Learning and memory in Lymnaea are negatively altered by acute low-level concentrations of hydrogen sulphide
Calgary Brain Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1
* Author for correspondence (e-mail: lukowiak{at}ucalgary.ca)
Accepted 4 May 2004
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Summary |
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Key words: hydrogen sulfide, Lymnaea stagnalis, operant conditioning, aerial respiratory behaviour, learning, memory
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Introduction |
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Our understanding of the effects of H2S on living organisms is
quite limited. H2S is itself an endogenous neurotransmitter/neural
modulator across all animal phyla (Abe and
Kimura, 1996; Julian et al.,
2002
; Eto and Kimura, 2003; Eto
et al., 2002
), and its actions have yet to be clearly delineated.
A simpler model system is needed to directly determine where and how
H2S alters learning and memory. For example, although
H2S exposure may result in memory loss in rodents, at physiological
concentrations H2S facilitates long-term potentiation (LTP) in
neuronal structures thought to be necessary for memory formation in an
activity- and dose-dependent manner (Abe
and Kimura, 1996
; Kimura,
2000
). We therefore have made use of a model system that was
initially developed to elucidate the causal neuronal mechanisms of learning
and memory (Lukowiak et al.,
2003b
) to examine the effects of low-level exposure to
H2S on a relatively simple, adaptable behaviour.
Our model system, the pond snail Lymnaea stagnalis, is a bimodal
breather that satisfies its oxygen needs either cutaneously, via
diffusion across the skin, or aerially, by opening the pneumostome (the
respiratory orifice) at the water surface. It is therefore possible to
modulate one of its respiratory behaviours while leaving the other unaffected.
We make use of a non-declarative, operant (i.e. instrumental) conditioning
paradigm to decrease the occurrence of aerial respiratory behaviour (Lukowiak
et al., 1996,
1998
,
2000
,
2003a
). These snails can still
breathe cutaneously and thus our procedure is not harmful to the animals.
Naïve snails when placed in hypoxic pondwater preferentially perform
aerial respiration (Lukowiak et al.,
1996). Snails placed in hypoxic pondwater are thus `motivated' to
perform aerial respiration and we operantly (instrumentally) condition snails
by applying a tactile stimulus to the pneumostome area as they begin to open
their pneumostome. Snails associatively learn not to perform aerial
respiration and are capable of committing this learning into long-lasting
non-declarative memory (Lukowiak
et al., 2003a
). By varying the length and number of the training
sessions given, different forms of memory can be developed
(Lukowiak et al., 2000
).
Intermediate term memory (ITM) lasts for only a few hours and requires
only the translation of pre-existing mRNA, whereas long-term memory
(LTM) lasts for at least 24 h and requires both the transcription of
new mRNA along with its translation
(Sangha et al., 2003a
).
A major advantage of our model system is that the underlying neural
circuitry controlling the behavior has been well characterized. A three-neuron
central pattern generator (CPG) has been found to be both necessary and
sufficient for controlling aerial respiration (Syed et al.,
1990,
1992
). Since non-declarative
memories are stored within the neuronal circuit mediating the behaviour
(Milner et al., 1998
), and
since the circuit that drives aerial respiratory behaviour is known as well as
or better than any other neuronal circuit, we possess a clearcut advantage in
determining the causal mechanisms of learning and its consolidation into
memory. Neural correlates of learning and LTM have been shown in one of the
CPG neurons, RPeD1 (Spencer et al.,
1999
,
2002
), which has been directly
demonstrated to be a necessary site for LTM formation, reconsolidation and
extinction (Scheibenstock et al.,
2002
; Sangha et al.,
2003b
,c
).
The experimental advantages of our model system may allow us to directly
determine how H2S affects important homeostatic behaviours such as
respiration and, importantly, how learning and its ability to be consolidated
into memory are affected at the level of a single neuron.
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Materials and methods |
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All experiments were performed in a 1 liter beaker filled with 500 ml of hypoxic water. Experiments were performed under hypoxic conditions in order to increase the occurrence of the aerial respiration behaviour (i.e. opening and closing of the pneumostome, the respiratory orifice). Under eumoxic conditions there is no real need for the snails to surface and open their pneumostomes to breathe, so they cannot be easily conditioned. The water was made hypoxic (<0.1 ml O2 l1) by bubbling nitrogen through it for at least 20 min prior to the beginning of the experiment.
The H2S environment
The H2S environment was created by dissolving a stock liquid
Na2S solution (iodometrically titrated to 75 mmol
l1), into 500 ml of water, until the desired concentration
was reached. The Na2S ionizes in the water to form one third
H2S and two thirds HS (it is unknown which of
these is the physiologically active form). The addition of the Na2S
solution did not alter the pH of the water. A fresh solution of the stock
Na2S and water was created for each session just prior to the
commencement of acclimatization (see below) to ensure consistent
concentrations. In these experiments conditions are said to be `standard'
(i.e. just hypoxic) when there is no H2S present in the solution.
During experiments involving H2S, N2 was only bubbled
prior to the addition of H2S to prevent its dissipation. Oxygen
levels were tested to ensure that there was no significant difference between
sessions that had continual nitrogen bubbling and those that only had the
pre-bubbling.
Water oxygen levels
The oxygen content of the water under the various paradigms was determined
using a Polarographic amplifier (A-M Systems model 1900, Sequin, WA, USA) and
electrode. Readings (in nA) were taken while the water was saturated with
either nitrogen or pure oxygen to act as controls, closely approximating 0%
and 100% dissolved oxygen, respectively (the beakers were covered with
Parafilm during these readings to prevent air from mixing with the water).
Readings were then taken under experimental conditions including: (1) after
bubbling N2 for 30 min while still bubbling N2, (2)
after removing the air stone after 30 min of N2 bubbling (i.e. no
N2 bubbling at time of measurement), (3) after adding the
Na2S (100 µmol l1 final concentration) to the
water that had been bubbled with N2 for 30 min, (4) 45 min after
Na2S had been added to the water, and (5) water that is at
equilibrium with the atmosphere. A linear relationship was created with the
two standards, and was used to determine the approximate % oxygen saturation
in the water.
Breathing observations
Snails were placed in a 1 liter beaker filled with 500 ml of hypoxic water
for an initial acclimatization period of 10 min; they were then gently pushed
just below the surface at the beginning of the observation period. The aerial
respiratory behavior was monitored continuously for 45 min. After this initial
observation period the snails were returned to eumoxic water for 1 h. A second
45 min observation period was then performed in hypoxic water using 100
µmol l1 of H2S. A final 45 min breathing
observation was performed 1 h later in standard hypoxic water. The time at
which each snail opened and closed its pneumostome was recorded. From these
recordings the number of pneumostome openings, total breathing time, and
average breathing time per opening were calculated for each snail. Breathing
observations that were carried out with H2S were done at a
concentration of 100 µmol l1, and a plastic cover was
used to limit any gas dissipation.
A second set of breathing observations following a more `intense hypoxic
challenge' were also performed. In these observations the snails' breathing
behaviour was first observed for 45 min in eumoxia. Following a 1 h interval
in eumoxia they were placed under a barrier in hypoxic pondwater for a period
of 45 min. They could not perform aerial respiratory behaviours for this
period of time prior to the start of the second observation period. In these
experiments there was no third observation period as previously we have shown
that the breathing parameters return to control levels following this
procedure (McComb et al.,
2002). The 45 min submersion in hypoxic water significantly
increases aerial breathing behaviour, presumably due to the accumulation of an
oxygen deficit.
Operant conditioning
For training, snails are placed in 500 ml of hypoxic water. The snails are
allowed a 10 min acclimatization period prior to the training session. The
snails are then pushed below the surface of the water just before the training
begins. Training involves the application of a tactile stimulus to the
pneumostome of the snail when it attempts to perform aerial respiration. The
time of each attempted opening is recorded. The training periods last for 30
or 45 min, with 1 h between sessions. Two training sessions are performed with
a memory test 24 h later. The memory test (session 3) follows the same
procedure as the training sessions. The operant conditioning training
procedure used produces a long-term memory (LTM) that persists for at least 24
h (Lukowiak et al.,
2003a,b
).
Snails were subjected to H2S at one of three times: (1) for 1 h prior to the first training session only; (2) during all training and memory test sessions; (3) for 1 h after each training session.
Another cohort of snails was also subjected to the 45 min hypoxia submersion procedure prior to both the operant conditioning training sessions and the test for savings 24 h later. These experiments were performed to determine if it was still possible for the snails to learn even with their increased motivation for aerial respiration.
Operational definitions of learning and memory
We have operationally defined memory as previously described (Lukowiak et
al., 1996,
2003b
; Sangha et al.,
2003b
,c
).
Learning was present if the number of attempted pneumostome openings in the
last training session was significantly less than the number of attempted
openings in the first training session. In order to be defined as memory, two
criteria had to be met: (1) the number of pneumostome openings in the test for
savings was significantly lower than that of the first training session, and
(2) the number of pneumostome openings in the test for savings was not
significantly higher than that of the last training session.
Yoked controls
Yoked controls were performed under standard hypoxic conditions, and
hypoxic conditions with H2S at a concentration of 75 µmol
l1. These experiments were performed as previously described
(Lukowiak et al., 1996). In
sessions 1 and 2 for the yoked control procedure the animal received a tactile
stimulus to the pneumostome area every time the snail to which it was `yoked'
attempted to open its pneumostome (i.e. in these sessions snails did not
receive a reinforcing stimulus contingent on when they open their
pneumostome). However, in the third session (i.e. memory test, MT), these
yoked control snails now received a tactile stimulus each time they attempted
to open their pneumostome (i.e. they received contingent stimulation). We
compared the number of attempted openings in MT of the yoked control snails
with the number of attempted openings in MT of the operantly conditioned
snails. If the observed change in behaviour is due to an associative process
(i.e. due to contingent presentation of the tactile stimulus to the
pneumostome as it attempted to open), the number of attempted openings in the
yoked control cohort should be significantly greater than the number of
attempted openings in the operantly conditioned group.
Assignment of marks
Snails were given grades on an individual basis to show how well (or how
poorly) they learned. The following grading scheme was used to assess
learning: a snail that showed a 50% or greater reduction in attempted
pneumostome openings from the first session to the second session was given an
A, B was a 3549.99% reduction, C was a 2034.99% reduction, and F
was assigned when a reduction of less than 20% was observed (see
Lukowiak et al., 2003b).
Statistics
To determine whether the number of attempted pneumostome openings was
significantly altered as a result of operant conditioning, repeated-measures
one-way analysis of variance (ANOVA) was performed. If the ANOVA was
significant (P<0.05), a post hoc Fisher's LSD
t-test was performed to show which sessions were significantly
different. The same test was performed in determining the significance of the
yoked controls. A 2-test statistic was used to determine if
the assigned marks were different for cohorts of snails exposed to
H2S or the `more intense hypoxic challenge'. Correlated (paired)
t-tests were performed to determine whether or not breathing
behaviour was significantly altered by H2S. A session difference
was considered statistically significant if P<0.05. A
t-test for separate groups was used to determine if the increase in
respiration caused by H2S was equivalent to the increase caused by
the `more intense hypoxic challenge' procedure.
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Results |
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Snails showed a statistically significant increase in the number of pneumostome openings, total breathing time and the average breathing time per pneumostome opening in the H2S condition compared to standard hypoxia (Fig. 1A, Table 1). These observations also exemplify that aerial respiratory behaviour was not permanently altered by exposure to H2S. That is, aerial respiratory behaviour recovered to pre-exposure levels following exposure to H2S [NSD (no significant difference) session 1 vs 3, P>0.05; session 2 significantly different from sessions 1 and 3, P<0.01, for both comparisons].
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It was possible that the increase in aerial respiratory behaviour in the hypoxic-H2S condition was due not to an effect of H2S but rather due to a decrease in O2 content of the pondwater. Thus, oxygen levels of the solutions were measured to ensure that the increase in breathing behaviour observed under H2S conditions, were not due to a chemical reaction that reduced the oxygen concentration in the water (Table 2). The recordings show that the addition of Na2S to the water does not alter the oxygen levels of the water. The % O2 saturation of the water does also not significantly increase 45 min after the cessation of N2 bubbling.
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We conclude from these experiments that acute exposure to H2S significantly increases aerial respiratory behaviour of snails under hypoxic conditions, and that the breathing behaviour returns to normal afterward. Breathing behaviour was also significantly increased in the snails that were subjected to the `more intense-hypoxic challenge' (mean total breathing time increased to 645.5 s from 266.2 s) compared to standard hypoxia (P>0.01). However breathing behaviours were not statistically different between this group and the snails subjected to hypoxia + H2S (P=0.1714). Further, the elevated breathing was not due to H2S decreasing the amount of oxygen in the water.
Having demonstrated that H2S significantly but reversibly increased aerial respiratory behaviour we wished to determine if H2S affected the capability of snails to learn and/or form memory. Snails trained in the standard hypoxic condition exhibited learning and memory (Fig. 2A). We then determined the effect of operant conditioning training (two 45 min training sessions separated by a 1 h interval) on snails in 100 µmol l1 H2S-hypoxic pondwater (Fig. 2C). Snails trained under such conditions neither learned nor formed memory. That is, when we trained snails in 100 µmol l1 H2S-hypoxic pondwater there was no significant difference in the number of attempted pneumostome openings between session 1 and session 2. On the following day we tested the unlikely possibility that these snails formed memory. They did not. That is, the number of attempted pneumostome openings in session 3 was not significantly different from session 1. Notice in these experiments that snails still had the capacity to perform aerial respiration; however, they did not have the capability of making an association (i.e. learn) between opening the pneumostome and receiving a noxious stimulus.
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It might be argued that the inability to learn and form LTM in the 100 µmol l1 H2S-hypoxic pondwater was due to the increased need to perform aerial respiration. To control for this possibility we trained snails that were subjected to the `more intense-hypoxic challenge'. These snails performed aerial respiration at the same level as the snails in 100 µmol l1 H2S-hypoxic pondwater did (Table 1). However, snails subjected to the `more intense-hypoxic challenge' still have the capacity to learn and to form LTM (Fig. 2B). That is, the number of attempted pneumostome openings in session 2 was significantly less than in session 1 (i.e. learning demonstrated). Moreover, as the number of attempted openings in the savings test session was not statistically greater than the number in session 2, but was significantly less than the number in session 1, memory was shown. Thus the learning impairment caused by H2S is not simply due to the fact that there is an increased drive for respiration.
We next asked whether a lower concentration of H2S would similarly affect a snail's ability to learn and form memory. We therefore repeated the conditioning experiments in 50 and 75 µmol l1 H2S-hypoxic pondwater respectively (Fig. 3). In contrast to the results obtained with 100 µmol l1 H2S-hypoxic pond water, we found that snails exposed to either 50 (Fig. 3A) or 75 µmol l1 H2S (Fig. 3B) could both learn and form memory. That is, both the cohort exposed to 50 µmol l1 and the cohort exposed to 75 µmol l1 H2S demonstrated learning (i.e. the last training session was significantly lower (P<0.01) than the first). Both groups also demonstrated memory [i.e. the memory test session (MT) was significantly lower (P<0.01) than the session 1 and was not significantly greater than session 2]. To show that these changes in behaviour were bona fide examples of associative learning we performed yoked control experiments. The yoked control snails did not show any LTM formation in either group (i.e. the standard hypoxic (N=11), and hypoxic 75 µmol l1 H2S (N=11) group; Fig. 4).
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To determine whether there was a diminished capability to learn and form
memory in the H2S challenged snails we gave each individual snail a
`grade' (see Materials and methods) and then determined if there were
significant differences in the number of A grades (etc.) between the different
cohorts (standard hypoxic; `more intense-hypoxic challenge'; 50 µmol
l1 H2S-hypoxic; 75 µmol l1
H2S-hypoxic; and 100 µmol l1
H2S-hypoxic, respectively, Fig.
5). As the concentration of H2S is increased, the
percentage of snails with `A' grades decreases and more received an `F' grade,
which would be consistent with the hypothesis of a dose-dependent deleterious
effect of the H2S on the snails' ability to learn. A
2-statistical analysis was performed to determine if the
snails trained under standard conditions and H2S conditions showed
a significantly different number of A and F grades (i.e. P<0.05).
In this test the grades (A grades and F grades) of the snails trained under
the standard conditions were used as the expected frequencies, and the snails
trained in the H2S and the `more intense hypoxic challenge' were
used as the observed frequencies to compare the two (such as in a placebo
group vs a treatment group). There was no significant difference in
the frequency of A grades and F grades between the standard hypoxic condition
and 50 µmol l1 H2S-hypoxic group
(P=0.081), but there was a significant difference between the
standard hypoxic condition and 75 µmol l1
H2S-hypoxic group (P<0.01). Not surprisingly there was
also a significant difference between the standard hypoxic group and the 100
µmol l1 H2S group (P<0.01). Thus
as the concentration of H2S in the hypoxic pondwater increases
there is an increase in the frequency of snails that receive a failing grade.
Finally, when we performed this analysis on the snails given the `more
intense-hypoxic challenge' we found that statistically they were the best
learners and had the best memory. That is, they received statistically more A
grades and fewer F grades than any of the other groups (P<0.01
when compared to the standard hypoxic group).
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The finding that relatively low levels of H2S in pondwater
significantly diminished the ability of snails to learn prompted us to
hypothesize that H2S exposure would interfere with the memory
consolidation process. Memory consolidation can be interfered with by a 1
h-cooling period (to 4°C) if it is applied within 10 min after the last
training session (Smyth et al.,
2002; Sangha et al.,
2003a
). We therefore exposed snails trained in standard hypoxic
conditions to H2S for 1 h immediately (within 30 s) after session
2. Following the 1 h exposure to H2S the snails were returned to
their home aquaria (Fig. 6).
When we tested memory retention on the following day (MT) we found that snails
had memory. That is, the number of attempted openings in MT was significantly
different from the number of attempted openings in session 1 and was not
significantly greater than the number of attempted openings in session 2.
Thus, the memory consolidation process was not impeded by exposure to
H2S during the consolidation period.
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Discussion |
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Typically, in eumoxic conditions snails rely on cutaneous respiration to
satisfy their respiratory requirements and perform aerial respiration only
intermittently (Taylor and Lukowiak, 2001;
Taylor et al., 2003). When
snails encounter a hypoxic environment there is a significant increase in
aerial respiratory behaviour. In other words, in a hypoxic environment
cutaneous respiration is insufficient to meet the respiratory needs of the
snail, and thus aerial respiration must be increased to satisfy the snails'
oxygen requirements. We found that exposure to H2S (100 µmol
l1) in the hypoxic environment significantly and reversibly
increased aerial respiratory behaviour, while having no effect on the oxygen
content of the water. A similar increase in respiratory drive occurred when
snails were maintained in a hypoxic environment where they were unable to
perform aerial respiratory behaviour for a period (i.e. the `more
intense-hypoxic challenge') as shown here and previously
(McComb et al., 2002
). In that
situation, snails develop an oxygen debt and significantly increase aerial
respiratory behaviour to `pay back' this debt. There are a number of possible
explanations for the increase in aerial respiratory drive seen with the
H2S-challenge: (1) H2S interferes with cutaneous
respiration; (2) H2S increases the O2 requirements of
the snail; (3) H2S has a direct stimulatory effect on the
respiratory CPG. At present we cannot distinguish between these possibilities;
however, with the in vitro semi-intact preparation
(Inoue et al., 1996
;
McComb et al., 2003
) it may be
possible to determine directly whether H2S alters CPG activity.
Such experiments will be initiated shortly.
Because the increase in aerial respiratory behaviour to the
H2S-challenge was reversible, any alteration in the efficiency of
cutaneous respiration that could be attributed to the H2S-challenge
has to be short lived (i.e. <1 h). Likewise, if the
H2S-challenge causes an increase in the snails' O2
requirements this increase would also have to be relatively short lived. We
have not determined how long the increase in respiratory behaviour can be
maintained in the H2S-environment. Nor have we attempted to
determine how long Lymnaea can remain viable in this concentration of
H2S. For example, will much longer H2S exposure times
lead to irreversible changes in aerial respiratory behaviour? These
experiments will also be initiated in the future. Since CPG activity that
drives aerial respiratory behaviour is easily modifiable
(Taylor and Lukowiak, 2000) we
presently favor the hypothesis that H2S has direct effects on CPG
output. That is, we expect to find that H2S directly alters the
activity of the CPG neurons so that rhythmogenesis is increased, as it is
following `more intense-hypoxic challenge', thus driving an increase in aerial
respiratory behaviour.
We also hypothesized that an H2S-challenge would affect the ability of Lymnaea to associatively learn. Our data show that there is a concentration-dependent effect of H2S on the acquisition of operant conditioning, a form of associative learning. As the concentration of H2S was increased from 50 µmol l1 to 100 µmol l1 there was a corresponding decrement in the snails' ability to acquire learning. At the lowest concentration tested here (50 µmol l1) the ability of the cohort of snails to acquire learning was not any different from the control cohort. However, at 75 µmol l1 H2S there was a significant decrease in the ability of the cohort of snails to acquire learning and at 100 µmol l1 H2S the cohort was incapable of learning. When we further analyzed each individual of the various cohorts the effects of the H2S-challenge became even more apparent. We found that only 4% of snails challenged with the 100 µmol l1 H2S obtained a mark of A, whilst over 10 times that number received an A in the control standard hypoxic environment or the group following `more intense-hypoxic challenge'. At the other end of the spectrum in the standard hypoxic situation, approximately 20% of snails received an F grade, whilst the majority (52%) of snails challenged with 100 µmol l1 H2S received an F grade. Similarly, examining the marks of individual snails challenged with 75 µmol l1 H2S we found that only 18% received an A grade, whilst 45% received an F. Again, these marks are indicative of a detrimental effect of 75 µmol l1 H2S on learning ability. We do not believe that the impairment in learning is caused by an increased need for aerial respiration as snails that receive the `more intense-hypoxic challenge' had the most A grades and fewest F grades. That is, snails challenged with a procedure, that increases their respiratory needs, show no learning or memory deficits. Since yoked control snails did not show a change in aerial respiratory behaviour our data are consistent with the hypothesis that the detrimental effects of H2S on learning and memory formation are the result of changes caused by H2S on molecular processes in the neurons that are necessary for learning and memory.
Our data, however, did not support our final hypothesis, which was that an
H2S challenge would block the memory consolidation process.
Learning and memory are not a unitary process; rather they are separate but
related processes (Dudai,
2003). Following the acquisition of a new behaviour (i.e.
learning) there is a time period during which the learned behaviour is
committed to memory (i.e. the consolidation process). When first acquired,
memory is sensitive to disruption by external events. With the passage of
time, however, storage becomes more permanent and less susceptible to
disruption (White and Salinas,
1998
). Brain injury, electroconvulsive shock, cooling and protein
synthesis inhibitors can disturb memory, and even new learning if applied
during the consolidation period (McGaugh,
1999
;
2000
).
In order to form a long-lasting memory (>5 h) in Lymnaea, as in
all other animals, altered gene activity and new protein synthesis are
required (Dudai, 2002). Thus,
in Lymnaea the application of a transcription or translation
inhibitor (Actinomycin D and Anisomycin, respectively) or quickly cooling the
snail for 1 h at 4°C immediately after the last training session, prevents
the formation of LTM (Sangha et al.,
2003a
,b
).
We therefore exposed snails to 100 µmol l1 H2S
for 1 h immediately after the last operant conditioning training session. This
procedure did not interfere with the formation of memory. That is, these
snails still had the capability of consolidating the new learned behaviour
into a memory that was not different from control. Thus, 100 µmol
l1 H2S exposure, which blocked learning, did not
block the consolidation processes that underlies the formation of memory. We
did not test whether a longer exposure to 100 µmol l1
H2S would block memory formation. We therefore conclude that in
Lymnaea a 100 µmol l1 H2S challenge
only affects the learning process and not the memory consolidation process.
Similar findings were also reported by Partlo et al.
(2001
), when they tested rats
after exposure to H2S. Thus an H2S-challenge alters both
declarative learning (spatial learning) in rats and non-declarative memory
(operant conditioning) in snails.
In the rat, endogenous levels of H2S (50160 µmol
l1) have been shown to facilitate the formation of LTP;
however, at higher concentrations (320 and 640 µmol l1)
this effect is no longer seen as population spikes and field EPSPs (excitatory
post synaptic potentials) become suppressed
(Abe and Kimura; 1996). Also
lethal levels of sulfide have also been shown to be less than two times those
of endogenous levels, indicating that the doseresponse curve for
H2S is very steep (Warenycia et
al; 1989
). These phenomena may help to explain the results seen in
our experiments. The lowest level of H2S exposure used here (50
µmol l1) had little or no effect on aerial respiratory
behaviour or learning ability. This may be due to the fact that there is not
enough accumulation of H2S, or it can be metabolized quickly enough
so it does not reach a level where it can be detrimental to the learning
process. However, at the higher concentrations used here (75 µmol
l1 and 100 µmol l1) the accumulation of
H2S may be sufficient to alter the synaptic interactions and/or
endogenous membrane properties of the central pattern generator (CPG) in such
a manner as to increase aerial respiratory behaviour and have a negative
influence on the ability of the CPG to undergo the changes in neuronal
activity that constitute the neural substrates of learning. It appears, for
example, that there is an optimal range of RPeD1 (one of the three CPG
neurons) activity within the respiratory CPG in Lymnaea that is
conducive for optimizing aerial respiratory behaviour
(McComb et al., 2003
). Any
increase or decrease in RPeD1's activity outside its optimal range has
detrimental affects on the ability of this neural network to produce a
respiratory rhythm.
Higher levels of H2S have been hypothesized to cause the
`metabolic intoxication' seen in the mammalian brain
(Wang, 2002). A similar
`intoxication' may be occurring in the CNS of Lymnaea, inhibiting the
ability of the snail to acquire a learned response. In any case we have shown
here that our Lymnaea model system can be used to study the effects
of toxic gas on the ability to learn and form memory. Because we have shown
that learning and memory formation require the soma of RPeD1, one of the three
CPG neurons, we may be able to specify how H2S alters learning and
memory ability at the single neuron level.
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Acknowledgments |
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