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The Journal of Neuroscience, August 1, 1998, 18(15):5988-5998
Cellular Correlates of Long-Term Sensitization in
Aplysia
Leonard J.
Cleary,
Wai L.
Lee, and
John H.
Byrne
W. M. Keck Center for the Neurobiology of Learning and Memory,
Department of Neurobiology and Anatomy, University of Texas Houston
Medical School, Houston, Texas 77225
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ABSTRACT |
Although in vitro analyses of long-term changes in
the sensorimotor connection of Aplysia have been used
extensively to understand long-term sensitization, relatively little is
known about the ways in which the connection is modified by learning
in vivo. Moreover, sites other than the sensory neurons
might be modified as well. In this paper, several different biophysical
properties of sensory neurons, motor neurons, and LPl17, an identified
interneuron, were examined. Membrane properties of sensory neurons,
which were expressed as increased excitability and increased spike
afterdepolarization, were affected by the training. The biophysical
properties of motor neurons also were affected by training,
resulting in hyperpolarization of the resting membrane potential and a
decrease in spike threshold. These results suggest that motor neurons
are potential loci for storage of the memory in sensitization. The
strength of the connection between sensory and motor neurons was
affected by the training, although the connection between LPl17 and the
motor neuron was unaffected. Biophysical properties of LPl17 were
unaffected by training. The results emphasize the importance of
plasticity at sensory-motor synapses and are consistent with the idea
that there are multiple sites of plasticity distributed throughout the
nervous system.
Key words:
sensitization; facilitation; excitability; plasticity; tail withdrawal; Aplysia
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INTRODUCTION |
Sensitization is a form of
nonassociative learning in which an animal's response to a weak
stimulus is enhanced after another strong, usually noxious, stimulus.
This form of learning has important advantages for studying the
cellular mechanisms underlying behavioral modifications. For example,
sensitization affects behaviors that are mediated by well defined
neural circuits. Consequently, it is possible to identify major sites
of plasticity. Moreover, changes in the properties of circuit neurons
are likely to contribute directly to the behavioral modification.
The marine mollusk Aplysia californica has been used
extensively as a model system to study cellular mechanisms underlying sensitization. Two defensive withdrawal reflexes have been studied most
intensively, the siphon-induced siphon-gill withdrawal reflex and the
tail-induced tail-siphon withdrawal reflex (for review, see Cleary et
al., 1995 ). The monosynaptic components of circuits underlying both of
these reflexes have been identified, as have several polysynaptic
components (Cleary et al., 1995). The duration of sensitization depends
on the training protocol (Pinsker et al., 1973; Frost et al., 1985;
Scholz and Byrne, 1987). Short periods of training result in behavioral
modifications that last for a relatively short time (15-60 min),
whereas multiple training sessions occurring over longer periods of
time result in more persistent modifications (1-14 d). Although the
duration of sensitization appears to be graded, there is a key
mechanistic difference between the two forms of sensitization. Only the
long-term form is blocked by inhibitors of protein synthesis
(Castellucci et al., 1989) (see also Davis and Squire,
1984).
To understand fully the mechanisms underlying learning, it is critical
to study changes in the nervous systems of behaving animals, i.e.,
animals that have been trained in their intact, living state. Ideally,
one would use noninvasive techniques to assess neuronal properties of
individual animals before and after training. At present, however, a
more practical approach is to look for correlates of sensitization by
comparing the properties of neurons in trained animals with those in
untrained animals. A potential problem with the design of these
experiments is the large number of possible changes that can occur in
the nervous system and the difficulty of interpreting which among these
is necessary for the behavioral modification. By using
Aplysia as a model system, we took advantage of the large
body of evidence gathered from in vitro preparations to
focus our attention on critical elements of the neural circuit
mediating the tail-siphon withdrawal reflex and their most important
biophysical properties.
In this paper we examined several different biophysical properties of
sensory neurons, motor neurons, and LPl17, an identified interneuron,
in animals that had been subjected to long-term sensitization training.
As observed in other studies, membrane properties of sensory neurons
were affected by the training. The biophysical properties of motor
neurons also were affected by training, which suggests that these
neurons are potential loci for storage of the memory in sensitization.
As in previous studies, the strength of the connection between sensory
and motor neurons was affected by the training, although the connection
between LPl17 and the motor neuron was unaffected.
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MATERIALS AND METHODS |
Behavioral training
Aplysia californica weighing 125-500 gm were
obtained from Marine Specimens Unlimited (Pacific Palisades, CA) and
Alacrity Marine Biological (Redondo Beach, CA). Animals were
parapodectomized to permit full visualization of the siphon. The
strength of the tail-induced siphon withdrawal reflex was tested by
delivery of a weak electrical stimulus through Teflon-coated silver
electrodes (type Ag 5T, Medwire) implanted under the skin in the
posterior region of the tail, as described previously (Fig.
1) (Scholz and Byrne, 1987; Goldsmith and
Byrne, 1993). Each test stimulus consisted of a 20 msec AC shock
provided by a Variac for which the output was gated by a relay. The
output of the Variac passed through a resistance of 20.9 k to
provide a quasi constant current. The maximum short-circuit current
that could be passed through this circuit for testing stimuli was ~6
mA. The stimulus voltage was set at two times the threshold intensity
level necessary to elicit a siphon withdrawal. Ten tests were delivered
at intervals of 5 min to alternating sides (i.e., five tests were
delivered per side). The duration of the siphon withdrawal was measured
from the start of siphon contraction until the start of relaxation. Relaxation was defined as the reextension of the siphon to its original
position. These weak tail stimuli also elicited a withdrawal of the
tail, but this response was difficult to quantify and was not measured
in these studies. Several criteria were in place at this stage of the
experiment to detect and exclude unsuitable animals: inking while
acclimating to the test bowl (n = 4), inking in
response to test stimuli (n = 0), and threshold
exceeding 3 mA (n = 0).
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Figure 1.
Behavioral training protocol. A dorsal view of the
animal is illustrated in A. Thin silver electrodes were
implanted bilaterally under the skin of the tail for delivery of weak
test stimuli. Strong sensitizing stimuli were delivered to one side of
the body wall (hatched area) through a hand-held
spanning electrode. Baseline responsiveness of the siphon withdrawal
component of the reflex was determined during a pretraining test period
(B). Test stimuli consisted of a single weak AC
shock of 20 msec duration. These were delivered alternately to the left
and right sides of the animal at 5 min intervals, totaling five tests
per side. During the subsequent training session, sensitizing stimuli
were delivered as four trains of strong AC shocks separated by 30 min.
Each train consisted of 10 pulses of 500 msec duration delivered at 1 Hz. At 24 hr after the training session the testing protocol was
repeated.
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For analysis the five responses were combined to provide an average
withdrawal duration. For tests before training the 5 min interval was
sufficient to prevent frequency-dependent changes in the response
during the pretraining session. After training, however, the responses
at the end of the posttraining session were somewhat greater than those
earlier in the session. This probably did not contribute to lateralized
enhancements induced by training, because both sides exhibited the
frequency-dependent enhancement.
Sensitizing stimuli were applied to the region of the body wall lateral
to the parapodium (Fig. 1). This region was anterior to the area in
which the test electrodes were implanted and excluded receptive fields
of tail sensory neurons (Walters et al., 1983; Dulin et al., 1995). A
hand-held spanning electrode (1 mm gap, 1 mm electrode length) was used
to deliver four trains of strong AC shocks. Each train consisted of 10 shocks of 500 msec duration at 1 Hz. The output of the Variac was
passed to the spanning electrode through a resistance of 940 and
was set to deliver a short-circuit current of 60 mA. Shocks of this
strength reliably produced an inking response. The amount of ink that
was released tended to decrease during the training session as the
store was depleted. A single strong shock of 500 msec duration did not
produce any visible sign of damage to the skin either immediately after
training or 24 hr later. To minimize any possible cumulative damage
over the 10 successive shocks, we systematically moved the
electrode to 10 different locations on the lateral surface of the body
wall. Each training session consisted of four trains of 10 shocks
delivered at intervals of 30 min. Only one side of the animal received
training; the contralateral side served as control. The side to be
trained was determined by coin flip at the start of the session.
At a period 24 hr later the strength of siphon withdrawal was tested
again, using the protocol described above. The strengths of the testing
stimuli were exactly the same as those used the previous day. The
individual who performed the siphon withdrawal tests did not know which
side of the animal had been sensitized.
Intracellular recordings
Immediately after the post-training test the animals were
anesthetized with isotonic MgCl2, and the left
pleural pedal ganglia were removed. Therefore, animals in the
sensitized group were trained on the left side of the animal, and
animals in the control group were sensitized on the right side. Then
the ganglia were treated with 0.5% glutaraldehyde in artificial
seawater for 40 sec to immobilize the connective tissue (Mirolli and
Gorman, 1968; Byrne et al., 1979). The sheath overlying the pleural
pedal ganglia was removed surgically in a solution containing 50%
isotonic MgCl2 and 50% artificial seawater. Intracellular
recordings were made through glass microelectrodes with a resistance of
3-10 M. Sensory and motor neurons innervating the tail were
identified by their size and position in the pleural pedal ganglia
(Walters et al., 1983). Sensory neurons used in this study were
localized in the medial portion of the ventrocaudal cluster of the
pleural ganglion, where sensory neurons innervating the tail are
concentrated (Fig. 2) (Walters et al.,
1983; Zhang et al., 1993). Neurons in this region also have a high
probability of forming monosynaptic connections with tail motor neurons
in the pedal ganglion. Sensory neurons were impaled with one electrode;
motor neurons were impaled with two to control membrane potential.
LPl17 was identified by its position in the pleural ganglion and its
ability to evoke a characteristic slow EPSP in the tail motor neuron
(Cleary and Byrne, 1993). The individual performing the intracellular
recordings did not know which side of the animal had been sensitized
nor the results of the testing procedure.
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Figure 2.
Schematic representation of the neural circuits
underlying the tail-elicited tail-siphon withdrawal reflex.
Stimulation of the tail of the animal activates sensory neurons
(SN) located in the pleural ganglion. These
neurons activate motor neurons (MN) in the pedal
ganglion that produce tail withdrawal. In addition, sensory neurons
activate a polysynaptic pathway that projects to the abdominal
ganglion, resulting in siphon withdrawal. The interneuron
(IN) LPl17 is an element of this pathway.
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Testing protocol
The cellular properties of sensory, motor, and interneurons were
tested by a standardized protocol.
Sensory neurons and motor neurons. First, a sensory neuron
and a follower motor neuron were impaled. The resting membrane potentials of both cells were recorded after a brief period of stabilization (2-5 min). Then we held the membrane potential of the
motor neuron manually at  80 mV by passing current through the second
electrode. Depolarizing current pulses of gradually increasing
intensity (0.1 nA steps, 50 msec duration) were applied to the sensory
neuron until the cell fired, evoking an EPSP. The number of spikes
elicited by the short current pulse was recorded, as was the amplitude
of the afterdepolarization. The EPSP was evoked two additional times at
intervals of 5 min, and the amplitudes were averaged. This procedure
minimized the contribution of other activity-dependent phenomenon such
as posttetanic potentiation (PTP) or long-term potentiation,
because the pulse elicited more than one spike in only two cases.
Sensory neuron excitability was tested by passing a long depolarizing
current pulse (2 nA, 1 sec) and recording the number of evoked action
potentials. Input resistance of both sensory and motor neurons was the
next parameter to be measured. This parameter was measured by evoking
voltage transients with a series of hyperpolarizing current pulses. The amplitude of the hyperpolarizing steps was chosen on a cell-by-cell basis and depended on the input resistance. Generally, cells were tested in increments of 0.1-0.3 nA, resulting in voltage steps from
approximately 2 to 40 mV. The input resistance was calculated from
the slope of the linear portion of the resulting I-V
curve.
Interneurons and motor neurons. After the sensory-motor
series a second series of measurements was made for interneurons and motor neurons. The holding potential of the motor neuron was adjusted to 80 mV, and excitability was tested by measuring the threshold for
evoking an action potential. The amplitude of a depolarizing current
pulse was increased in steps (0.1 nA, 50 msec) until an action
potential was evoked. The threshold was recorded as the maximum level
of depolarization that did not evoke an action potential. After these
tests the excitatory interneuron LPl17 was identified by its production
of a slow EPSP in the motor neuron. After a 15 min rest period the
input resistance of the interneuron was tested with a series of
hyperpolarizing pulses as described above. Five minutes later the
amplitude of the slow EPSP evoked in the motor neuron by the
interneuron was measured. The interneuron was stimulated with a series
of depolarizing current pulses of increasing amplitude (1, 2, 4, 6, 8, 10, 12, 15, 18, and 21 nA). Because some interneurons evoked a fast
component of the EPSP, which could mask the peak of the slow component,
the value of the EPSP at 1 sec after the stimulus was used for
analysis. The magnitude of the EPSP was quantified by the slope of the
linear portion of the resulting stimulus-response curve.
Statistical analysis
Data were represented by the median and interquartile range
(IR). The Wilcoxon test (behavior) and the Mann-Whitney test (cellular correlates) were calculated by the Prism statistical package (GraphPad Software, San Diego, CA). More animals were tested behaviorally than
were tested physiologically, because the cellular experiments could not
be completed for all ganglia. In some cases this was attributable to
the inability to find a monosynaptic connection. In others it was
attributable to recorded cells not surviving the testing protocol.
Similarly, more preparations were used for analysis of the
sensory-motor connection than the LPl17-motor neuron connection,
primarily because the success rate for LPl17 identification was
lower.
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RESULTS |
Long-term sensitization training produced a lateralized
enhancement of the tail-induced siphon withdrawal reflex
Before training, the median duration of siphon withdrawal was
~3.5 sec for both sides of the animal (control: 3.44 sec, 3.0 IR;
sensitized: 3.55 sec, 2.5 IR). The difference was not significant (T42 = 340; p = 0.16).
The response of the siphon to weak tail shock was tested 24 hr after
sensitization training. The response elicited by test stimuli on the
side of the animal ipsilateral to the training stimulus was markedly
enhanced as compared with the pretraining response (Fig.
3). The average ratio of post-test
response to pretest response was 186% (95 IR) for the sensitized side.
Conversely, the response elicited by test stimuli contralateral to the
trained side was not affected. The average ratio of post-test response to pretest response was 102% (41 IR). The enhancement on the
sensitized side of the animal was significantly greater than that on
the control side (T42 = 61; p < 0.0001). These results demonstrate that the protocol used in this study
produced long-term sensitization of the tail-induced siphon withdrawal
reflex. Moreover, the enhancement was lateralized; that is, it was
observed only when test stimuli were applied to the trained side of the
animal. From these results we infer that the tail withdrawal component
of the reflex was enhanced as well, although it was not measured
directly.
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Figure 3.
Long-term sensitization training increased the
duration of the tail-induced siphon withdrawal reflex. The duration of
siphon withdrawal observed during the post-training tests was expressed
as a percentage of the pretest duration. Learning was assessed by
comparing the normalized duration on the trained side of the animal
(Sensitized) with that on the contralateral untrained
side (Control).
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A critical step in elucidating the cellular mechanisms that mediate
this form of learning is to identify the sites of plasticity within the
nervous system. Key elements of the circuit mediating the reflex have
been identified (see Fig. 2). The pleural sensory neurons make
monosynaptic contacts to motor neurons in the pedal ganglion. They also
form polysynaptic pathways to motor neurons in the abdominal ganglion
that mediate the siphon withdrawal component of the response. The
interneurons that project to the abdominal ganglion have not been
characterized fully. An excitatory interneuron LPl17 has been shown to
excite interneurons and motor neurons in the abdominal ganglion
contributing to siphon withdrawal (Cleary and Byrne, 1993). This neuron
also forms a parallel circuit from tail sensory neurons to tail motor
neurons. The inhibitory interneuron RPl4 also forms a parallel circuit
to tail motor neurons (Xu et al., 1994), but its effects on siphon
motor neurons are unknown.
Modulation of the intrinsic cellular properties of any of these cells
could contribute to modulation of the reflex by altering their firing
properties. Perhaps more important is the possibility that modulation
of synaptic strength anywhere in the circuit, including sensory-motor,
sensory-interneuron, and interneuron-motor, could have a large effect
on motor neuron output (White et al., 1993). Therefore, we examined the
properties of identified neurons in each of these classes and the
connections between them.
Long-term sensitization training affects excitability of the tail
sensory neurons
Previous studies demonstrated that long-term sensitization
training affected biophysical properties of tail sensory neurons (Scholz and Byrne, 1987). Specifically, training reduced the net outward current elicited by a depolarizing voltage step, an observation consistent with modulation of the S-potassium current. As in short-term sensitization, modulation of the S-current would be expected to increase the excitability of tail sensory neurons (Klein et al., 1986;
Baxter and Byrne, 1989). This expectation was confirmed in the present
study.
Sensory neuron excitability was measured as the number of action
potentials elicited by a depolarizing current pulse of constant amplitude and duration (2 nA, 1 sec). The number of action potentials elicited per pulse increased from a median of seven spikes (7 IR) in
ganglia from the control side of the animal to 14 spikes (8.5 IR) in
ganglia from the sensitized side (Fig.
4A). This difference was statistically significant (U19,19 = 267;
p < 0.01). The increase in excitability was expressed
primarily as antiadaptation. Sensory neurons in ganglia from the
control side stopped firing early in the pulse, whereas sensory neurons
in ganglia from the sensitized side fired throughout the pulse.
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Figure 4.
Long-term sensitization produced changes in three
biophysical properties of tail sensory neurons: excitability
(A), the amplitude of the afterdepolarization
that follows a 1 sec, 2 nA depolarization (B),
and the amplitude of the afterdepolarization that follows a 50 msec,
0.5 nA depolarization (C). In Panels
1 (top), recordings from both control and
sensitized animals are shown. In Panels 2
(bottom), the group data are shown. In B
and C, the examples were chosen to emphasize the
differences.
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Another biophysical property of sensory neurons contributing to
excitability is the afterdepolarization. The amplitude of the
afterdepolarization that follows both the long pulse described above
and a short (50 msec) pulse was examined also. In both of these
circumstances the amplitude of the afterdepolarization was increased in
ganglia from the sensitized side. After the long pulse (1 sec) the
afterdepolarization increased from a median of 0.8 mV (1.8 IR) in
ganglia from the control side to 2.6 mV (4.2 IR) in ganglia from the
sensitized side (U19,19 = 260; p < 0.02) (Fig. 4B). The afterdepolarization that
follows a single spike was affected, as well (Fig. 4C). The
afterdepolarization increased from a median of 1.3 mV (1.7 IR) in
ganglia from the control side to 2.5 mV (3.8 IR) in ganglia from the
sensitized side (U18,21 = 260; p < 0.01).
Long-term sensitization training had no significant effect on the
resting membrane potential or input resistance of the tail sensory
neurons (Table 1).
Long-term sensitization training affects two biophysical properties
of tail motor neurons
Little is known about the effects of long-term sensitization
training on follower motor neurons. Therefore, we examined several biophysical properties of the tail motor neuron. Resting membrane potentials were recorded 5 min after impalement of the neuron with the
microelectrode. On average, motor neurons were hyperpolarized after
long-term sensitization training (Fig.
5A). The resting membrane
potential decreased from a median of 59 mV (9 IR) in ganglia from the
control side to a median of 63 mV (6 IR) in ganglia from the
sensitized side (U19,21 = 254;
p < 0.04).
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Figure 5.
Long-term sensitization produced changes in two
biophysical properties of tail motor neurons: resting membrane
potential (A) and threshold for spike initiation
(B). In Panels 1
(top), recordings from both control and sensitized
animals are shown. In Panels 2 (bottom),
the group data are shown. In B1, the threshold for each
neuron is illustrated by a solid line. The solid
line in the Control trace was extended by a
dashed line to facilitate comparison. Note that
threshold was defined as the largest depolarization that did not
initiate a spike. Therefore, threshold was not determined from these
traces.
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The effects on membrane potential are counterintuitive, because
hyperpolarization would tend to make the motor neuron more difficult to
fire under circumstances when it should be firing more intensely. The
hyperpolarization may be balanced in part by a decrease in the
threshold for spike generation. The threshold was determined by
gradually increasing the strength of a depolarizing current pulse in
0.1 nA steps at intervals of 1 sec from a fixed holding potential of
80 mV. The threshold was defined as the membrane potential reached by
the largest current pulse that did not elicit a spike. Long-term
sensitization decreased the threshold for spike initiation (Fig.
5B). The average threshold decreased from a median of 44
mV (9 IR) to a median of 46 mV (6 IR) (U15,16 = 171; p < 0.05).
The decrease in spike threshold suggested that membrane excitability of
the motor neuron was enhanced. This possibility was tested more
directly in some motor neurons by delivering depolarizing current
pulses of 3 sec duration in a series of increasing amplitudes of 0.5 nA
steps at intervals of 30-70 sec. Then the number of resulting spikes
was counted. Excitability was defined as the slope of the linear
portion of the input-output curve. Although there was a strong trend
toward increased motor neuron excitability, the enhancement was not
statistically significant (Table 2). Moreover, there was a trend toward decreased input resistance of the
tail motor neuron after long-term sensitization training, but this
modulation was also not statistically significant (Table 2).
Long-term sensitization training increased the strength of the
sensorimotor synapse
The efficacy of sensory neuron transmission was assessed by
measuring EPSPs evoked in tail motor neurons. EPSPs were measured with
the membrane potential of the motor neuron held at 80 mV to prevent
the motor neuron from firing and to control for possible effects of
long-term sensitization on the resting potential of motor neurons (Fig.
6A). After long-term
sensitization training, the EPSP increased from a median of 6.9 mV (7.3 IR) in ganglia from the control side to a median of 11 mV (7.2 IR) in
ganglia from the sensitized side, but this difference was not
statistically significant (U17,20 = 212;
p = 0.21). To reduce the variability, we calculated
synaptic currents by dividing the amplitude of the EPSP by the input
resistance of the motor neuron. The EPSC increased from a median of
0.53 nA (0.86 IR) in ganglia from the control side to a median of 1.01 nA (1.06 IR) in ganglia from the sensitized side
(U16,18 = 202; p < 0.05) (Fig.
6B).
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Figure 6.
Long-term sensitization training increased
synaptic strength between sensory and motor neurons. Recordings of
EPSPs evoked in motor neurons by sensory neurons are shown in
A. In this experiment the motor neurons were held at
80 mV. For the purposes of comparing group data, the EPSC was
calculated by normalizing the EPSP by the input resistance of the motor
neuron.
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In a previous study the percentage of sensory neurons making
connections to motor neurons appeared to increase as a result of
long-term sensitization (Frost et al., 1985). The input of tail sensory
neurons onto a specific follower is not amenable to this type of
analysis, however. There are more neurons in the pleural sensory
cluster than in the abdominal LE cluster, and only a small percentage
converges onto an individual tail motor neuron.
In ganglia from the sensitized side there was a correlation between the
amplitude of the EPSC and the duration of siphon withdrawal (r = 0.59, after log transformation of the data) (Fig.
7). This correlation did not hold for
ganglia from the control side. Therefore, the strength of the
behavioral response on the control side of the animal appeared to
depend on many factors. The enhanced response on the trained side
appeared to depend, at least in part, on enhanced transmission from
tail sensory neurons.
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Figure 7.
The amplitude of the EPSC from ganglia from the
sensitized side was correlated with the duration of withdrawal after
sensitization training (r = 0.59). Duration was
expressed as a percentage of the pretest duration. Values <100%
indicate that the animal was not sensitized.
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Synaptic connections from sensory neurons to interneurons probably were
modulated to the same extent. We attempted to test this hypothesis
directly by examining the amplitude of the EPSP evoked in the
interneuron LPl17 by the sensory neuron. The success rate was too low
for analysis, however.
Long-term sensitization training did not affect the excitatory
interneuron LPl17
LPl17 is an identified interneuron that has widespread effects
throughout the CNS (Cleary and Byrne, 1993). Perhaps most
importantly, it forms another pathway parallel to the monosynaptic
component of the tail withdrawal circuit. Therefore, modulation of
LPl17 output could have a significant impact on motor neuron activation (White et al., 1993). Also, it activates siphon motor neurons in the
abdominal ganglion. Therefore, it could be an additional site for
enhancement of siphon withdrawal beyond sensory neuron facilitation and
enhanced excitability. Three biophysical properties of LPl17 were
examined: resting membrane potential, input resistance, and
excitability (Table 3). None of these
properties was affected by long-term sensitization training. The
strength of the synaptic connection between LPl17 and the follower
motor was examined also. A characteristic feature of LPl17 is its
ability to evoke a slow EPSP in tail motor neurons when it is activated
by a series of action potentials (Fig.
8A,B). The amplitude of
the slow EPSP evoked in the motor neuron was not affected by
sensitization training (Fig. 8C).
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Figure 8.
Training had no effect on the amplitude of the
slow EPSP evoked in the motor neuron by LPl17. Recording of slow EPSPs
evoked in ganglia from control (top) and sensitized
(bottom) sides of the animal are illustrated in
A. Because it was difficult to control the number of
spikes generated in LPl17 by a given current pulse, several slow EPSPs
were evoked by depolarizations of different amplitudes. The amplitude
of the slow EPSP was plotted against the number of spikes elicited in
LPl17. The slope of the regression line (mV/spike) over the linear
range was used to compare ganglia from control and sensitized sides.
The relationships for the two preparations shown in A
are illustrated in B. The slopes of the regression lines
are 0.112 mV/spike (Control) and 0.123 mV/spike
(Sensitized). Group data are illustrated in
C.
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DISCUSSION |
A great deal of information has been obtained regarding defensive
withdrawal reflexes in Aplysia. Key emerging concepts are the important contribution of the monosynaptic component of the circuit
to the reflex and the important contribution of plasticity at the
sensory-motor synapse to modulation of the reflex by learning. These
concepts emerged primarily from study of the short-term forms of
learning such as sensitization, habituation, and classical conditioning. It is reasonable to argue that these same concepts also
hold for long-term forms of learning. There are, however, very few data
available to support that hypothesis.
A key site of plasticity appears to be the afferent limb of the circuit
for both short- and long-term forms of sensitization. After delivery of
sensitizing stimulation to the skin of a semi-intact preparation or
stimulation of the connectives that would convey this information,
several short-term changes in biophysical properties of the sensory
neurons, expressed as a depolarized membrane potential, increased
excitability, and increased action potential duration, have been
observed (Walters et al., 1983; Byrne et al., 1990). In addition, there
is an increase in the amplitude of the postsynaptic potential evoked in
the follower motor neuron by the sensory neuron (Walters et al., 1983;
Castellucci et al., 1989).
For long-term sensitization the link between behavioral sensitization
and enhancement of the sensorimotor EPSP is less well established.
There have been only two studies in which animals were trained and then
24 hr later ganglia were removed from the animal for
electrophysiological analysis (Frost et al., 1985; Walters, 1987). In
both of these studies the sensorimotor EPSP was enhanced 24 hr after
training. Several in vitro analogs of long-term
sensitization have been examined in isolated ganglia (Castellucci et
al., 1989; Emptage and Carew, 1993; Zhang et al., 1994, 1997) and cell
culture (Montarolo et al., 1986; Dale et al., 1987, 1988),
demonstrating changes in synaptic strength and excitability of sensory
neurons. Changes in sensory neuron morphology also appear to contribute
to long-term sensitization (Bailey et al., 1996). In the population of
animals studied here, no effects of sensitization training were
observed on sensory neuron morphology (Wainwright et al., 1997).
Ultrastructural changes were not ruled out, however.
Correlates of long-term habituation have been examined in
Aplysia. Synaptic input to motor neurons was reduced (Carew
and Kandel, 1973), as was the number of detectable synapses
(Castellucci et al., 1978). In the tail-induced siphon withdrawal
circuit we did not test for changes in the number of detectable
synapses (Frost et al., 1985) because the pleural sensory cluster is,
unlike the abdominal LE cluster, structurally heterogeneous, and
convergence onto identified motor neurons is low.
In this paper we report a more extensive analysis of the correlates of
long-term sensitization of the tail-siphon withdrawal reflex. Several
different biophysical properties of sensory neurons, motor neurons, and
LPl17, an identified interneuron, were examined in animals that had
been subjected to long-term sensitization training.
Effects of long-term sensitization training on tail
sensory neurons
Previous work using long-term sensitization training protocols,
and supported by in vitro analogs, suggested that tail
sensory neurons were an important site of plasticity. Tail sensory
neurons demonstrated a persistent decrease of net outward currents
after behavioral training (Scholz and Byrne, 1987). Moreover, the
amplitude of the sensorimotor EPSP was enhanced (Walters, 1987). In the present study both of these effects were confirmed.
Two biophysical properties of tail sensory neurons, membrane
excitability and amplitude of the afterdepolarization, were affected by
training. Modification of both of these properties is consistent with
previous work demonstrating a persistent (24 hr) decrease by training
in net outward currents that had kinetics and voltage-sensitivity similar to the S-potassium current (Scholz and Byrne, 1987). The S-current has a strong effect on membrane excitability (Baxter and
Byrne, 1989; Byrne et al., 1990) because of its relatively slow
kinetics. We do not know the ionic mechanisms of the
afterdepolarization and its modulation. One possibility is that the
reduction or elimination of the S-current would allow a late inward
current to express itself as a slow depolarization.
One consequence of potassium current modulation could be enhanced
action potential duration. We did not quantitate action potential
duration, because it is difficult to measure this feature accurately
when the spike is elicited by the recording electrode. Nevertheless,
there were no obvious qualitative changes in action potential
duration.
Our results were consistent with the enhancement of the EPSP reported
by others for the tail sensory-motor synapse (Walters, 1987) and the
siphon sensory-motor synapse (Frost et al., 1985), but the effect was
not statistically significant. One possible difference could be the
sampling procedures used. In one study only animals that reached a
performance criterion were tested (Frost et al., 1985). Moreover, in
that study the animals were trained for a longer period of time (4 d)
than in the protocol used here. In another study only the strongest
synaptic connections were included in the analysis (Walters, 1987).
Both of these procedures could increase the probability of encountering
an enhanced synapse in trained animals. We did not use any selection
criteria in our analyses, however, and the resultant variability was
too large for the difference to achieve statistical significance. When
variability was reduced by calculating the EPSC, we observed a
significant enhancement. There could be differences in the enhancement
induced in specific sensory neurons attributable to heterogeneous
modulatory input. For example, some sensory neurons receive denser
input from serotonergic pathways than others (Zhang et al., 1991).
One observation that was not confirmed was the occurrence of
regenerative bursting in tail sensory neurons (Walters, 1987). In our
sample only two cells in 18 ganglia fired multiple action potentials in
response to a brief (50 msec) intracellular depolarizing stimulus. One
possible explanation is that our training protocol minimized the risk
of injury to tail sensory neurons, which causes regenerative bursting
(Clatworthy and Walters, 1994). Alternatively, minor differences in the
details of the training protocol could have enhanced regenerative
spiking selectively.
Although training produced a number of changes, it was interesting to
take the analysis one step further and correlate the magnitude of a
neuronal property with the magnitude of the behavior. With a linear
regression analysis the amplitude of the calculated EPSC was
correlated significantly with the duration of the behavior. This is the
first study to our knowledge in which this correlation was examined for
long-term sensitization in Aplysia. The correlation itself
was relatively low (r = 0.59), which perhaps is
expected, considering the number of synapses in the circuit mediating
the tail-elicited siphon response. Surprisingly, the correlation held in trained animals, but not in control animals. This suggests that, in
control animals, there are a number of factors regulating siphon
withdrawal, but after sensitization the amplitude of the sensory-motor
EPSP plays a more prominent role. The causal relationship between EPSP
amplitude and duration of contraction is not fully understood. Sensory
neuron input appears to contribute only to the initial part of motor
neuron activation (Walters et al., 1983; Lieb and Frost, 1997), whereas
response duration appears to depend on the activation of interneurons
(Cleary and Byrne, 1993; White et al., 1993; Frost and Kandel, 1995;
Lieb and Frost, 1997). Therefore, the enhanced EPSP in the motor neuron
may reflect enhanced input to interneurons, prolonging the duration of
the response. At the behavioral level the amplitude of the gill
withdrawal reflex was correlated with the duration of the siphon
withdrawal response (Stopfer and Carew, 1987).
Other biophysical changes were not correlated with the duration of
siphon withdrawal (Table 4). The lack of
correlation does not necessarily rule out a causal role in long-term
sensitization, however. For example, lack of correlation with
excitability could be attributable to the fact that the 20 msec
behavioral test stimulus was too brief to activate the modified
processes underlying the enhanced excitability.
Effects of long-term sensitization on tail motor neurons
Previous studies of long-term sensitization found no effect of
training on the biophysical properties of the motor neuron. For
example, in siphon motor neurons both the resting membrane potential
and the input resistance were unaffected (Frost et al., 1985). In the
present study the input resistance of the motor neuron tended to
decrease, but the effect was not statistically significant. However,
the resting membrane potential was hyperpolarized in motor neurons from
the trained side. This effect would work against the increased motor
neuron activity that presumably underlies the enhanced withdrawal. The
hyperpolarization may be compensated for, in part, by a decrease in
spike threshold. Thus, modulation of these two biophysical properties
of motor neurons may cancel each other effectively. Nevertheless, these
observations are important because they demonstrate that the motor
neuron is also a site of plasticity. Although minor in themselves, they
could indicate that other, more significant, properties of the motor
neuron are affected. For example, there are some data to suggest that
the morphology of motor neurons is modified by long-term sensitization (Bailey and Chen, 1988). Because the motor neuron fires more readily after sensitization, we would expect to see an increase in
excitability. There was a tendency for increased excitability in motor
neurons from the sensitized side of the animal, but the increase was
not statistically significant.
The role of interneurons in long-term sensitization
Clearly, the monosynaptic component of the circuit plays a crucial
role in mediation and modulation of withdrawal reflexes. Nevertheless,
there is strong evidence in Aplysia for a distributed representation of the memory for short-term sensitization (Frost et
al., 1988; Trudeau and Castellucci, 1992; Fischer and Carew, 1995;
Frost and Kandel, 1995; Wright and Carew, 1995; Xu et al., 1995).
Virtually nothing is known of the role of interneurons in long-term
sensitization. In the siphon withdrawal circuit a higher percentage of
sensory neurons recruited interneurons after long-term sensitization, a
finding consistent with the increased strength of the sensorimotor
synapse (Frost et al., 1985). In the present study several biophysical
properties of the excitatory interneurons LPl17 were examined directly.
No effects of behavioral training were observed. Moreover, the strength
of the slow EPSP evoked in motor neurons by LPl17 was not affected by
long-term sensitization training.
Although these results suggest that LPl17 is not itself a site of
plasticity, enhancement of sensory neuron output to LPl17 still could
be an important mechanism for increasing motor neuron activation (White
et al., 1993). Furthermore, other identified interneurons could be
affected by long-term training. For example, modulation of the
inhibitory interneurons RPl4 and RPl5 (Buonomano et al., 1992; Xu et
al., 1994) may contribute to the behavioral modification. Indeed,
mathematical simulations have suggested that inhibition of RPl4 would
be an effective mechanism for increasing motor neuron activation
(Medina et al., 1994). These interneurons are affected by acute
application of 5-HT (Xu et al., 1995), raising the intriguing
possibility that they also may be a site for long-term sensitization.
In summary, these data confirm and extend the results of others,
demonstrating that sensory neurons are a site of plasticity in
long-term sensitization. In addition, this is the first study to
demonstrate that a second locus is affected by training, the motor
neurons. Although the biophysical modifications are modest, they could
represent more substantive cellular changes, relating to neurite
outgrowth, for example. The interneuron LPl17 was not affected by
training, but this does not rule out the possibility that other
interneurons, such as RPl4, are affected. Nevertheless, this work is
consistent with the idea that long-term sensitization, like the
short-term form, affects multiple sites of plasticity distributed
throughout the nervous system.
| |
FOOTNOTES |
Received Dec. 18, 1997; revised May 15, 1998; accepted May 15, 1998.
This work was supported by National Institutes of Health Grant R01
NS19895 and National Institute of Mental Health Award K05 MH00649 to
J.H.B. and by National Science Foundation Grant ISBN 9320549 to L.J.C.
We thank Michelle Aguirre, Jason Molpus, Andrew Tang, and Han Zhang for
technical assistance with the behavioral training procedures.
Correspondence should be addressed to Dr. John H. Byrne, Department of
Neurobiology and Anatomy, University of Texas Houston Medical School,
P.O. Box 20708, Houston, TX 77225.
| |
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J. Levenson, S. Endo, L. S. Kategaya, R. I. Fernandez, D. G. Brabham, J. Chin, J. H. Byrne, and A. Eskin
Long-term regulation of neuronal high-affinity glutamate and glutamine uptake in Aplysia
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E. Beaumont and P. Gardiner
Effects of daily spontaneous running on the electrophysiological properties of hindlimb motoneurones in rats
J. Physiol.,
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[Abstract]
[Full Text]
[PDF]
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Top
Abstract
Introduction
