| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
The Journal of Neurophysiology Vol. 88 No. 4 October 2002, pp. 1915-1923
Copyright ©2002 by the American Physiological Society
1Neuroscience and Respiratory Research Groups, Departments of Cell Biology and Anatomy, Physiology and Biophysics, Health Sciences Centre, Calgary, Alberta T2N 4N1; and 2Department of Biological Sciences, Brock University, St. Catharines, Ontario L2S 3A1, Canada
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Spencer, Gaynor E., Mustapha H. Kazmi, Naweed I. Syed, and Ken Lukowiak. Changes in the Activity of a CPG Neuron After the Reinforcement of an Operantly Conditioned Behavior in Lymnaea. J. Neurophysiol. 88: 1915-1923, 2002. We have previously shown that the aerial respiratory behavior of the mollusk Lymnaea stagnalis can be operantly conditioned, and the central pattern generating (CPG) neurons underlying this behavior have been identified. As neural correlates of operant conditioning remain poorly defined in both vertebrates and invertebrates, we have used the Lymnaea respiratory CPG to investigate neuronal changes associated with the change in behavior after conditioning. After operant conditioning of the intact animals, semi-intact preparations were dissected, so that changes in the respiratory behavior (pneumostome openings) and underlying activity of the identified CPG neuron, right pedal dorsal 1 (RPeD1), could be monitored simultaneously. RPeD1 was studied because it initiates the rhythmic activity of the CPG and receives chemo-sensory input from the pneumostome area. Pneumostome openings and RPeD1 activity were monitored both before and after a reinforcing training stimulus applied to the open pneumostome of operantly conditioned and yoked control preparations. After presentation of the reinforcing stimulus, there was a significant reduction in both breathing behavior and RPeD1 activity in operant preparations but not in yoked and naïve controls. Furthermore these changes were only significant in the subgroup of operantly conditioned animals described as good learners and not in poor learners. These data strongly suggest that changes in RPeD1 activity may underlie the behavioral changes associated with the reinforcement of operant conditioning of the respiratory behavior.
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The neuronal substrates of
associative learning and memory have not yet been fully elucidated
largely due to the complexity of the nervous systems and behaviors
studied (Martin et al. 2000
; Milner et al.
1998). Only in a few instances has the sufficiency and
necessity of a neural circuit for a specific behavior showing associative learning been demonstrated. In the pond snail Lymnaea stagnalis, not only has the three-neuron CPG, which controls
aerial respiratory behavior, been identified (Syed et al. 1990,
1992), but the respiratory behavior can also be operantly
conditioned (a form of associative learning) to produce long-term
memory (Lukowiak et al. 1996, 1998).
Aerial respiratory activity in Lymnaea increases
significantly when the oxygen content of the pond water is low
(hypoxic). Under these conditions, the animal moves to the air-water
interface where it opens and closes its respiratory orifice, the
pneumostome. During the operant conditioning procedure, the animal is
placed in a hypoxic environment to increase its respiratory drive, and a tactile stimulus is applied to the pneumostome area whenever the
animal attempts to breathe. We have operationally defined learning in
this model as a significant reduction in the number of attempted
pneumostome openings from session 1 to 5 and have demonstrated this to
be an example of associative learning (Lukowiak et al.
1996, Spencer et al. 1999).
The CPG controlling the aerial respiratory behavior of
Lymnaea is made up of three interneurons, right pedal dorsal
1 (RPeD1), visceral dorsal 4 (VD4) and input 3 interneuron (IP3I)
(Syed et al. 1990, 1991). IP3I controls pneumostome
opening (expiration), whereas VD4 controls pneumostome closing
(inspiration). Motor neurons (I/J cells) that innervate the pneumostome
and receive direct synaptic input from the CPG have also been
identified. We have previously shown that operant conditioning of
aerial respiration results in significant changes in CPG interneuron
and motor neuron activity patterns as well as synaptic connections, in
the isolated central ring ganglia (CNS) removed from conditioned
animals (Spencer et al. 1999). In these previous
studies, however, the peripheral input from the pneumostome area was
removed, and thus a direct relationship between the neuronal changes
and specific changes in behavior could not be deduced. Furthermore, the
input from the periphery significantly alters CPG rhythmicity
(Inoue et al. 2001). In this study, intact animals were
first operantly conditioned and subsequently prepared as semi-intact
preparations (CNS and its innervation of the pneumostome area remained
intact). This semi-intact preparation allowed us to directly monitor
changes in neuronal activity simultaneously with changes in behavior in response to the application of a reinforcing stimulus to the pneumostome.
Peripheral chemo-receptors are critical for the initiation of the
respiratory rhythm, and it is the dopaminergic CPG neuron, RPeD1, that
receives excitatory afferent chemo-sensory input from the
pneumostome-osphradial area (Inoue et al. 2001). Once
stimulated, RPeD1 activates IP3I, which in turn excites the pneumostome
opener motor neurons (I/J cells). Action potentials in the I/J cells produce 1:1 excitatory junction potentials in the pneumostome muscles,
producing pneumostome opening. We therefore chose to monitor the
activity of RPeD1, not only because it is the CPG neuron that receives
peripheral input, but also because its activity directly initiates the
respiratory rhythm (Lukowiak 1991; Syed et al.
1990). Because selective removal of RPeD1 also abolishes the
respiratory activity of freely moving animals (Haque
1999), RPeD1 is considered a likely locus for neuronal changes
associated with the conditioned change in behavior.
The specific aims of this study were to identify differences in behavior or neuronal activity between semi-intact preparations from operant and yoked control animals immediately after the five training sessions in the intact animal. We also wished to determine whether the presentation of the reinforcing stimulus to the semi-intact preparation induced behavioral and/or neuronal changes.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Specimens of L. stagnalis were laboratory bred and kept in well-aerated, artificial pond water and fed on lettuce. All animals used were between 26 and 30 mm in shell length.
Training procedures
Animals were trained according to the methods described
previously (Lukowiak et al. 1996; Spencer et al.
1999). Briefly, freely moving animals received five training
sessions, each 30 min in duration, over a period of 2.5 days (2 sessions a day, with at least a 1-h interval between sessions) in
hypoxic pond water. Before each training session, the animals were
given a 10-min acclimatization period during which they could freely
open their pneumostomes. At the end of this 10-min period, they entered
the 30-min training session. During each training session, the operant animals received a tactile stimulus to the pneumostome each time it
opened. This was a punishment conditioning paradigm with a positive
contingency between the pneumostome opening behavior and the
reinforcement. As a result of the reinforcement, the behavior was
reduced. That is, the tactile stimulus caused the pneumostome to close
but did not cause the animal to withdraw into its shell. Typically, the
animal stayed at the water's surface following tactile stimulation.
Because Lymnaea respire through the skin (cutaneous
respiration) and through their pneumostome (the respiratory orifice
connecting the lung to the atmosphere), inhibiting aerial respiration
behavior does not compromise the survival of the animal (Lukowiak et al. 1996).
Control procedures
The standard protocol used to test that yoked control animals neither exhibited a learned change in behavior nor short- or long-term memory was to monitor their number of attempted pneumostome openings 24 h before the start of the five yoked control training sessions (pretest), and at either 1 or 24 h after the final session (posttest).
For the behavioral analysis only, the yoked control group received
contingent stimulation of their open pneumostomes during the pretest
session. A single 30-min training session has previously been
determined not to result in any learning or long-term memory (Lukowiak et al. 2000). The yoked control animals were
then subjected to five yoked control sessions during which they
received noncontingent stimulation of the pneumostome area. That is,
the stimuli presented during these five yoked training sessions were
not contingent on the opening of the yoked animal's pneumostome but
rather on the pneumostome opening of the operant animal to which it was yoked.
To determine whether the yoked control animals showed any immediate changes in respiratory behavior (learning) after the five sessions, the number of attempted pneumostome openings were again tested 1 h after the final session. The number of attempted pneumostome openings were also tested 24 h after the final session to determine whether the yoked control animals demonstrated any long-term memory. During these posttest sessions, the yoked control animal again received stimulation contingent on its own pneumostome openings (similar to the pretest). For this reason, different sets of animals were tested in the 1- and 24-h posttest sessions. It was determined that no significant changes in pneumostome openings occurred in the yoked control animals, either 1 or 24 h after the yoked control sessions (see Fig. 1).
|
Yoked control animals dissected for electrophysiological analysis underwent the standard five, noncontingent yoked sessions (as described in the preceding text), but did not, however, receive the pre- or posttest contingent reinforcements.
A hypoxic control group was also used in which the animals were
maintained in the hypoxic pond water and prevented from surfacing for
the duration of the 30-min training periods. We have previously shown
that after this hypoxic treatment, animals do not demonstrate any
significant change in their respiratory behavior compared with the
yoked controls (Lukowiak et al. 1996). We also confirmed in this study using semi-intact preparations from hypoxic-maintained animals that there was no significant difference in behavior from yoked
control preparations (see following text).
A naïve control group of animals that were not subjected to hypoxia or stimulation were also dissected and tested for changes in both behavior and RPeD1 activity, and the numerical data incorporated in RESULTS.
Semi-intact preparation
One hour after the fifth training session, operantly conditioned and yoked control animals were anesthetized in Listerine. Listerine is a standard anesthetic used in Lymnaea studies, and its application has been shown not to affect memory. Operantly conditioned animals placed in Listerine and anesthetized between training sessions 4 and 5 continued to show a significant reduction in number of openings in session 5 (2.0 ± 1.0) compared with in session 1 (6.1 ± 1.2; t-test; P < 0.009; n = 6, data not shown). These data confirm that the use of Listerine does not affect the retention of memory after conditioning and is therefore suitable for use in this study.
All procedures for the preparation of semi-intact preparations were
carried out in a similar manner to that described previously (Inoue et al. 1996, 2001). Specifically, in the presence
of the anesthetic, the buccal mass and salivary ducts, reproductive
organs and head-foot musculature were carefully removed, exposing the CNS. The CNS, nerves innervating the pneumostome area, and the pneumostome itself were left intact. The preparation was then pinned in
a dish with the dorsal surface of the CNS and pneumostome area exposed
(see Fig. 2). The outer sheath of the CNS
was removed using fine forceps, and the ganglia briefly exposed to
protease to soften the inner sheath. The preparation was washed and
then bathed in hypoxic saline (92% nitrogen, 8% oxygen) to mimic the hypoxic conditions during the training sessions. The preparation was
placed in hypoxic conditions for a period of 10 min prior to commencing
data collection, and this was maintained throughout the experiment.
Previous experiments have demonstrated that the pneumostome will open
and close spontaneously in the semi-intact preparation, providing that
the saline level is maintained at or below the level of the pneumostome
(Syed et al. 1991). The saline level in these
experiments was therefore lowered below the level of the pneumostome
(but continued to bathe the CNS and remainder of the preparation).
|
Experimental procedure
Intracellular electrophysiological recordings were made from the CPG interneuron, RPeD1. Pneumostome openings were visually scored during the entire course of the electrophysiological recordings and were recorded electronically on a chart recorder. Control recordings of neuronal activity and pneumostome openings were obtained from preparations from both operant and control animals for a period of 10 min. This was called the prestimulus period. After this period, the reinforcing stimulus was presented to the pneumostome of both operant and control preparations, (using a sharpened wooden applicator similar to that used during the training sessions of the intact, freely moving animal) on their next attempted pneumostome opening (0-5 min latency). The neuronal activity and pneumostome openings were then monitored for a further 10-min period (see Fig. 2). This is called the poststimulus period. The mean firing frequency of RPeD1 was calculated over the entire 10 min pre- and poststimulus periods.
Pneumostome behavior was first monitored in a group of semi-intact preparations from the hypoxic control animals. There was no effect of the reinforcing stimulus on either the number (before: 11.1 ± 2.9, after: 10.9 ± 2.6) or duration of openings (before: 5.1 ± 0.6 s, after: 4.3 ± 0.5 s) of the hypoxic controls (n = 7, paired t-tests P > 0.05). Furthermore no significant changes were observed in either the number or duration of pneumostome openings, either before or after the reinforcing stimulus, compared with yoked controls (unpaired t-tests, P > 0.05, df = 31; data not shown). No further experiments were therefore performed on this particular group of control animals.
Data collection and analysis
Data from operant and control preparations were analyzed for the entire 10-min period both before (prestimulus period) and after (poststimulus period) the reinforcing stimulus. All data collection and analyses were performed blind (the animal's training category was unknown to the person performing the electrophysiological recording and analysis). All values were expressed as means ± SE. Unless otherwise stated, two-way ANOVA followed by Dunn's (Bonferroni corrected) post hoc test were used to determine significance.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Animals that are operantly conditioned using the well-established
training protocol (Lukowiak et al. 1996) exhibit a
significant reduction in their respiratory behavior (total breathing
time and pneumostome openings) at the end of their final training
session (Lukowiak et al. 1996, 1998, Spencer et
al. 1999). In previously published reports, we have also shown
that yoked control animals do not exhibit any significant changes in
their aerial respiratory behavior after five yoked control sessions
(Lukowiak et al. 1996, 1998). Because semi-intact
preparations used in this study were dissected immediately after the
final training session, it was first necessary to determine (using a
group of animals for behavioral analyses only) that the yoked procedure
used throughout this study did not produce a change in behavior of the
yoked control animals. An operant group (n = 30)
exhibited a significant reduction in the respiratory behavior over five
training sessions (number of attempted pneumostome openings: session 1, 11.7 ± 1.1, session 5, 2.7 ± 0.4; P < 0.01, data not shown). The number of attempted pneumostome openings of
the yoked control group were obtained 24 h before (pretest) the
five yoked control training sessions and again tested either at 1 or
24 h after the final session (posttest). It was found that there
was no significant difference in the respiratory behavior of the yoked
control animals either at 1 or 24 h after the final session (Fig.
1A). These results are consistent with all previously
published reports (Haney and Lukowiak 2001;
Lukowiak et al. 1996, 1998, 2000) and confirm that the
yoked control animals neither show learning or memory after the yoked
control procedure.
A separate group of animals was next used for the neurophysiological
studies in the semi-intact preparations. In these experiments, the
yoked control group was subjected to the five yoked control sessions
but did not receive any contingent reinforcement. Following the final
(5th) operant conditioning training session, the behavioral scores
(number of attempted pneumostome openings) were tabulated for all
operantly conditioned animals (n = 44), and a typical learning curve was produced (Fig. 1B). The training was
found to have a significant effect on the number of openings over the five training sessions [1-way ANOVA, F(43,4) = 52.3, P < 0.0001]; the number of openings in session 5 were
found to be significantly less than the number of openings in session 1 (Dunn's (Bonferroni corrected) post hoc test, P < 0.01). However, evaluation of the number of attempted openings of
individual animals in the operant group suggested that a number of
these animals did not exhibit learning. The criteria for a "good
learner" was previously described as an animal whose number of
attempted pneumostome openings were reduced by 
50% from session 1 to
5 (Spencer et al. 1999). In this present study, only 26 of the 44 conditioned animals demonstrated at least a 50% reduction in
behavior by the fifth training session (Fig. 1C). In this
group of good learners, the training sessions had a significant effect
on the number of pneumostome openings [1-way ANOVA,
F(25,4) = 107.4, P < 0.001] producing
a 70% overall reduction in respiratory behavior (Fig. 1C).
Of the remaining 18 conditioned animals, it was shown that even though
the training sessions did exert an effect on the number of openings
[1-way ANOVA, F(17,4) = 5.3, P < 0.05], these "poor learners" only demonstrated an overall 22%
reduction in behavior. To identify neural correlates of a behavioral
change following operant conditioning, it is first necessary to observe
that change in behavior. For instance, we previously found significant
changes in the isolated ganglia, only when animals categorized as good
learners were analyzed (Spencer et al. 1999). In this
study, we have analyzed all animals that received
conditioning regardless of their behavioral score. Because neuronal
changes can be subtle, however, in addition to grouping all conditioned
animals together for analysis, we also separately analyzed data from
good and poor learners.
Semi-intact preparations were dissected from all operantly trained animals as well as from yoked controls, following the final (5th) training session. Intracellular recordings were made from the CPG neuron, RPeD1, while pneumostome openings were simultaneously monitored. Initially, pneumostome opening movements and neuronal activity were monitored for a control period of 10 min (prestimulus period). The reinforcing training stimulus was presented to all preparations (including all controls) on the next attempted pneumostome opening (Fig. 2). After this stimulus, pneumostome opening movements and neuronal activity were monitored for a further 10 min (poststimulus period).
Operantly conditioned animals showed a significant reduction in total breathing time after the reinforcing stimulus
We first sought to determine whether the semi-intact preparation would respond to a reinforcing training stimulus and, if so, whether the preparations from operantly conditioned animals responded differently to controls. To determine whether the reinforcing stimulus induced a change in respiratory behavior in the semi-intact preparations, the total breathing time was calculated for both the prestimulus and poststimulus periods in operant, yoked control, and naïve animals. A two-way ANOVA revealed a significant interaction [F(77,3) = 9.0, P = 0.0003] between different groups and stimulus treatment. It was found that only the operantly conditioned preparations showed a significant reduction in total breathing time following the reinforcing stimulus [Dunn's (Bonferroni corrected) post hoc test; P < 0.01; Fig. 3A]. The yoked control (Fig. 3, A and B) and naïve preparations (before: 101.4 ± 19.5 s; after: 116.6 ± 22.6 s; n = 11, data not shown) did not show any significant change (P > 0.05) in their respiratory activity after application of the reinforcing stimulus.
|
We also compared the change in respiratory activity induced by the reinforcing stimulus in poor and good learners and found that only the good learners exhibited a significant reduction in their total breathing time after the stimulus (P < 0.01; Fig. 3B). These data demonstrate that application of a reinforcing stimulus to semi-intact preparations dissected from operantly conditioned animals resulted in a significant change in respiratory activity. In summary, after dissection, the semi-intact preparations maintained their memory for the operantly conditioned behavior.
Pneumostome opening movements of operant preparations were significantly different in the prestimulus period
Having determined that only the operantly conditioned preparations showed a significant change in breathing activity after the reinforcing stimulus, we next aimed to determine whether there were any significant changes in the pneumostome opening parameters between preparations from operantly conditioned and yoked control animals during the initial 10-min recording (prestimulus period). That is, were any changes in respiratory behavior initially apparent following dissection of the semi-intact preparation (after the 5 training sessions of the intact animal)? We measured both the number and duration of each individual pneumostome opening during the prestimulus recording period.
In the prestimulus period, it was found that both the number and duration of pneumostome openings were significantly different in operant preparations compared with yoked controls. The number of pneumostome openings in the operantly conditioned group were significantly lower (Fig. 4A), whereas the duration was significantly increased compared with yoked controls (Fig. 4C). Furthermore, when we compared the good and the poor learner preparations, we found that only the good learners showed a significant difference in the number (Fig. 4B) and duration (Fig. 4D) of openings.
|
These data demonstrate that despite no significant difference in total breathing time in the prestimulus period, significant differences in both number and duration of openings were evident between yoked control and operantly conditioned preparations. These differences were only evident in the good learners. Interestingly, poor learners showed no significant changes in pneumostome opening movements from yoked controls.
Reinforcement of conditioning induced significant changes in the number of pneumostome openings in operantly conditioned preparations, compared with yoked controls
Following application of the reinforcing stimulus, the number of pneumostome openings was significantly less in the operant preparations than in the yoked control preparations (Fig. 5A). However, the number of openings were only significantly reduced in the good learners and not in the poor learners (Fig. 5B). No significant differences in the duration of openings were found between operant and yoked control preparations (Fig. 5, C and D). These data show that the significant changes in pneumostome openings evident in the operant preparations following the reinforcing stimulus were exhibited only by the good learners but not by the poor learners.
|
In summary, differences in pneumostome openings parameters were found between operantly conditioned and yoked control preparations both immediately after dissection (prestimulus period) and after application of the reinforcing stimulus (poststimulus period). Despite these changes in opening parameters, there was no difference in total breathing time between operant and yoked preparations before application of the stimulus. Only following the reinforcement was total breathing time significantly reduced in the operant preparations.
RPeD1 activity was reduced after the reinforcing stimulus in operant preparations
We next aimed to determine whether the changes in respiratory behavior induced by the reinforcing stimulus were reflected in the properties of the CPG neuron, RPeD1. Representative examples of the electrophysiological recordings obtained from the semi-intact preparations from a yoked control (A) and operantly conditioned (B) animal are shown in Fig. 6.
|
We first determined whether there were any changes in the resting
membrane potential of RPeD1. The resting membrane potential of RPeD1 in
semi-intact preparations from operantly conditioned animals (
59 ± 1 mV; n = 38) was not significantly different from that of yoked control animals (58 ± 2 mV; n = 24, Student's t-test, P > 0.05, df = 60).
The firing frequency of RPeD1 was monitored both before and after presentation of the reinforcing stimulus to operantly conditioned and control preparations. The mean frequency of RPeD1 firing of the operant group in the prestimulus period was 0.1651 ± 0.0325 Hz (n = 44), which was not significantly different from that of the yoked control preparations (0.1737 ± 0.0693 Hz, n = 26; P > 0.05). However, after presentation of the reinforcing stimulus, the RPeD1 frequency was significantly reduced in preparations from operantly conditioned animals to 0.1078 ± 0.0233 Hz, n = 26 P < 0.01; Fig. 7A). The preparations from yoked control animals did not, however, exhibit any significant change in RPeD1 firing frequency after the stimulus (0.1744 ± 0.0637 Hz; n = 26; P > 0.05). A comparison of poor and good learners with control preparations revealed that only the good learner preparations showed a significant reduction in RPeD1 firing activity after the reinforcing stimulus (P < 0.01; Fig. 7B). Neither poor learners nor yoked controls showed a significant reduction in RPeD1 firing. Likewise, naïve preparations did not exhibit any significant change in firing activity after the reinforcing stimulus (before: 0.2013 ± 0.045 Hz; after: 0.2259 ± 0.0532 Hz; n = 11; P > 0.05, data not shown).
|
In summary, these data demonstrate that the reinforcing stimulus in the semi-intact preparation induced a significant reduction in the total breathing time of operant preparations. These behavioral changes were accompanied by a significant reduction in the operant group of the mean firing frequency of the CPG neuron RPeD1. When the operant group was analyzed based on the performance of the intact animal, both total breathing time and RPeD1 firing were significantly reduced in the good learners but not in the poor learners. These data strongly suggest that the reduced activity in RPeD1 is a neural correlate of associative learning of the conditioned response in Lymnaea.
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
In this study, we used semi-intact preparations from intact animals that had undergone the operant conditioning or yoked control training procedure and investigated changes in the activity of a neuron, RPeD1, known to be necessary for the conditioned behavior. We found significant differences between conditioned and yoked control semi-intact preparations at both the behavioral and neuronal level.
Among the many advantages of invertebrate preparations for the study of
learning and memory is the fact that it is possible to use the
semi-intact preparations to simultaneously monitor behavior and
neuronal activity. For example, semi-intact preparations were used to
operantly condition the leg-positioning in insects using
shock-avoidance paradigms (Horridge 1962;
Woollacott and Hoyle 1977) and the operant conditioning
of a single neuron in Helix was based on the withdrawal
reflex of a semi-intact preparation (Tsitolovsky and Shvedov
1998). Furthermore, semi-intact preparations have also been
used to investigate the neuronal mechanisms underlying classical
conditioning of withdrawal reflexes (Antonov et al. 2001; Colebrook and Lukowiak 1988;
Lukowiak 1986; Lukowiak and Sahley 1981),
long-term sensitization in Aplysia (Cleary et al. 1998), as well as the feeding behavior in Lymnaea
(Staras et al. 1999). While semi-intact preparations
have the potential to reveal whether a change in behavior is
accompanied by a change in neuronal properties, few studies have
advanced our understanding of how learning and memory after operant
conditioning are encoded in the nervous system. One possible reason for
this is that thorough knowledge of the neuronal circuitry that mediates
the conditioned behavior is lacking in many preparations
(Lukowiak 1986). For example, after the classical
conditioning of appetitive feeding in Lymnaea, a neural
correlate of the learning and its memory was monitored in a motor
neuron. Although the motor neuron activity may reflect CPG activity, it
is not currently known to be involved in the generation or modulation
of the feeding response (Staras et al. 1999). If changes
are observed in a neuron that is a necessary component of the
expression of a particular behavior, then it may be possible to
eventually determine the causal neuronal mechanisms of associative
learning and memory. In our study, we have recorded from a respiratory
CPG neuron that receives sensory information from the pneumostome area,
activates the rhythmic activity of the respiratory CPG, and is
necessary for CPG function and aerial respiratory behavior
(Haque 1999; Lukowiak 1991; Syed
et al. 1990).
It has previously proven difficult to prepare a semi-intact preparation
from conditioned animals that will continue to exhibit learning and
memory "in the dish." For example, while 89% of intact Lymnaea exhibited appetitive classical conditioning, only
54% of the semi-intact preparations made from these in vivo
preparations continued to exhibit the learned behavior (Staras
et al. 1999). In our study, we have demonstrated that memory of
the conditioned behavior was not compromised by the dissection
procedure and that the semi-intact preparations dissected from
operantly conditioned animals responded to the reinforcing stimulus.
After the reinforcing stimulus, the semi-intact preparations from
operant animals exhibited a significant reduction in overall breathing
time. This reduction in total breathing time has also been demonstrated
in the posttraining period of intact animals after conditioning
(Lukowiak et al. 1996). The reduction in breathing time
was, however, only evident in good-learner preparations and not in the
poor learners. These data support the notion that semi-intact
preparations retain their memory after dissection, respond in a similar
manner to the reinforcing stimulus as the intact, operantly conditioned
animals and thus make suitable preparations in which to directly study
the neuronal mechanisms of associative learning and memory.
In addition to the significant changes in the behavior of the
semi-intact preparations from operantly conditioned animals (compared
with controls), we also showed significant differences in the activity
of RPeD1. However, it is noteworthy that these activity changes in
RPeD1 were not evident in the prestimulus period but only in the
poststimulus period. The fact that we saw significant differences in
RPeD1 activity only after presentation of the reinforcing stimulus
supports the concept that the reinforcing stimulus itself plays a major
role in eliciting altered neuronal activity during learning and/or
memory. Stopfer and Carew (1996) demonstrated that
stimulation of a reduced preparation caused a change in sensory neuron
activity during habituation of a reflex behavior. Nargeot et al.
(1999a), using an analog of operant conditioning, observed a
similar alteration in neuronal activity after presentation of the
reinforcing stimulus. They demonstrated the importance of the
association between the reinforcement and the centrally emitted
neuronal activity in the induction of neuronal plasticity. Similar
effects have also been observed in classical conditioning studies in a
reduced preparation (Lechner et al. 2000). That is, neural correlates (extracellular activity recorded from nerves mediating the various phases of feeding behavior and integrated synaptic input to the motor neurons) of the associative learning were
only seen after electrical stimulation of the nerve that mimicked the
conditioned stimulus. In future studies in Lymnaea, it will
be interesting to test the necessity of presenting the reinforcing
stimulus to the pneumostome during its opening, to alter RPeD1
activity. That is, if the stimulus is presented to a closed
pneumostome, will changes in RPeD1 activity still occur?
In our previous study using isolated ganglia (where no peripheral input
was present), there were no significant changes in the mean firing
frequency of RPeD1 between trained and yoked control preparations
(Spencer et al. 1999). The data obtained from the current study therefore emphasize the importance of using semi-intact preparations in the elucidation of neuronal activity associated with
behavioral changes. Furthermore, the peripheral input from the
pneumostome area and osphradial ganglion modulates respiratory CPG
activity (Inoue et al. 2001). The frequency of CPG
activity in isolated ganglionic preparations in eumoxic conditions is
significantly greater than it is in intact or semi-intact preparations.
Thus it is more difficult to ascertain changes in RPeD1 frequency in isolated ganglionic preparations. In naïve semi-intact
preparations, bathing the pneumostome area with hypoxic saline results
in increased firing of RPeD1 compared with eumoxic conditions. In this
study, we have shown that presentation of a reinforcing stimulus
produced a subsequent decrease in RPeD1 activity in operantly
conditioned preparations only.
An important consideration for conditioning studies is that specific
changes in synaptic efficacy cannot always be correlated with behavior
changes. Colebrook and Lukowiak (1988) demonstrated that
a significant change in behavior was seen without a change in the
synaptic efficacy at the synapse where the change was predicted to
occur. Conversely, significant changes in synaptic efficacy were
sometimes observed at this site without a concomitant change in
behavior. An additional consideration is that changes at specific neuronal sites that accompany or cause the learning and its memory may
be subtle and not necessarily numerically significant. For example,
Lechner et al. (2000) show increases in neuronal
activity of a multifunctional neuron in preparations from conditioned
animals, but these changes were not significantly different from
control animals. In this study, in addition to analyzing the operant
group as a whole, we subdivided the group into good and poor learners for further analysis. The reason for this subdivision was that we had
previously found significant changes in the isolated ganglia only when
animals categorized as good learners were analyzed separately (Spencer et al. 1999). This subdivision was based on
whether an individual intact animal demonstrated a 50% (or greater)
reduction in its attempted pneumostome openings in the fifth training
session, compared with the first session. In our present study,
analysis of the operant group as a whole showed significant changes in both respiratory behavior and RPeD1 activity in response to the application of the reinforcing stimulus to the semi-intact preparation. However, further analysis showed that only the group of good learners demonstrated these significant changes. The group characterized as poor
learners did not show any significant changes compared with yoked
controls. This group of poor learners received contingent stimulation
of their open pneumostomes during the conditioning of the intact animal
but did not demonstrate the same degree of reduced respiratory behavior
as the group of good learners. These poor learners therefore serve as
an additional control group in that they received contingent
stimulation but did not show the same degree of learning. It is not
known as to why some intact animals did not learn (poor learners) as
well as others (good learners), but the data clearly show that the
semi-intact preparations from these poor learners did not exhibit
behavioral differences and subsequently did not show a significant
change in RPeD1 activity. These studies therefore show that not only is
the behavior of the intact animal reflected in the behavior of the
semi-intact preparation but further strengthen the hypothesis that
changes in the activity of RPeD1 are directly associated with learning and/or memory of the conditioned response.
The data presented here strongly suggest that RPeD1 plays an important
role in mediating learning or recall. However, because changes in RPeD1
activity were only evident after presentation of the reinforcing
stimulus, it is likely that its altered activity is only associated
with the subsequent change in behavior directly after
reinforcement. Dopamine released by RPeD1 activates the respiratory
rhythm (Syed et al. 1990) and hence reduced RPeD1 activity after reinforcement may act to reduce the overall output of
the respiratory CPG. It is likely, however, that the changes in
neuronal activity that accompanied the learned behavioral change were
not confined to just RPeD1 and may occur at multiple loci (Colebrook and Lukowiak 1988; Lukowiak
1986; Staras et al. 1999). Other changes in the
activity and/or synaptic connections of respiratory CPG interneurons or
motoneurons may exist, and this possibility requires further
investigation. For example, the effect of reduced IP3 activity (as a
result of reduced pneumostome openings) on the firing frequency of
RPeD1 was not evaluated in this study. Furthermore whether changes in
the efficacy of dopaminergic innervation of other CPG neurons or motor
neurons by RPeD1 occurred after operant conditioning (or its
reinforcement) was not determined in this study. Dopaminergic synapses
have, however, been shown to mediate the neuronal changes induced by
reinforcement in an analogue of operant conditioning, both in
Aplysia (Nargeot et al. 1999b) as well as in
mammals (Waelti et al. 2001).
In summary, this study used a semi-intact preparation to demonstrate that decreases in the activity of a CPG neuron were directly associated with behavioral changes following reinforcement of operant conditioning. This preparation will allow us to further investigate the cellular mechanisms that underlie operant conditioning of a patterned rhythmic behavior and to identify how, when, and where the cellular changes associated with learning might occur.
| |
ACKNOWLEDGMENTS |
|---|
We thank R. Cotter, D. Krygier, and N. Adatia for contributions toward animal training, Drs R. Hawkes and R. Wilson for critical comments on an earlier version of the manuscript, and Dr. D. DiBattista and S. Mercier for statistical advice.
This work was supported by Canadian Institutes of Health Research (CIHR) to K. Lukowiak and National Sciences and Engineering Research Council (Canada) to G. E. Spencer. G. E. Spencer was supported by Alberta Heritage Foundation for Medical Research and Neuroscience Canada Foundation Fellowships and is a Parker B. Francis Fellow for Pulmonary Research.
| |
FOOTNOTES |
|---|
Address reprint requests to: G. E. Spencer (E-mail: gspencer{at}spartan.ac.brocku.ca).
Received 2 August 2001; accepted in final form 20 June 2002.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
This article has been cited by other articles:
|
|
 |
|
  K. Lukowiak, K. Martens, D. Rosenegger, K. Browning, P. de Caigny, and M. Orr The perception of stress alters adaptive behaviours in Lymnaea stagnalis J. Exp. Biol., June 1, 2008; 211(11): 1747 - 1756. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. V. Orr and K. Lukowiak Electrophysiological and Behavioral Evidence Demonstrating That Predator Detection Alters Adaptive Behaviors in the Snail Lymnaea J. Neurosci., March 12, 2008; 28(11): 2726 - 2734. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Cheung, M. Moghaddasi, H. L. Hall, J. J. B. Smith, L. T. Buck, and M. A. Woodin Excitatory actions of GABA mediate severe-hypoxia-induced depression of neuronal activity in the pond snail (Lymnaea stagnalis) J. Exp. Biol., November 15, 2006; 209(22): 4429 - 4435. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Carter, K. Lukowiak, J. O. Schenk, and B. A. Sorg Repeated cocaine effects on learning, memory and extinction in the pond snail Lymnaea stagnalis J. Exp. Biol., November 1, 2006; 209(21): 4273 - 4282. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Hawkins, G. A. Clark, and E. R. Kandel Operant Conditioning of Gill Withdrawal in Aplysia J. Neurosci., March 1, 2006; 26(9): 2443 - 2448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Lowe and G. E. Spencer Perturbation of the activity of a single identified neuron affects long-term memory formation in a molluscan semi-intact preparation J. Exp. Biol., February 15, 2006; 209(4): 711 - 721. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. McComb, N. Varshney, and K. Lukowiak Juvenile Lymnaea ventilate, learn and remember differently than do adult Lymnaea J. Exp. Biol., April 15, 2005; 208(8): 1459 - 1467. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Parvez, O. Stewart, S. Sangha, and K. Lukowiak Boosting intermediate-term into long-term memory J. Exp. Biol., April 15, 2005; 208(8): 1525 - 1536. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sakakibara, T. Aritaka, A. Iizuka, H. Suzuki, T. Horikoshi, and K. Lukowiak Electrophysiological Responses to Light of Neurons in the Eye and Statocyst of Lymnaea stagnalis J Neurophysiol, January 1, 2005; 93(1): 493 - 507. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Rosenegger, S. Roth, and K. Lukowiak Learning and memory in Lymnaea are negatively altered by acute low-level concentrations of hydrogen sulphide J. Exp. Biol., July 1, 2004; 207(15): 2621 - 2630. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sangha, A. Scheibenstock, R. Morrow, and K. Lukowiak Extinction Requires New RNA and Protein Synthesis and the Soma of the Cell Right Pedal Dorsal 1 in Lymnaea stagnalis J. Neurosci., October 29, 2003; 23(30): 9842 - 9851. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sangha, A. Scheibenstock, and K. Lukowiak Reconsolidation of a Long-Term Memory in Lymnaea Requires New Protein and RNA Synthesis and the Soma of Right Pedal Dorsal 1 J. Neurosci., September 3, 2003; 23(22): 8034 - 8040. [Abstract] [Full Text] [PDF] |
,
n = 26) and poor learners (
,
n = 18).
