in
the strength of the synaptic connection made by the sensory neuron that
received paired training (CS+ sensory neuron) is
compensated for by decreases in some (or possibly all) of the other
sensory neurons that synapse with the motor neuron. The algebraic sum
of all of these synaptic decreases is approximately equal to the
inverse of the training-induced increase in strength of the synapse
made by the CS+ sensory neuron. Further experimental
work will be required to test the validity of such a model for
classical conditioning in Aplysia.
Various cellular mechanisms contributing to classical conditioning
in Aplysia
The present data, together with those of our earlier study (Murphy
and Glanzman, 1997), suggest that classical conditioning of the
siphon-withdrawal reflex comprises at least three different cellular
mechanisms. First, there is a sensitization-related, nonassociative
component (Carew et al., 1971). This component results, in part, from
presynaptic facilitation of sensorimotor synapses (Byrne and Kandel,
1996) attributable to 5-HT released by serotonergic interneurons
activated by tail shock (Glanzman et al., 1989; Mackey et al., 1989).
The approximate time course of the nonassociative enhancement under the
conditions of our experiments is indicated by the
CS data in the nondifferential conditioning
experiments (Fig. 6A; Murphy and Glanzman, 1997). The
nonassociative component is maximal shortly after training but is still
present at 1 hr after training, as indicated by the significant
difference in the nondifferential data for the 60 min post-test between
the amplitude of the (normalized) CS EPSP and the
amplitude of the EPSP in preparations that received only test stimuli
(test-alone preparations; see Murphy and Glanzman, 1997, their Fig.
2D). The second cellular component is a hypothesized competitive mechanism that operates whenever different synapses on the
same motor neuron receive different types of plasticity-inducing stimulation. We infer the presence of synaptic competition among sensory neurons from the difference between the nondifferential and
differential data with respect to the CS results
at 15 min after training (Fig. 6; also compare the
CS results in Fig. 3B for the 15 min
post-test with those of Murphy and Glanzman, 1997, their Fig.
2C). The outcome of the proposed synaptic competition
appears to interact with Hebbian, NMDA receptor-dependent potentiation,
because it is blocked by the NMDA receptor antagonist APV (Fig. 4;
Schacher et al., 1997). Furthermore, the effects of the putative
competition between the CS+ and
CS sensory neurons appear to have their onset
relatively early, because they are apparent at 15 min after training in
the differential conditioning experiments (Figs. 3, 6). The third
component of conditioning is a purely associative process, which we
believe to be Hebbian potentiation. As indicated by the lack of a
significant difference between the CS+ and
CS data on the 15 min post-test in the
nondifferential experiments (Fig. 6A, Murphy and
Glanzman, 1997, their Fig. 2D), the associative component of conditioning appears to have a delayed onset. It is
robust, however, by 60 min after training, as indicated by the
significant difference between the CS+ and
CS data in the nondifferential experiments (Fig.
6A).
We propose that the specific outcome of behavioral conditioning in
Aplysia is determined, at least in part, by a parametric interaction among the above three cellular components. For example, the
significant difference between the CS+ and
CS groups on the 15 min post-test in the present
experiments appears to be attributable predominately to the effect of
synaptic competition between the CS+ and
CS sensory neurons. The difference between these
two groups on the 60 min post-test, however, appears to be attributable
to the associative component and, possibly, the competitive mechanism
as well.
At present the nature of the associative cellular mechanism recruited
by classical conditioning in Aplysia is controversial. Originally, this associative mechanism was thought to be exclusively presynaptic (Hawkins et al., 1983; Walters and Byrne, 1983; Carew et
al., 1984; Abrams, 1985; Buonomano and Byrne, 1990). Given our data
(Murphy and Glanzman, 1996, 1997; present study), together with the
other data (Schacher et al., 1997; Bao et al., 1998), however, this
idea is no longer tenable. One recent proposal is that
conditioning-related associative plasticity of sensorimotor synapses is
attributable to a combination of presynaptic and postsynaptic coincidence detectors (Bao et al., 1998; Lechner and Byrne, 1998). Alternatively, it is possible that detection of the coincident occurrence of the CS and US occurs postsynaptically, but that presynaptic mechanisms are critically involved in the persistent expression of the synaptic change. Further experiments will be required
to delineate the relative roles of presynaptic and postsynaptic mechanisms in the associative strengthening of sensorimotor synapses during classical conditioning in Aplysia.
| |
FOOTNOTES |
Received March 17, 1999; revised Aug. 31, 1999; accepted Sept. 23, 1999.
This work was supported by National Institutes of Health Grant NS29563
and National Science Foundation Grant IBN-9410579 to D.L.G., as well as
National Institute of Mental Health Grant F31-MH11136 to G.G.M. We
thank Drs. M. Barish, D. Buonomano, M. Fanselow, and W. Wright for
helpful comments.
Correspondence should be addressed to Dr. David L. Glanzman,
Departments of Physiological Science and Neurobiology, 695 Young Drive
South, Room 2506C, Box 951761, University of California, Los Angeles,
CA 90095-1761. E-mail: dglanzman{at}physci.ucla.edu.
Dr. Murphy's present address: Department of Neurobiology, School of
Medicine, University of California, Los Angeles, CA
90095-1761.
| |
REFERENCES |
-
Abrams TW
(1985)
Activity-dependent presynaptic facilitation: an associative mechanism in Aplysia.
Cell Mol Neurobiol
5:123-145[ISI][Medline].
-
Bao JX,
Kandel ER,
Hawkins RD
(1998)
Involvement of presynaptic and postsynaptic mechanisms in a cellular analog of classical conditioning at Aplysia sensory-motor neuron synapses in isolated cell culture.
J Neurosci
18:458-466[Abstract/Free Full Text].
-
Buonomano DV,
Byrne JH
(1990)
Long-term synaptic changes produced by a cellular analog of classical conditioning in Aplysia.
Science
249:420-423[Abstract/Free Full Text].
-
Buonomano DV,
Merzenich MM
(1998)
Cortical plasticity: from synapses to maps.
Annu Rev Neurosci
21:149-186[ISI][Medline].
-
Byrne JH,
Kandel ER
(1996)
Presynaptic facilitation revisited: state and time dependence.
J Neurosci
16:425-435[Abstract/Free Full Text].
-
Byrne J,
Castellucci V,
Kandel ER
(1974)
Receptive fields and response properties of mechanoreceptor neurons innervating siphon skin and mantle shelf in Aplysia.
J Neurophysiol
37:1041-1064[Free Full Text].
-
Carew TJ,
Castellucci VF,
Kandel ER
(1971)
An analysis of dishabituation and sensitization of the gill-withdrawal reflex in Aplysia.
Int J Neurosci
2:79-98[Medline].
-
Carew TJ,
Walters ET,
Kandel ER
(1981)
Classical conditioning in a simple withdrawal reflex in Aplysia californica.
J Neurosci
1:1426-1437[Abstract].
-
Carew TJ,
Hawkins RD,
Kandel ER
(1983)
Differential classical conditioning of a defensive withdrawal reflex in Aplysia californica.
Science
219:397-400[Abstract/Free Full Text].
-
Carew TJ,
Hawkins RD,
Abrams TW,
Kandel ER
(1984)
A test of Hebb's postulate at identified synapses which mediate classical conditioning in Aplysia.
J Neurosci
4:1217-1224[Abstract].
-
Collingridge GL,
Kehl SJ,
McLennan H
(1983)
Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus.
J Physiol (Lond)
334:33-46[Abstract/Free Full Text].
-
Dale N,
Kandel ER
(1993)
L-Glutamate may be the fast excitatory transmitter of Aplysia sensory neurons.
Proc Natl Acad Sci USA
90:7163-7167[Abstract/Free Full Text].
-
Frost WN,
Kandel ER
(1995)
Structure of the network mediating siphon-elicited siphon withdrawal in Aplysia.
J Neurophysiol
73:2413-2427[Abstract/Free Full Text].
-
Frost WN,
Clark GA,
Kandel ER
(1988)
Parallel processing of short-term memory for sensitization in Aplysia.
J Neurobiol
19:297-334[ISI][Medline].
-
Glanzman DL
(1994)
Postsynaptic regulation of the development and long-term plasticity of Aplysia sensorimotor synapses in cell culture.
J Neurobiol
25:666-693[ISI][Medline].
-
Glanzman DL
(1995)
The cellular basis of classical conditioning in Aplysia californicait's less simple than you think.
Trends Neurosci
18:30-36[ISI][Medline].
-
Glanzman DL,
Mackey SL,
Hawkins RD,
Dyke AM,
Lloyd PE,
Kandel ER
(1989)
Depletion of serotonin in the nervous system of Aplysia reduces the behavioral enhancement of gill withdrawal as well as the heterosynaptic facilitation produced by tail shock.
J Neurosci
9:4200-4213[Abstract].
-
Glanzman DL,
Kandel ER,
Schacher S
(1991)
Target-dependent morphological segregation of Aplysia sensory outgrowth in vitro.
Neuron
7:903-913[ISI][Medline].
-
Hawkins RD
(1998)
Learning and the sensorimotor synapse in Aplysia (in Technical Comments)
Science
281:619a[Free Full Text]. Full text at www.sciencemag.org/cgi/content/full/281/5377/619a.
-
Hawkins RD,
Abrams TW,
Carew TJ,
Kandel ER
(1983)
A cellular mechanism of classical conditioning in Aplysia: activity-dependent amplification of presynaptic facilitation.
Science
219:400-405[Abstract/Free Full Text].
-
Hickie C,
Walters ET
(1995)
Motor neuronal control of tail-directed and head-directed siphon responses in Aplysia californica.
J Neurophysiol
74:307-321[Abstract/Free Full Text].
-
Lechner HA,
Byrne JH
(1998)
New perspectives on classical conditioning: a synthesis of Hebbian and non-Hebbian mechanisms.
Neuron
20:355-358[ISI][Medline].
-
Lin XY,
Glanzman DL
(1994a)
Long-term potentiation of Aplysia sensorimotor synapses in cell culture: regulation by postsynaptic voltage.
Proc R Soc Lond B Biol Sci
255:113-118[Medline].
-
Lin XY,
Glanzman DL
(1994b)
Hebbian induction of long-term potentiation of Aplysia sensorimotor synapses: partial requirement for activation of an NMDA-related receptor.
Proc R Soc Lond B Biol Sci
255:215-221[Medline].
-
Lin XY,
Glanzman DL
(1997)
Effect of interstimulus interval on pairing-induced LTP of Aplysia sensorimotor synapses in cell culture.
J Neurophysiol
77:667-674[Abstract/Free Full Text].
-
Mackey SL,
Glanzman DL,
Small SA,
Dyke AM,
Kandel ER,
Hawkins RD
(1987)
Tail shock produces inhibition as well as sensitization of the siphon-withdrawal reflex of Aplysia: possible behavioral role for presynaptic inhibition mediated by the peptide Phe-Met-Arg-Phe-NH2.
Proc Natl Acad Sci USA
84:8730-8734[Abstract/Free Full Text].
-
Mackey SL,
Kandel ER,
Hawkins RD
(1989)
Identified serotonergic neurons LCB1 and RCB1 in the cerebral ganglia of Aplysia produce presynaptic facilitation of siphon sensory neurons.
J Neurosci
9:4227-4235[Abstract].
-
Marcus EA,
Nolen TG,
Rankin CH,
Carew TJ
(1988)
Behavioral dissociation of dishabituation, sensitization, and inhibition in Aplysia.
Science
241:210-213[Abstract/Free Full Text].
-
Miller KD
(1996)
Synaptic economics: competition and cooperation in synaptic plasticity.
Neuron
17:371-374[ISI][Medline].
-
Murphy GG,
Glanzman DL
(1996)
Enhancement of sensorimotor connections by conditioning-related stimulation in Aplysia depends upon postsynaptic Ca2+.
Proc Natl Acad Sci USA
93:9931-9936[Abstract/Free Full Text].
-
Murphy GG,
Glanzman DL
(1997)
Mediation of classical conditioning in Aplysia californica by LTP of sensorimotor synapses.
Science
278:467-471[Abstract/Free Full Text].
-
Murphy GG,
Glanzman DL
(1998)
Learning and the sensorimotor synapse in Aplysia (in Technical Comments).
Science
281:619a. Full text at www.sciencemag.org/cgi/content/full/281/5377/619a.
-
Schacher S,
Wu F,
Sun ZY
(1997)
Pathway-specific synaptic plasticity: activity-dependent enhancement and suppression of long-term heterosynaptic facilitation at converging inputs on a single target.
J Neurosci
17:597-606[Abstract/Free Full Text].
-
Trudeau LE,
Castellucci VF
(1992)
Contribution of polysynaptic pathways in the mediation and plasticity of Aplysia gill and siphon withdrawal reflex: evidence for differential modulation.
J Neurosci
12:3838-3848[Abstract].
-
Walters ET,
Byrne JH
(1983)
Associative conditioning of single sensory neurons suggests a cellular mechanism for learning.
Science
219:405-408[Abstract/Free Full Text].
-
Walters ET,
Byrne JH,
Carew TJ,
Kandel ER
(1983)
Mechanoafferent neurons innervating tail of Aplysia. II. Modulation by sensitizing stimuli.
J Neurophysiol
50:1543-1559[Abstract/Free Full Text].
-
Wright WG,
Yong R,
Glanzman DL
(1999)
Synaptic competition at the Aplysia sensorimotor synapse: tetanic stimulation of one presynaptic input depresses a second presynaptic input.
Soc Neurosci Abstr
25:1314.
Copyright © 1999 Society for Neuroscience 0270-6474/99/192310595-08$05.00/0
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ABSTRACT
INTRODUCTION
