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First published online March 12, 2009
Journal of Experimental Biology 212, 922-933 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.023069
Novel neural correlates of operant conditioning in normal and differentially reared Lymnaea
Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St Catharines, Ontario, Canada L2S 3A1
* Author for correspondence (e-mail: gspencer{at}brocku.ca)
Accepted 14 December 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: learning, memory, invertebrate, aerial respiration, central pattern generator, semi-intact preparation, mollusc
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Specifically, the aerial respiratory behaviour of
Lymnaea can be operantly conditioned and resultant neural changes are
manifested within the neural network, the respiratory central pattern
generator (CPG) regulating this behaviour
(Spencer et al., 1999;
Spencer et al., 2002;
McComb et al., 2005;
Lowe and Spencer, 2006).
Lymnaea is a bimodal breather. When challenged with hypoxia, the
animal supplements cutaneous respiration with aerial respiration, which
involves migrating to the air–water interface and opening and closing
its respiratory orifice, the pneumostome
(Jones, 1961).
Lymnaea's aerial respiratory behaviour can be operantly conditioned
to demonstrate long-term memory (LTM)
(Lukowiak et al., 1996;
Lukowiak et al., 1998). This
is accomplished by the application of an aversive stimulus to the pneumostome
area with each attempted pneumostome opening. This results in the immediate
closure of the pneumostome and cessation of aerial respiration. Thus, with
operant conditioning, the animal learns to suppress hypoxia-induced
ventilatory behaviour in response to a `punishing' stimulus and therefore
demonstrates a reduction in aerial respiratory behaviour
(Lukowiak et al., 1996).
Like most rhythmic behaviours, this aerial respiratory behaviour is
controlled by a CPG, the cellular components of which have been identified
(Syed et al., 1990;
Syed and Winlow, 1991). This
particular respiratory CPG consists of three monosynaptically connected
interneurons located within the central ganglionic ring: right pedal dorsal 1
(RPeD1), input 3 (IP3) interneuron, and visceral dorsal 4 (VD4). The
sufficiency and necessity of this three-cell CPG in generating the coordinated
patterned firing activity has been demonstrated in isolated brain preparations
and also in cell culture (Syed et al.,
1990; Syed et al.,
1991; Syed and Winlow,
1991). RPeD1 is a large neuron and chemosensory information
concerning oxygen partial pressure is thought to be relayed to RPeD1
(Bell et al., 2008;
Inoue et al., 2001;
Syed and Winlow, 1991;
Wedemeyer and Schild, 1995).
IP3 interneuron activity produces a pneumostome opening via
monosynaptic, excitatory connections with the pneumostome opener motorneurons,
the visceral I and J (VI/J) cells. VD4 activity produces pneumostome closing.
The respiratory CPG cycle is initiated by hypoxia-induced patterned activity
in RPeD1 (Inoue et al.,
2001).
With no known detriment to their health, Lymnaea can be raised in
an environment in which they are prevented from rising to the water's surface
to perform aerial respiration. Hermann and Bulloch
(Hermann and Bulloch, 1998)
have previously demonstrated in this way, that aerial respiratory behaviour in
Lymnaea develops independently of experience. However, it has not yet
been determined whether the ability of the CPG to change during operant
conditioning is independent of previous behaviour and experience. One main aim
of this study was to investigate the plasticity of the respiratory CPG in
differentially reared animals, that is, in animals reared with no prior
experience of the aerial respiratory behaviour. We sought to determine if the
ability to exhibit higher-order plasticity (i.e. associative learning) is
dependent on experience during development.
Previous operant conditioning studies in Lymnaea have identified a
number of neuronal changes occurring within the respiratory CPG. In
particular, RPeD1 has been identified as an important locus of learning and
memory, and there is evidence that gene transcription in RPeD1 is necessary
for formation of LTM (Scheibenstock et
al., 2002). Lowe and Spencer
(Lowe and Spencer, 2006) also
demonstrated that artificially silencing RPeD1 in a semi-intact preparation
reduced the number of training sessions required to produce LTM. However, it
is clear from previous studies that the neural correlates underlying this
behaviour are dispersed throughout the network and may include changes in
other CPG interneurons such as the IP3 interneuron, as well as motorneuron
connections (Spencer et al.,
1999; McComb et al.,
2005). The second main goal of this study was to utilize the
semi-intact preparation, in which both pneumostome behaviour and CPG neural
activity can be simultaneously monitored
(Lowe and Spencer, 2006), to
identify novel neural correlates of operant conditioning of the aerial
respiratory behaviour.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Differentially reared Lymnaea
Differentially reared animals were unable to perform aerial respiration
throughout development. Clear, plastic breeding containers with fine mesh
walls were submerged in well-aerated, artificial pond water aquariums. Special
care was taken to ensure that no air bubbles were trapped in the enclosures. A
single egg sack was hatched under each enclosure and snails were raised to
adulthood (3–6 months) without ever experiencing aerial respiration. The
snails were kept on the same light:dark cycle and the same diet as normally
reared animals. These snails were age-matched (3–6 months) to normally
reared snails but as reported previously, some were slightly smaller in size
(Hermann and Bulloch, 1998).
All animals used for training and electrophysiology were between 18 and 25 mm
in shell length. No differences in behaviour between the smaller and larger
adults were found in this study, as also found previously by Hermann and
Bulloch (Hermann and Bulloch,
1998).
Procedures
Operant conditioning
Snails were selected and randomly assigned to one of three groups:
naïve, yoked or conditioned. During the training sessions and the memory
test, naïve animals were allowed to freely perform aerial respiration.
The operantly conditioned group received a punishing tactile stimulus that was
contingent on the animal opening its pneumostome at the air–water
interface to perform aerial respiration. The stimulus was of sufficient
intensity to cause immediate pneumostome closure. Yoked animals also received
the tactile stimuli, but these were not contingent on pneumostome opening.
Instead, they were applied to the pneumostome area and timed according to the
stimulus given to the conditioned animal to which it was yoked.
Snails were individually identified by a series of markings on their shells
and placed in an 800 ml beaker with well-aerated pond water. The snails were
given 10 min to habituate and explore the new environment. 100% nitrogen gas
was bubbled into the water for 10 min prior to and for the duration of each
session in order to induce the hypoxic ventilatory response. Generally, there
was a 10-fold reduction in oxygen content after the 10 min period. After the
acclimatization period, 300 ml of water were siphoned out of the beaker at
which point the snails entered the 30 min pre-observation session, during
which all animals were allowed to freely perform aerial respiration. The
number of pneumostome openings and total breathing time were monitored and
calculated for each animal. We did not wish the differentially reared animals
to surface between training sessions as this has been shown to affect learning
and memory (Sangha et al.,
2003). Thus, the beaker was capped for both normally reared and
differentially reared animals between training sessions, preventing them from
surfacing. Thus, in this modified procedure, the discriminative stimulus was
aerial respiration and not the application of the first contingent physical
stimulus to signify the beginning of each training session. One hour after the
pre-observation period, the animals entered the first of four 30 min training
sessions. The number of attempted pneumostome openings was recorded for the
conditioned animals while the number of pneumostome openings and total
breathing time were determined for both naïve and yoked control groups.
Each of the four training sessions was separated by 1 h to allow consolidation
of memory (Lukowiak et al.,
2000). Eighteen hours after the final training session, the snails
entered the memory test, which was procedurally similar to the training
sessions. One hour following the memory test, the snails entered the
post-observation period in which all of the snails were again allowed to
freely perform aerial respiration and the post-training number of pneumostome
openings and total breathing time were determined for all animals.
Dissection of semi-intact preparations
A similar approach to that of Lowe and Spencer
(Lowe and Spencer, 2006) was
used to dissect the semi-intact preparations. Briefly, snails were
anaesthetized in a Lymnaea saline solution (composition in mmol
l–1: 51.3 NaCl, 1.7 KCl, 4.1 CaCl22H2O;
1.5 MgCl26H2O, 5 Hepes buffer, pH to 7.9 with NaOH)
containing 30% Listerine (Pfizer Canada, Toronto, Canada) for 3 min. The
anaesthetic agent in Listerine is menthol and its use does not affect memory
formation (Spencer et al.,
2002). The outer shell was removed and the body was pinned dorsal
side up. The pneumostome was propped on a small piece of Sylgard to easily
view the openings. A medial incision from the base of the mantle to the head
was made to expose the inner cavity. The oesophagus and the reproductive
organs were excised and Sylgard was positioned under the CNS. The commissure
linking the left and right pedal ganglia was severed and the CNS was pinned to
the Sylgard. All preparations were then given 20–30 min to recover from
surgery prior to electrophysiological and behavioural analysis.
Electrophysiological recordings
Intracellular recordings were simultaneously obtained from the RPeD1 and
the VI cell in semi-intact preparations using standard electrophysiological
techniques (Spencer et al.,
1999; Spencer et al.,
2002). Located on the dorsal surface, one VI cell is
morphologically distinct and is easily identified as the largest of the
pneumostome opener motorneurons. Cell penetration was aided by proteolytic
enzymatic treatment (Protease, Type IX; Sigma-Aldrich, MO, USA) over the
surface of the right pedal and the visceral ganglia. Glass microelectrodes
with a resistance of 20–60 M
were pulled on a Kopf electrode
puller (Model 730; David Kopf Instruments, CA, USA) and back filled with
saturated K2SO4. Signals were obtained using a Neuro
Data IR283A amplifier (Cygnus Technology, PA, USA) connected to a PowerLab/4SP
digital acquisition system (AD Instruments, CO, USA) and Chart recording
software (v4.2; AD Instruments).
Data and statistical analysis
In the intact animal, the respiratory parameters were quantified across all
sessions. For intact animal data, statistical analysis incorporated a repeated
measures design. A two-way repeated measures analysis of variance (two-way
RM-ANOVA) was used to test for a possible interaction effect between two
independent variables: the treatment group and the training sessions. All
post-hoc analyses were carried out using a Tukey–Kramer
test.
In the semi-intact preparation, all parameters were quantified over a 5–10 min recording session and significance was established using a two-way ANOVA design with rearing conditions as one factor and training as the second factor. Tukey–Kramer post-hoc tests were conducted to determine significance and results were considered significantly different if a P value of less than 0.05 was achieved. All data analyses were carried out using GB-Stat (Dynamic Microsystems, MD, USA). In all figures, the error bars represent the standard error of the mean.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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A two-way RM-ANOVA of all intact animal groups (naïve, yoked and conditioned) revealed a significant interaction effect between the two independent variables (treatment group and session) for both the number of openings (F(5,90)=2.96; P=0.01) and total breathing time (F(5, 90)=7.43; P<0.0001) for the pre-test session and post-test session. Further post-hoc analysis indicated significant differences in various groups as discussed below.
Analysis of the aerial respiratory behaviour in normal and differentially reared naïve animals
Hermann and Bulloch (Hermann and
Bulloch, 1998) have previously shown that differentially reared
Lymnaea are capable of performing aerial respiration when permitted
to do so. We first sought to determine what differences existed in the aerial
respiration of normally and differentially reared L. stagnalis using
our experimental set-up. Aerial respiration was quantified in naïve
animals to determine baseline respiratory behaviour under hypoxic
conditions.
Post-hoc analysis of the respiratory parameters in naïve
animals indicated a significant difference in the response between the two
groups. Normally reared naïve animals performed aerial respiration more
often than their differentially reared naïve counterparts in the
pre-observation session (P<0.01;
Fig. 1A). Normal naïve
animals also performed aerial respiration for a longer duration compared with
the naïve differentially reared animals in the pre-observation session
(P<0.01; Fig. 1B).
This behaviour was consistent across the four `training' sessions and the
memory test, and did not change from the pre- to the post-observation session
for either group (P>0.05). However, there were no differences in
the qualitative nature of the pneumostome openings between normally and
differentially reared animals [as also previously shown by Hermann and Bulloch
(Hermann and Bulloch, 1998)].
Taken together, in hypoxic conditions, differentially reared animals performed
aerial respiration significantly less often than normally reared animals.
Effects of operant conditioning on aerial respiration of intact animals
We next aimed to determine how the ability to modify aerial respiratory
behaviour was affected by rearing conditions. Post-hoc analysis
indicated that prior to training, again, normally reared animals exhibited
more respiratory behaviour than the differentially reared animals
(pre-observation session: P<0.01;
Fig. 1C). The same was true
when total breathing time was considered (P<0.01;
Fig. 1D). Importantly, only the
normally reared conditioned animals showed a significant reduction in the
number of pneumostome openings (P<0.05) and total breathing time
(P<0.01) from the pre- to the post-observation session. The
differentially reared conditioned animals did not show a similar reduction in
aerial respiratory behaviour as a result of the operant conditioning. There
were no significant changes in the number of pneumostome openings
(Fig. 1C) or total breathing
time (Fig. 1D) in
differentially reared animals from the pre- to the post-observation session.
There were no significant differences in the yoked control groups,
demonstrating that the non-contingent stimulus did not cause any reduction in
aerial respiratory behaviour.
The associative learning demonstrated by the normally reared conditioned
group was further analyzed by the construction of a learning curve
(Fig. 2). Learning has been
operationally defined in this model as the significant reduction in number of
attempted pneumostome openings from training session 1 (TS1) to TS4
(Lukowiak et al., 1996;
Spencer et al., 1999;
Spencer et al., 2002;
Lowe and Spencer, 2006). If
learning occurred, then LTM is defined as the significant reduction in
attempted pneumostome openings from TS1 to the memory test (MT)
(Lukowiak et al., 1996;
Lowe and Spencer, 2006). The
two-way RM-ANOVA showed a significant interaction effect
(F(20,360)=11.69; P<0.0001). Post-hoc
analysis showed that the normally reared conditioned animals demonstrated both
learning and LTM with our training procedure (P<0.01). However,
the differentially reared animals did not show a significant reduction in the
number of attempts to open their pneumostome from TS1 to TS4 or to the MT
(Fig. 2). Therefore, by our
definition, these animals were unable to learn and form LTM using the same
training as used for the normally reared conditioned animals.
Interestingly, however, the normally reared conditioned animals reduced their
aerial respiratory activity following training to the initial pre-training
level observed in the differentially reared animals. In other words, there
were no significant differences in the level of respiration between the
normally reared animals in the post-observation sessions and the
differentially reared animals in the pre-observation sessions. These
data suggest that the level of respiration observed following training in the
normally reared animals but prior to training in the differentially
reared animals may be a minimum level of respiration required in this hypoxic
environment.
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Behavioural and neural correlates of learning and memory in the semi-intact preparation
Despite the demonstration above that differentially reared animals were
unable to show a reduction in their respiratory behaviour following the
operant conditioning, it is possible that their initial level of respiratory
activity was too low to be further reduced by the training procedure.
Therefore, when analyzing the neural correlates of the normally reared animals
in a semi-intact preparation, we chose also to analyze the differentially
reared animals in order to determine, despite the lack of behavioural change,
whether they demonstrated any evidence of neural changes associated with the
training. According to the parameters defined above, there was no evidence to
support LTM formation in the intact differentially reared conditioned animals.
However, it was possible that conditioning produced partial effects,
behavioural and/or neural, that would become evident when examined in the
semi-intact preparation.
Following the post-observation session, all animals were immediately
dissected into a semi-intact preparation and the CPG, motorneuron and
pneumostome activity was simultaneously monitored. In this experiment, VI
motorneuron and RPeD1 cell activity were monitored. RPeD1 is the cell that
receives excitatory input from the peripheral chemoreceptor cells in the
pneumostome/osphradial area and initiates the CPG respiratory rhythm
(Inoue et al., 2001;
Bell et al., 2007;
Bell et al., 2008). Since the
pneumostome opener cell, IP3 interneuron, is located on the ventral surface of
the CNS and RPeD1 is located on the dorsal surface, IP3 interneuron activity
was indirectly assessed as characteristic bursting patterns in the VI
motorneuron. Input 3 was first described by Benjamin and Winlow
(Benjamin and Winlow, 1981) as
a characteristic synaptic input onto various cells, despite the unknown
identity of the source of the input. Syed et al.
(Syed et al., 1990) later
identified the IP3 interneuron and demonstrated that activity recorded in this
cell produced the characteristic activity previously described as input 3 onto
the identified follower cells. Since then, the VI cell has been widely used to
indirectly monitor the activity of the IP3 interneuron
(Syed et al., 1990;
Syed et al., 1991;
Spencer et al., 2002;
McComb et al., 2005), as
simultaneous recordings from RPeD1 and IP3 interneuron are not possible
because of their locations on different surfaces of the CNS. The
characteristic bursting activity monitored in the VI cell is a result of
synaptic input from the IP3 interneuron
(Syed et al., 1990) and here
will be defined as `input 3 events'.
The experimental protocol in the semi-intact preparation was designed to
observe both the neural and behavioural correlates of the respiratory
behaviour and to also monitor changes following presentation of a contingent,
punishing stimulus to an open pneumostome
(Spencer et al., 2002) in all
groups.
Analysis of pneumostome openings and IP3 events in naïve semi-intact preparations
The first aim was to determine the baseline differences in behavioural and
neural activity between normally and differentially reared naïve
preparations over an initial 5 min period of recordings. We investigated
pneumostome openings and corresponding IP3 events in the VI cell in the
naïve semi-intact preparations in order to determine whether the
behaviour correlated with cellular activity.
Fig. 3A,B illustrates the
number of pneumostome openings and IP3 events in the naïve animals during
the 5 min session. As expected, the differentially reared naïve
preparations displayed significantly fewer pneumostome openings than the
normally reared naïve preparations (P<0.05;
Fig. 3A). Accordingly, the
number of IP3 events recorded from the VI cell was also significantly reduced
in the differentially reared naïve preparations compared with normally
reared preparations (P<0.01;
Fig. 3B).
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From these data, we observed that the semi-intact preparations behaved in a manner similar to the intact animals from which they were derived. That is, as in the intact animal, differentially reared naïve preparations performed aerial respiration less often than normally reared preparations.
Analysis of pneumostome openings and IP3 events in conditioned semi-intact preparations
Having analyzed respiratory activity and associated IP3 events in
naïve semi-intact preparations, and having determined that this
semi-intact preparation was an appropriate representation of the behaviour of
the intact animal, conditioned and yoked animals were next dissected to
determine the effects of operant conditioning on the pneumostome opening
parameters and cellular activity.
We found that in the semi-intact preparations, pneumostome opening behaviour and IP3 events reflected operant conditioning in the intact animal (Fig. 3C,D). A two-way ANOVA revealed a significant interaction effect between rearing condition and training, for both the number of openings (F(1,47)=6.29; P<0.05) and the number of IP3 events (F(1,47)=6.02; P<0.05). The normally reared conditioned preparations opened their pneumostome significantly less often than their yoked controls (P<0.01), but not significantly less than the differentially reared yoked and conditioned animals (Fig. 3C). The number of IP3 events (recorded in the VI cell) that produce pneumostome openings was also significantly reduced in the normally reared conditioned preparations compared with their yoked controls (P<0.05). As found in the intact animals, there were no significant differences between differentially reared yoked and conditioned preparations in either the number of pneumostome openings (Fig. 3C) or IP3 events (Fig. 3D). Overall, these results suggested that the semi-intact preparation is a good model for studying the behavioural and neural correlates of learning and memory. As in the whole animal, normally reared conditioned preparations performed aerial respiration less than their yoked controls. Concurrently, IP3 events were also reduced in these preparations.
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) in which modifications induced by learning are dispersed
throughout the neural circuitry mediating the behaviour. Therefore, a number
of novel cellular parameters were analyzed to determine where neuronal changes
that underlie behavioural changes may occur in this system, and to compare
such changes between normally reared and differentially reared
preparations.
IP3 events recorded from the VI cell
IP3 activity (and IP3 events recorded in the VI cell), result in
pneumostome opening (Syed et al.,
1991; Syed and Winlow,
1991). We have already shown that the number of IP3 events
recorded in the VI cell is reduced in conditioned preparations. We next
analyzed three other IP3 event parameters and found them to be altered in
normally reared conditioned preparations, compared with yoked controls. These
parameters included the intensity of the IP3 events (intra-burst spike
frequency), the latency from the IP3 event to pneumostome opening, and the
coincident IP3 event and pneumostome activity.
Although we did not use a tension transducer to measure the force of each pneumostome opening, the intra-burst frequency of the IP3 event was used as an indirect measure of the intensity of the pneumostome opening. Generally, a high intra-burst frequency results in a qualitatively large pneumostome opening, whereas a low intra-burst spike frequency produces a smaller pneumostome opening. Owing to the aversive nature of the stimuli, we hypothesized that conditioned preparations would demonstrate a reduction in the intra-burst spike frequency of the IP3 event. Firstly, there was no significant difference in intra-burst spike frequency of the IP3 event between the normally reared (6.2±0.4 Hz) and differentially reared (6.6±0.4 Hz) naïve preparations (P>0.05), as well as no qualitative difference in the pneumostome opening. However, a two-way ANOVA revealed a significant interaction between rearing condition and training (F(1,47)=4.90; P<0.05). There was a significant difference between the conditioned and yoked normally reared preparations (P<0.01; Fig. 4A). The training affected the intensity of the IP3 event such that intra-burst spike frequency was significantly reduced in the normally reared conditioned preparations compared with the normally reared yoked preparations. This difference was not evident in the preparations of the differentially reared animals (P>0.05).
Latency from the IP3 event in the VI cell, to pneumostome opening
There is a direct monosynaptic connection between the VI motorneurons and
the pneumostome opener muscles (Syed et
al., 1991). We next wanted to measure the latency from the IP3
event in the VI cell to the resulting pneumostome opening, to see if it was
affected by conditioning. Wolpaw (Wolpaw,
1997) has previously shown a reduction in motorneuron conduction
velocity following operant conditioning of the H-reflex. We, therefore,
hypothesized an increase in the latency in conditioned preparations. In the
naïve groups, there was no difference in this latency between the
normally reared preparations (0.8±0.2 s) and the differentially reared
preparations (0.4±0.5 s). However, on analysis of the conditioned and
yoked control groups, a two-way ANOVA revealed a significant effect of
training only on the latency (F(1,46)=8.40;
P<0.01), but no significant interaction between training and
rearing (P>0.05). In the normally reared conditioned animals,
there was a significant difference in the pneumostome response
(Fig. 4B). In the yoked
controls, a pneumostome opening was recorded as soon as the IP3 event was
observed, while in the conditioned groups, there was a significant lag time in
the pneumostome opening. The differentially reared conditioned preparations
also demonstrated a similar increase in this latency compared with their
controls. This was the first significant result that indicated that the
differentially reared animals showed neural changes in response to the
conditioning.
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). Thus, IP3 events were also scored for coincident
pneumostome activity in the semi-intact preparation. In other words, we asked
the question: if IP3 events were observed in the VI cell and/or RPeD1, was
there a corresponding pneumostome opening? Although all normally and
differentially reared naïve and yoked preparations demonstrated >90%
coincident activity, the number of IP3 events that did not result in
pneumostome openings was significantly increased in conditioned preparations.
A two-way ANOVA revealed a significant effect of training
(F(1,117)=29.91; P<0.0001), although the
interaction between training and rearing did not reach significance
(P=0.06). Interestingly, post-hoc analysis revealed a
significant reduction in coincident activity in conditioned preparations from
both normally and differentially reared animals, again showing that
differentially reared animals exhibited neural changes associated with the
operant conditioning (Fig.
5). The motorneuron firing frequency (inclusive of IP3 events) was next determined for the 5 min recording session. Statistical analysis indicated no differences in VI frequency between the normally and differentially reared naïve preparations and no difference in activity between yoked and conditioned preparations (normally reared naïve: 3.0±0.2 Hz, normally reared yoked: 3.2±0.3 Hz, normally reared conditioned: 2.8±0.2 Hz, differentially reared naïve: 3.6±0.2 Hz, differentially reared yoked: 3.7±0.3 Hz, differentially reared conditioned: 3.6±0.3 Hz; P>0.05). Thus, the overall motorneuron activity was not affected either by operant conditioning or differential rearing.
Taken together, IP3 events were influenced by both rearing conditions and operant conditioning. With respect to the rearing conditions, the only difference observed was that differentially reared naïve preparations demonstrated fewer IP3 events in the VI cell compared with normally reared naïve preparations. This was reflective of the fewer number of pneumostome openings in both the intact animal and the semi-intact preparation of differentially reared animals. Operant conditioning affected the intensity (intra-burst frequency) of the IP3 events, the latency from IP3 events to pneumostome response, and coincident IP3 events and pneumostome activity. Furthermore, whereas the differentially reared conditioned preparations did not demonstrate any behavioural evidence of learning and memory, the cellular parameters strongly suggested aspects of neural plasticity associated with operant conditioning.
Neural and behavioural changes following application of the punishing stimulus
Following the 5 min recording session, a punishing stimulus was applied to
all preparations (naïve, yoked and conditioned) on the next pneumostome
opening. The recordings were then continued for a further 5 min, and
pneumostome behaviour and neural activity were analyzed in the post-stimulus
session and compared with the pre-stimulus session. This analysis determined
whether certain behavioural or neural parameters were affected by presentation
of the punishing contingent stimulus.
Only conditioned preparations demonstrated reduced pneumostome openings and reduced IP3 events following the punishing stimulus
A two-way ANOVA revealed a significant effect of training on the number of
openings (F(1,47)=4.36; P<0.05), as well as
the number of IP3 events (F(1,47)=6.42;
P<0.05) after presentation of the punishing stimulus, although
there was no significant interaction effect between rearing condition and
training (P>0.05). Following the application of the punishing
stimulus, it was shown that conditioned preparations from both normally and
differentially reared animals showed an overall reduction in the number of
pneumostome openings as well as a corresponding overall reduction in the
number of IP3 events (Fig. 6).
Yoked control groups did not show this reduction in activity. These data
indicated that despite the lower incidence of overall respiratory activity in
the differentially reared animals, they behaved in a similar manner to the
normally reared animals in response to the punishing stimulus. That is,
despite no overall change in behaviour as a result of the conditioning in the
intact animals, they responded to the punishing stimulus in a similar manner
to the normally reared conditioned group in the semi-intact preparation.
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). The
latency to the `next' pneumostome opening following the application of the
punishing stimulus was then determined for the conditioned and yoked
preparations. This parameter was used as an indication of the behavioural
response to the stimulus. Owing to the aversive nature of the stimulus, we
hypothesized that the conditioned groups would demonstrate a greater latency
than their controls.
There were no significant differences in the response to the punishing stimulus amongst the naïve preparations (normally reared naïve: 63±12 s, differentially reared naïve: 68±25 s; P>0.05). However, analysis of the conditioned and yoked groups using a two-way ANOVA revealed a significant effect of training on the latency from the stimulus to the next opening (F(1,47)=11.34; P<0.005), though there was no significant interaction effect between rearing conditions and training (P>0.05). Post-hoc analysis indicated that the normally reared conditioned preparations displayed a significantly greater latency to the next pneumostome opening than their yoked preparations (P<0.05; Fig. 7A). The differentially reared conditioned preparations also demonstrated a trend towards an increased latency compared with their yoked preparations, although the difference did not reach significance. These results were again indicative, though not conclusive, of behavioural plasticity in the differentially reared Lymnaea. Taken together, conditioned preparations suppressed the hypoxic ventilatory drive longer than controls following the application of the contingent punishing stimulus.
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), so we
hypothesized an increase in latency to resumed RPeD1 activity in conditioned
preparations. Indeed, a two-way ANOVA again revealed a significant effect of
training on the latency to resumed RPeD1 activity
(F(1,47)=9.01; P<0.005), though there was no
significant interaction effect between rearing conditions and training
(P>0.05). Normally reared conditioned preparations demonstrated a
significantly increased recovery time in RPeD1 impulse activity compared with
their respective yoked controls (P<0.05;
Fig. 7B). With respect to
differentially reared conditioned preparations, they too demonstrated an
increased RPeD1 quiescence compared with the differentially reared yoked
preparations (P<0.05). Thus, differentially reared conditioned
preparations demonstrated similar neural responses to the punishing stimulus
as the normal preparations.
Summary of semi-intact data
Overall, further analysis of the neural network properties in the
semi-intact preparation revealed changes associated with conditioning
dispersed throughout the CPG controlling aerial respiration. Operant
conditioning produced a reduction in both the number and intensity of IP3
events in the VI motorneuron, a change in the motor program controlling
pneumostome opening as well as RPeD1 impulse activity. With respect to
differentially reared preparations, the strongest evidence for plasticity was
demonstrated by the significant difference between differentially reared yoked
and conditioned preparations in latency to pneumostome opening following an
IP3 event, correlated pneumostome activity and IP3 events, and the duration of
RPeD1 quiescence following the punishing stimulus.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Hermann and Bulloch (Hermann and
Bulloch, 1998) have previously shown that Lymnaea could
be raised from eggs to adulthood without ever experiencing aerial respiration.
Lymnaea is a pulmonate mollusc
(Harris, 2003), in which
respiration occurs partially across the somatic epidermis (cutaneous
respiration) but also via a primitive lung (aerial respiration)
(Syed et al., 1991). Thus,
during differential rearing, cutaneous respiration appeared sufficient to meet
the metabolic demands of these animals and maintain homeostasis in normoxic
(aerated) pond water. When allowed to do so, differentially reared
Lymnaea surfaced and performed aerial respiration, showing that the
behaviour is genetically programmed and is activity and experience independent
(Hermann and Bulloch, 1998).
They did, however, show qualitative and quantitative differences in aerial
respiration; the most relevant to this study being that in hypoxic conditions,
the normally reared animals demonstrated the hypoxic ventilatory response
(increased aerial respiratory behaviour) whereas the differentially reared
animals did not. Likewise, in our study, we found that the differentially
reared snails exhibited a significant reduction in both number of pneumostome
openings and total breathing time in hypoxic conditions, compared with
normally reared animals. Hermann and Bulloch
(Hermann and Bulloch, 1998)
reasoned that differentially reared animals did not experience hypoxic
conditions during development as their rearing tanks were aerated and
normoxic, and the oxygen saturation of haemocyanin, the haemolymph oxygen
carrier in Lymnaea (Dawson and
Wood, 1982; Dawson and Wood,
1983), was estimated to be 100%. However, it is known that
developmental hypoxia in vertebrates reduces the adult hypoxic ventilatory
response, so we cannot rule out that developmental hypoxia led to the reduced
hypoxic ventilatory response of the differentially reared animals shown here.
It has previously been suggested that developmental hypoxia may lead to
aberrant function or number of chemoafferant neurons
(Erickson et al., 1998;
Joseph et al., 2000), so it is
also possible that in the differentially reared animals, developmental hypoxia
may have led to reduced peripheral sensory input to RPeD1.
The reasons and underlying mechanisms as to why the differentially reared
animals exhibited a reduced hypoxic ventilatory response are currently
unknown. One reason may be a reduced metabolic requirement for oxygen,
possibly due to increased anaerobic metabolism in the differentially reared
animals. Another may be a change in synaptic functioning or network properties
of the hypoxic-sensing neurons as a result of the differential rearing and/or
presumed CPG network inactivity. Bell et al.
(Bell et al., 2007) recently
showed that peripheral chemoreceptor cells in the osphradium of
Lymnaea are oxygen-sensing neurons with monosynaptic connections with
RPeD1. Furthermore, this synapse exhibits hypoxia-induced short-term
facilitation, at least in vitro. It has been shown that both
lesioning of the osphradial nerve (Bell et
al., 2007) as well as RPeD1 nerve crush
(Haque et al., 2006) reduces
aerial respiratory drive as well as reduces movements to the water surface. It
is thus plausible that disruptions in the synaptic connections of these cells
during differential rearing might underlie the reduced hypoxic response.
Another explanation may involve nitric oxide (NO) production, as NOS
inhibitors have also been shown to prevent or reduce the hypoxic ventilatory
response in Lymnaea (Taylor et
al., 2003). Whether the reduced hypoxic response in differentially
reared Lymnaea results from a reduced requirement for oxygen, or
changes to the oxygen-sensing pathway [as previously proposed by Hermann and
Bulloch (Hermann and Bulloch,
1998)], remain to be determined.
The CPG aerial respiratory rhythm is also driven by mechano-sensory
excitatory inputs to RPeD1 as a result of the pneumostome breaking the surface
of the water (Haque et al.,
2006). We hypothesize that during differential rearing of
Lymnaea, these excitatory inputs to RPeD1 were silent and the
respiratory CPG was inactive. Presumably, the CPG connections were not
subjected to any activity-dependent modulation during development. In this
study, we thus sought to determine if previous experience and CPG activity
during development was necessary for plasticity of the network in the adult.
This was assessed by operant conditioning of the aerial respiratory behaviour
in differentially reared animals and both behavioural and neural monitoring in
the semi-intact preparation.
The first important observation was that operant conditioning of
differentially reared intact animals did not produce a significant reduction
in the aerial respiratory behaviour. That is, there were no significant
changes in the number of pneumostome openings or the total breathing time from
the pre- to the post-observation session, or the number of attempted
pneumostome openings from TS1 to TS4 and the MT. Thus, using the criteria we
had previously established, differentially reared Lymnaea did not
demonstrate learning or LTM when conditioned with the same procedure
as normally reared Lymnaea
(Lukowiak et al., 1996;
Spencer et al., 2002). We
cannot rule out that this may have resulted from muscle weakness in the
pneumostome as a result of differential rearing, but we consider this
unlikely, as there were no qualitative differences in the pneumostome openings
of this group compared with normally reared animals [as also previously shown
by Hermann and Bulloch (Hermann and
Bulloch, 1998)]. It can, however, be argued that aerial
respiration could not be further reduced in the differentially reared animals
following conditioning since they started with such a low level of behavioural
expression. This notion is further strengthened by the fact that the baseline
level of respiration in the differentially reared animals prior to
conditioning is the same level of respiration that normally reared animals
maintain following conditioning. It is also possible (though less
likely), that because they did not attempt aerial respiration as often as
respiring animals, the differentially reared animals may not have received
sufficient punishing stimuli to produce the operant response
(Papini and Bitterman, 1990;
Terry, 2003). Although Martens
et al. (Martens et al., 2007)
have recently demonstrated long-term memory lasting at least 24 h following a
single trial of conditioning of aerial respiratory behaviour, their protocol
was designed to evoke the whole-body withdrawal response, an event considered
more significant than the tactile stimulation of the open pneumostome used
here.
Semi-intact preparations derived from the differentially reared conditioned animals did not initially demonstrate any significant differences in number of pneumostome openings compared with their yoked controls. However, despite this, there were behavioural indications of operant conditioning in these differentially reared semi-intact preparations. For example, statistical analysis revealed a significant effect of training on the change in opening behaviour after the punishing stimulus. Both the normally reared and differentially reared conditioned preparations demonstrated a reduction in aerial respiratory behaviour after the punishing stimulus. These modifications were the first indications of behavioural plasticity in the differentially reared animals.
Significant differences in neural activity as a result of operant conditioning were observed in three parameters in the differentially reared preparations: the latency from IP3 events (recorded in the motorneuron) to pneumostome opening, the level of coincident IP3 events and pneumostome opening, and the duration of RPeD1 quiescence following the application of the contingent stimulus. These significant changes were also found in the normally reared conditioned preparations (but not the yoked control groups) and are thus indicative of network plasticity as a result of operant conditioning. These results are significant in that they demonstrate that neural plasticity can occur in differentially reared preparations following the operant conditioning procedure. Importantly the neural plasticity occurred both in the absence of a significant change in behaviour of the intact animal, and in the presumed absence of experience-dependent activity during development. Even though neural changes were not observed in all parameters measured in the differentially reared preparations, the fact that some were identified, may suggest that the differentially reared intact animals may have formed memory traces with less punishing stimuli than the normally reared group (though it is not known what the minimum number of stimuli are required to produce the same neuronal changes in the normally reared group). The fact that the presence of neural changes in the semi-intact preparation did not appear to correlate with behavioural changes in the differentially reared intact animals is probably due to the initially low level of behavioural expression as intact animals.
The number of novel changes that were identified in the network in this
study further supports the notion that changes underlying learning and memory
are generally not confined to a single locus, but rather occur at multiple
sites within a neural network (Benjamin et
al., 2000; Brembs,
2003; Lukowiak and Colebrook,
1988; Spencer et al.,
2002). RPeD1 is the CPG neuron that receives both chemosensory
(Inoue et al., 2001) and
mechanosensory input (Haque et al.,
2006) from the periphery and which triggers the CPG rhythm
(Syed et al., 1990). It has
previously been defined as an important site for LTM in operant conditioning
of the aerial respiratory behaviour. Specifically, expression of new genes in
the soma of RPeD1 is required for LTM formation
(Scheibenstock et al., 2002)
and activity levels of RPeD1 in isolated CNSs taken from trained animals are
significantly reduced following conditioning
(Spencer et al., 1999). Here,
we showed for the first time that the immediate response of RPeD1 to
a punishing stimulus is also significantly altered; RPeD1 was quiescent for
significantly longer following the punishing stimulus in conditioned
preparations compared with yoked preparations, regardless of rearing
conditions. It is known that a physical stimulus to an open pneumostome in a
semi-intact preparation produces temporary cessation of RPeD1 activity and
interestingly, Lowe and Spencer (Lowe and
Spencer, 2006) recently showed that artificially silencing RPeD1
activity between training sessions augmented LTM formation, possibly as a
result of altered gene expression induced by the experimental
hyperpolarization. Thus, we hypothesize from our results that RPeD1 quiescence
immediately following the stimulus may represent a neural encoding of LTM.
Though changes in synaptic efficacy are well accepted to play a role in the
cellular encoding of learning and memory, many have suggested that altered
neuronal excitability and firing behaviour of neurons is also an important or
essential mechanism for learning and memory
(Giese et al., 2001;
Daoudal and Debanne, 2003).
Indeed, invertebrate models of conditioning appeared to have led the way for
this hypothesis (Alkon et al.,
1982; Alkon, 1984;
Brembs et al., 2002;
Jones et al., 2003). However,
most cellular correlates of learning have previously been associated with an
increase in neuronal excitability, though there are also examples of reduced
excitability and firing playing a role
(Burrell et al., 2001;
Lowe and Spencer, 2006). Our
findings here suggest that activity changes in RPeD1 occur as a direct result
of the punishing stimulus, and our previous studies suggest that this
reduction in firing is a long-lasting change
(Spencer et al., 1999) that is
directly correlated to the reduction in behaviour
(Spencer et al., 2002).
Though RPeD1 plays an important role in LTM formation, other network
parameters are also probably affected
(Spencer et al., 1999;
McComb et al., 2005) and it is
unlikely that all neural changes associated with the operant conditioning of
aerial respiratory behaviour in Lymnaea have been identified. Here we
have further identified significant changes within the CPG and its motor
output and now provide evidence to suggest that IP3 events, responsible for
pneumostome opening, are also important in the formation or expression of LTM.
Conditioned preparations demonstrated significantly reduced intra-burst spike
frequency of IP3 events in the VI cell, that produced qualitatively smaller
pneumostome openings in the conditioned semi-intact preparations, compared
with yoked controls. These findings strongly suggest either a change in the
firing frequency of the IP3 interneuron, or a change in synaptic efficacy
between the IP3 cell and the VI motorneuron. McComb et al.
(McComb et al., 2005)
previously identified reduced duration of IP3 events in conditioned
preparations, also supporting these hypotheses. Unfortunately, it is difficult
to confirm either of these hypotheses as IP3 lies on the opposite surface of
the CNS and cannot be directly accessed at the same time as either RPeD1 or
the VI motorneurons (Syed et al.,
1990).
Another important finding of this study, suggesting a change in synaptic
efficacy within the network, was that coincident IP3 events and pneumostome
openings were significantly reduced in the conditioned preparations. In other
words, IP3 events did not necessarily lead to pneumostome openings. Since IP3
events were observed in the VI cell, which is monosynaptically connected to
the pneumostome opener muscles (Bell et
al., 2008), this may indicate a change in neuromuscular
transmission, such as decreased transmitter release from the VI cell or
decreased excitability of the postsynaptic pneumostome opener muscle cells
(Kandel, 2001). Such a change
in synaptic efficacy between the VI motorneuron and the pneumostome muscles is
again supported by previous findings of McComb et al.
(McComb et al., 2005), who
showed that artificial depolarization of VI failed to elicit a
pneumostome opening in conditioned semi-intact preparations. Lukowiak and
Colebrook (Lukowiak and Colebrook,
1988) also showed that the ability of a gill motorneuron to elicit
gill withdrawal in Aplysia was significantly altered following
classical conditioning, although in this case, a potentiation (rather than a
reduction) was observed. The change in synaptic efficacy between the VI
motorneuron and muscle may also be the cause of the increased latency observed
when the IP3 event in the VI cell did produce a pneumostome opening,
though we cannot rule out a reduction in conduction velocity in the
motorneuron as the cause of this effect, as has been previously observed in
the operant conditioning of the vertebrate H-reflex
(Wolpaw, 1997). Taken
together, these results demonstrate synaptic remodelling throughout the CPG as
a result of conditioning and that changes in IP3 events and/or neuromuscular
transmission may occur concomitant with changes in RPeD1 activity.
In summary we have identified novel neural correlates of operant
conditioning in Lymnaea stagnalis at the single cell level, to
provide evidence that there is encoding of LTM not only within the respiratory
CPG but also within the VI motorneurons and their connections with the
pneumostome opener muscles. These finding are consistent with recent
literature that documented the occurrence of modulation at all levels of the
nervous system, including the CPG, the sensory, motor, and facilitating
neurons, the sensory organs and the musculature
(Baxter and Byrne, 2006;
Harris-Warrick and Marder,
1991). Furthermore, we have demonstrated for the first time that
network changes associated with operant conditioning of the aerial respiratory
behaviour can occur in differentially reared animals that did not experience
aerial respiration during development. These data strongly suggest that
network plasticity in the adult CNS is not dependent on experience-dependent
activity of the same network during development.
|
Footnotes |
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|
References |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Alkon, D. L. (1984). Calcium-mediated reduction
of ionic currents: a biophysical memory trace. Science
226,1037
-1045.
Alkon, D. L., Lederhendler, I. and Shoukimas, J. J.
(1982). Primary changes of membrane currents during retention of
associative learning. Science
215,693
-695.
Baxter, D. A. and Byrne J. H. (2006). Feeding
behaviour of Aplysia: a model system for comparing cellular
mechanisms of classical and operant conditioning. Learn.
Mem. 13,669
-680.
Bell, H. J., Inoue, T., Shum, K., Luk, C. and Syed, N. I. (2007). Peripheral oxygen-sensing cells directly modulate the output of an identified respiratory central pattern generating neuron. Eur. J. Neurosci. 25,3537 -3550.[CrossRef][Medline]
Bell, H. J., Inoue, T. and Syed, N. I. (2008). A peripheral oxygen sensor provides direct activation of an identified respiratory CPG neuron in Lymnaea. Adv. Exp. Med. Biol. 605,25 -29.[CrossRef][Medline]
Benjamin, P. and Winlow, W. (1981). The distribution of three wide-acting synaptic inputs to identified neurons in the isolated brain of Lymnaea stagnalis (L.). Comp. Biochem. Physiol. 70,293 -307.
Benjamin, P. R., Staras, K. and Kemenes, G.
(2000). A systems approach to the cellular analysis of
associative learning in the pond snail Lymnaea. Learn.
Mem. 7,124
-131.
Brembs, B. (2003). Operant conditioning in invertebrates. Curr. Opin. Neurobiol. 13,710 -717.[CrossRef][Medline]
Brembs, B., Lorenzetti, F. D., Reyes, F. D., Baxter, D. A. and
Byrne, J. H. (2002). Operant reward learning in
Aplysia: neuronal correlates and mechanisms.
Science 296,1706
-1709.
Burrell, B. D., Sahley, C. L. and Muller, K.
(2001). Non-associative learning and serotonin induce similar
bi-directional changes in excitability of a neuron critical for learning in
the medicinal leech. J. Neurosci.
21,1401
-1412.
Daoudal, G. and Debanne, D. (2003). Long-term
plasticity of intrinsic excitability: learning rules and mechanisms.
Learn. Mem. 10,456
-465.
Dawson, A. and Wood, E. J. (1982). Equilibrium and kinetic studies of oxygen binding to the haemocyanin from the freshwater snail Lymnaea stagnalis. Biochem. J. 207,145 -153.[Medline]
Dawson, A. and Wood, E. J. (1983). Equilibrium and kinetic studies of oxygen binding to fragments of Lymnaea stagnalis (freshwater snail) haemocyanin obtained by proteolytic digestion. Biochem. J. 209,519 -526.[Medline]
Erickson, J. T., Mayer, C., Jawa, A., Ling, L., Olson, E. B.,
Jr, Vidruk, E. H., Mitchell, G. S. and Katz, D. M. (1998).
Chemoafferent degeneration and carotid body hypoplasia following chronic
hyperoxia in newborn rats. J. Physiol.
509,519
-526.
Giese, K. P., Peters, M. and Vernon, J. (2001). Modulation of excitability as a learning and memory mechanism: a molecular genetic perspective. Physiol. Behav. 73,803 -810.[CrossRef][Medline]
Haque, Z., Lee, T. K. M., Inoue, T., Luk, C., Hasan, S. U., Lukowiak, K. and Syed, N. I. (2006). An identified central pattern-generating neuron co-ordinates sensory-motor components of respiratory behavior in Lymnaea. Eur. J. Neurosci. 23, 94-104.[CrossRef][Medline]
Harris, C. L. (2003). Concepts in Zoology. New York: Harper Collins.
Harris-Warrick, R. M. and Marder, E. (1991). Modulation of neural networks for behavior. Annu. Rev. Neurosci. 14,39 -57.[CrossRef][Medline]
Hermann, P. M. and Bulloch, A. G. M. (1998). Developmental plasticity of repiratory behavior in Lymnaea. Behav. Neurosci. 112,656 -667.[CrossRef][Medline]
Inoue, T., Haque, Z., Lukowiak, K. and Syed, N. I.
(2001). Hypoxia-induced respiratory patterned activity in
Lymnaea originates at the periphery. J.
Neurophysiol. 86,156
-163.
Jones, J. D. (1961). Aspects of respiration in Planorbis corneus L. and Lymnaea stagnalis. L. (Gastropoda: Pulmonata). Comp. Biochem. Physiol. 4, 1-29.[Medline]
Jones, N. G., Kemenes, I., Kemenes, G. and Benjamin, P. R. (2003). A persistent cellular change in a single modulatory neuron contributes to associative long-term memory. Curr. Biol. 13,1064 -1069.[CrossRef][Medline]
Joseph, V., Soliz, J., Pequignot, J., Sempore, B., Cottet-Emard,
J. M., Dalmaz, Y., Favier, R., Spielvogel, H. and Pequignot, J. M.
(2000). Gender differentiation of the chemoreflex during growth
at high altitude: functional and neurochemical studies. Am. J.
Physiol. Regul. Integr. Comp. Physiol.
278,R806
-R816.
Kandel, E. R. (2001). The molecular biology of
memory storage: a dialogue between genes and synapses.
Science 294,1030
-1038.
Lowe, M. R. and Spencer, G. E. (2006).
Perturbation of the activity of a single identified neuron affects long-term
memory formation in a molluscan semi-intact preparation. J. Exp.
Biol. 209,711
-721.
Lukowiak, K. and Colebrook, E. (1988).
Classical conditioning alters the efficacy of identified gill motor neurons in
producing gill withdrawal movements in Aplysia. J. Exp.
Biol. 140,273
-285.
Lukowiak, K., Ringseis, E., Spencer, G., Wildering, W. and Syed, N. (1996). Operant conditioning of aerial respiratory behaviour in Lymnaea stagnalis. J. Exp. Biol. 199,683 -691.[Abstract]
Lukowiak, K., Cotter, R., Westly, J., Ringseis, E., Spencer, G. and Syed, N. (1998). Long-term memory of an operantly conditioned respiratory behaviour pattern in Lymnaea stagnalis. J. Exp. Biol. 201,877 -882.[Abstract]
Lukowiak, K., Adatia, N., Krygier, D. and Syed, N.
(2000). Operant conditioning in Lymnaea: evidence for
intermediate- and long-term memory. Learn. Mem.
7, 140-150.
Martens, K., Amarell, M., Parvez, K., Hittel, K., De Caigny, P., Ito, E. and Lukowiak, K. (2007). One-trial conditioning of aerial respiratory behaviour in Lymnaea stagnalis. Neurobiol. Learn. Mem. 88,232 -242.[CrossRef][Medline]
McComb, C., Rosenegger, D., Varshney, N., Kwok, H. Y. and Lukowiak, K. (2005). Operant conditioning of an in vitro CNS-pneumostome preparation of Lymnaea. Neurobiol. Learn. Mem. 84,9 -24.[CrossRef][Medline]
Papini, M. R. and Bitterman, M. E. (1990). The role of contingency in classical conditioning. Psychol. Rev. 97,396 -403.[CrossRef][Medline]
Sangha, S., McComb, C. and Lukowiak, K. (2003).
Fogetting and the extension of memory in Lymnaea. J. Exp.
Biol. 206,71
-77.
Scheibenstock, A., Krygier, D., Haque, Z., Syed, N. and
Lukowiak, K. (2002). The soma of RPeD1 must be present for
long-term memory formation of associative learning in Lymnaea. J.
Neurophysiol. 88,1584
-1591.
Spencer, G. E., Syed, N. I. and Lukowiak, K.
(1999). Neural changes after operant conditioning of the aerial
respiratory behaviour in Lymnaea stagnalis. J.
Neurosci. 19,1836
-1843.
Spencer, G. E., Kazmi, M. H., Syed, N. I. and Lukowiak, K.
(2002). Changes in the activity of a CPG neuron after the
reinforcement of an operantly conditioned behavior in Lymnaea. J.
Neurophysiol. 88,1915
-1923.
Syed, N. I. and Winlow, W. (1991). Respiratory behavior in the pond snail Lymnaea stagnalis. II. Neural elements of the central pattern generator (CPG). J. Comp. Physiol. A 169,557 -568.
Syed, N. I., Bulloch, A. G. and Lukowiak, K.
(1990). In vitro reconstruction of the respiratory
central pattern generator of the mollusk Lymnaea.Science 250,282
-285.
Syed, N. I., Harrison, D. and Winlow, W. (1991). Respiratory behavior in the pond snail Lymnaea stagnalis. I. Behavioral analysis and the identification of motor neurons. J. Comp. Physiol. A 169,541 -555.
Taylor, B. E., Harris, M. B., Burk, M., Smyth, K., Lukowiak, K. and Remmers, J. E. (2003). Nitric oxide mediates metabolism as well as respiratory and cardiac responses to hypoxia in the snail Lymnaea stagnalis. J. Exp. Zool. 295A,37 -46.[CrossRef]
Terry, W. S. (2003). Learning and Memory: Basic Principles, Processes, and Procedures. 2nd Edn. USA: Pearson Education.
Wedemeyer, H. and Schild, D. (1995). Chemosensitivity of the osphradium of the pond snail Lymnaea stagnalis.J. Exp. Biol. 198,1743 -1754.[Medline]
Wolpaw, J. R. (1997). The complex structure of a simple memory. Trends Neurosci. 20,588 -594.[CrossRef][Medline]
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P<0.01) in the pre-observation period than
normally reared naïve animals (6.0±0.6). (B) Differentially reared
animals also showed a reduced total breathing time (37±7 s;
