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The Journal of Neuroscience, September 15, 1999, 19(18):8094-8103
Levels of Serotonin in the Hemolymph of Aplysia
Are Modulated by Light/Dark Cycles and Sensitization Training
Jonathan
Levenson1,
John H.
Byrne2, and
Arnold
Eskin1
1 Department of Biology and Biochemistry, University of
Houston, Houston, Texas 77204-5513, and 2 Department of
Neurobiology and Anatomy, University of Texas-Houston Medical School,
Houston, Texas 77225
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ABSTRACT |
Serotonin (5-hydroxytryptamine, 5-HT) modulates the behavior and
physiology of both vertebrate and invertebrate animals. Effects of
injections of 5-HT and the morphology of the serotonergic system of
Aplysia indicate that 5-HT may have a humoral, in
addition to a neurotransmitter, role. To study possible humoral roles
of 5-HT, we measured 5-HT in the hemolymph. The concentration of 5-HT
in the hemolymph was ~18 nM, a value close to previously reported thresholds for eliciting physiological responses. The concentration of 5-HT in the hemolymph expressed a diurnal rhythm. In
addition, electrical stimulation that leads to long-term sensitization significantly increased levels of 5-HT in the hemolymph during training, 1.5 hr after training, and 24 hr after training. Moreover, levels of 5-HT in the hemolymph were significantly correlated with the
magnitude of sensitization. The half-life of an increase in 5-HT in the
hemolymph was ~0.5 hr. Therefore, the persistent increase of 5-HT in
the hemolymph 24 hr after sensitization training indicates that
training caused a long-lasting increase in the release of 5-HT. This
long-lasting increase in 5-HT in the hemolymph was blocked by treatment
with an inhibitor of protein synthesis during training. Based on the
levels of 5-HT in the hemolymph and its regulation by environmental
events, we propose that 5-HT has a humoral role in regulation of the
behavioral state of Aplysia. In support of this
hypothesis, we found that increasing levels of 5-HT in the hemolymph
led to significant alterations in feeding behavior. Increasing levels
of 5-HT during the daytime when they were normally low increased the
latency to assume feeding posture from daytime to nighttime values.
Key words:
Aplysia; mollusk; serotonin; hemolymph; sensitization; diurnal rhythms; learning; feeding; emetine
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INTRODUCTION |
Serotonin influences behavioral
state in a number of vertebrates and invertebrates (for review, see
Edwards and Kravitz, 1997 ). In Aplysia, 5-HT is involved in
mediating sensitization of defensive withdrawal reflexes (Brunelli et
al., 1976; Bernier et al., 1982; Klein et al., 1982; Walters et al.,
1983; Glanzman et al., 1989; Hammer et al., 1989; Schacher
et al., 1990; Trudeau and Castellucci, 1993; Emptage et al., 1996) and
regulating the phase of the ocular circadian rhythm (Corrent and Eskin,
1982), cardiac output (Liebeswar et al., 1975; Sawada et al., 1984),
muscle tone (Weiss et al., 1975; Cooper et al., 1989; McPherson and
Blankenship, 1992), bag cell afterdischarge (Kaczmarek et al., 1978),
locomotor activity (Mackey and Carew, 1983), swimming behavior (Parsons
and Pinsker, 1989; McPherson and Blankenship, 1991; Laurienti and
Blankenship, 1997), and the sensitivity of photoreceptors and
mechanoreceptors (Eskin and Maresh, 1982; Billy and Walters, 1989).
Feeding behavior is also modulated by 5-HT released by the metacerebral
cell (Weiss et al., 1975; Kupfermann et al., 1979; Rosen et al., 1983).
Moreover, activity of the serotonergic metacerebral cell directly
correlates with central arousal in Aplysia (Kupfermann and
Weiss, 1982).
Several lines of evidence indicate that 5-HT may have a humoral role in
Aplysia. 5-HT has widespread physiological effects, and
injections of 5-HT into the hemocoel increase the number and frequency
of a variety of spontaneous behaviors for a short period of time
(Palovcik et al., 1982; Mackey and Carew, 1983; Parsons and Pinsker,
1989). Also, the projections of serotonergic fibers indicates that 5-HT
may be released into the hemocoel. For example, serotonergic fibers
innervate the aorta and other arteries, and also form a basket around
the eye (Takahashi et al., 1988; Alevizos et al., 1989; Skelton and
Koester, 1992).
As a first step to further examine the possible humoral role of 5-HT in
Aplysia, we investigated whether 5-HT was present in the
hemolymph and, if so, whether its levels were regulated. In this study,
we demonstrate that 5-HT is present in the hemolymph of
Aplysia and that its concentration is near threshold for a number of physiological effects of 5-HT. We show that levels of 5-HT in
the hemolymph are regulated by light/dark cycles and by electrical
stimulation that leads to long-term sensitization. Furthermore, we show
that perturbations of 5-HT in the hemolymph affect aspects of
appetitive feeding behavior.
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MATERIALS AND METHODS |
Aplysia californica (100-150 gm) were obtained from
Marinus (Long Beach, CA). Animals were maintained in Instant Ocean
(Aquarium Systems, Mentor, OH) at 15°C under a 12 hr light/dark (LD)
cycle. Animals were allowed to adapt to laboratory conditions for 3 d before use except in the experiment measuring the 5-HT levels of
animals just after their arrival in the laboratory. Animals used for
sensitization training were kept in individual plastic cages in
recirculating seawater tanks. They were fed dried seaweed every 2-3 d.
All other animals, except those used in feeding latency experiments
(described below), were placed directly in recirculating seawater tanks
(100 gal) and fed romaine lettuce every 2-3 d. Animals were housed in
constant darkness (DD) by transfer to a 30 gal recirculating seawater
tank kept in light-tight housing.
Measurements of levels of 5-HT in hemolymph of Aplysia
living in the field were performed on animals (100-150 gm) captured from tidal pools along the coast of Southern California and immediately sampled (see below). All animals were captured and sampled during the
day in mid-October. Conditions at the site of capture were calm, and
water temperature was ~19°C.
To measure levels of 5-HT in the hemolymph of animals immediately after
arrival into the laboratory, animals were removed from original
shipping containers and equilibrated for 15-30 min. During this
equilibration, animals remained in individually sealed plastic bags,
which were placed in seawater tanks maintained at 15°C. After
equilibration, bags were opened, and hemolymph was immediately sampled
(see below). Animals were then fed (lettuce), transferred to individual
plastic cages, and maintained under standard environmental conditions
(see above).
Long-term sensitization produced by electrical stimulation was
performed as previously described (Goldsmith and Byrne, 1993; Cleary et
al., 1998). Briefly, animals were administered 4 blocks of 10 shocks
over 1.5 hr to one randomly selected side of the animal. Each shock
consisted of a 500 msec pulse of 60 mA applied to the body wall.
Release of ink and opaline, defensive secretions caused by noxious
stimuli, were reliably elicited with this training protocol (Goldsmith
and Byrne, 1993). Baseline hemolymph samples were withdrawn 0.5 hr
before the first stimulus. Other samples were withdrawn at times
indicated in the text. Single animals were sampled multiple times
during training (as noted in Results), but in all other cases hemolymph
was sampled only once from an animal. Control animals did not receive
electrical stimulation, but were handled identically to sensitized
animals in all other respects.
Siphon withdrawal duration was measured, and hemolymph was subsequently
sampled to correlate changes in duration of siphon withdrawal with
changes in levels of 5-HT in the hemolymph. Teflon-coated silver wire
electrodes were implanted into the posterior region of the tail, as
previously described (Scholz and Byrne, 1987; Goldsmith and Byrne,
1993), for delivery of electrical stimuli (20 msec, AC shock). Baseline
hemolymph samples were withdrawn 30 min before threshold determination.
Several test stimuli were administered to determine threshold current
required for eliciting siphon-withdrawal. Siphon withdrawal was
assessed at two-times the threshold current required to elicit the
reflex. Immediately after determining the baseline siphon withdrawal
duration for each side, electrical stimulation was administered over
one side of the body wall as above. Siphon withdrawal duration was
measured 24 hr after the end of electrical stimulation, and then
hemolymph was withdrawn immediately afterward. Hemolymph was withdrawn
from the side of the animal that did not receive electrical
stimulation. Withdrawal of hemolymph did not induce long-term
sensitization. Control animals did not receive electrical stimulation,
but were handled identically to sensitized animals in all other
respects. The experimenter who measured siphon withdrawals was blind to whether the animals received electrical stimulation and the side from
which hemolymph was withdrawn.
To measure the half-life of 5-HT in the hemolymph of animals that were
electrically stimulated once 5-HT was elevated, individual Aplysia were sensitized by the standard long-term training
protocol and then placed in 3 l of seawater that contained 250 µM 5-HT (creatine-sulfate complex; Sigma, St.
Louis, MO) for 0.5 hr (Alberini et al., 1994). Hemolymph was sampled
before, immediately after, and then 0.5, 1, and 6 hr after treatment.
Animals were only sampled once during this experiment. Sensitized
animals were used in this experiment because the turnover of 5-HT or
its diffusion through the skin might be altered by the training procedure.
Collection of hemolymph from animals used in this study was performed
using the following protocol. Hemolymph samples (0.5 ml) were withdrawn
from animals using a sterile 16 gauge needle and 1 cc syringe.
Hemolymph was withdrawn from the animal at a level between the
rhinophores and parapodia near the foot. Care was taken to minimize
trauma to the animal. Withdrawal of hemolymph only required ~10 sec
of handling. In addition, removal of hemolymph only elicited inking in
10% of the animals used in this study. Subsequent samples of hemolymph
from animals indicates that withdrawal of hemolymph did not produce
elevation of 5-HT in the hemolymph.
Hemolymph was centrifuged at 4°C for 2 min to pellet hemocytes.
Previous studies have shown that the hemocytes of various invertebrates, including Aplysia, contain 5-HT (Stefano et
al., 1989; Ottaviani and Cossarizza, 1990; Ottaviani et al., 1992; Levenson and Eskin, unpublished observations). Centrifugation did not
elicit release of 5-HT from Aplysia hemocytes, because the
level of 5-HT measured from centrifuged hemolymph was nearly identical
to the level measured from hemolymph diluted with isotonic MgCl2 (180 mM final
concentration) or hemolymph that was passed through a 0.22 µm filter
(data not shown). Cell-free hemolymph was aliquoted, immediately frozen
in liquid N2, and stored at  80°C. Assays were
always performed within 5 d of collection of hemolymph. 5-HT was
measured using a commercially available ELISA (ICN Biochemicals, Costa
Mesa, CA). Hemolymph was processed as plasma according to the
manufacturer's protocol. After acylation, the resulting solution was
diluted with 200 µl of assay buffer. During processing, hemolymph was
exposed to 14% acetone to precipitate proteins. This treatment should
denature any proteins that may bind 5-HT. Thus, total 5-HT in the
hemolymph was measured in all of our assays.
Several control experiments were done to verify that the ELISA was
valid, because the primary antiserum used actually binds to
N-acyl-5-HT. Hemolymph that was not exposed to the acylation reagent did not have detectable levels of 5-HT. No 5-HT was present in
seawater that had been exposed to acylation. Moreover, the ELISA could
accurately measure known amounts of 5-HT added to seawater or hemolymph
(data not shown).
The role of protein synthesis in the long-lasting increase in hemolymph
5-HT caused by electrical stimulation was assessed via injection of
emetine into the intact animal. Baseline hemolymph samples were
withdrawn from animals 0.5 hr before receiving an injection of emetine.
Animals were injected with 1 ml, 9 mM emetine [dihydrochloride; Sigma; dissolved in buffered-filtered seawater (BFSW) with 3% ethanol] per 140 gm body weight 30 min before
electrical stimulation. Injections were estimated to yield ~100
µM emetine in the hemolymph, assuming 65% of the weight
of the animal is hemolymph. This concentration was sufficient to
significantly inhibit protein synthesis (see Results; Schwartz et al.,
1971; Castellucci et al., 1986; Montarolo et al., 1986). Animals
injected with emetine responded to electrical stimulation similar to
control animals; copious amounts of ink and opaline were secreted, and animals withdrew from the site of shock. Animals injected with emetine
became flaccid near the site of injection, but appeared to recover
~3-4 hr after injection. Control animals received a vehicle
injection (3% ethanol in BFSW) in place of emetine. The vehicle did
not appear to affect behavior and/or muscle tone. Electrical
stimulation was performed as described above. Hemolymph was collected
24 hr after the end of electrical stimulation.
The effect of emetine injections on protein synthesis was assayed by
measuring incorporation of [3H]leucine
into protein in both pleural-pedal and abdominal ganglia as previously
described (Raju et al., 1991). In each experiment, two experimental
animals were injected with emetine, and two control animals were
injected with the vehicle (as above). This experiment was repeated
three times. Thirty minutes after injection, pleural-pedal and
abdominal ganglia were removed and exposed to 40 µCi/ml
[3H]leucine (1 mCi/ml; ICN Biochemicals)
in BFSW for 1 hr at 15°C. After exposure to leucine, ganglia were
rinsed 6 times with ice-cold BFSW, blotted dry, and frozen in liquid
N2. Ganglia were homogenized, and incorporation
of leucine into protein was measured as TCA-precipitable counts. This
value was normalized to total counts in the homogenate to adjust for
differences in uptake of leucine by the ganglia.
Latency to feeding posture was measured following the technique of
Kupfermann (1974). After adaptation to the laboratory, animals were fed
to satiation with dried seaweed (Kupfermann, 1974), housed in
individual plastic cages, and fasted for 5 d. On the fifth day,
latency to feeding posture was tested with a piece of seaweed ~2.5
mm2 in area and 0.25 gm in weight. Animals
were only included in this study that had an initial behavioral state
of 0 or 1, as defined by Kupfermann (1974), just before
experimentation. The 0 and 1 states are characterized by an immobile
animal. Rhinophores were brushed with a piece of seaweed, and the
seaweed was then kept in the seawater near its surface. Latency to
feeding posture was designated as the time elapsed between brushing the
rhinophores with seaweed and the display of characteristic "heads-up
feeding posture" (Kupfermann, 1974). Latencies measured at night,
zeitgeber time 18 (ZT18), were performed under dim red light.
Experimental animals received an injection of 1 ml, 3 µM
5-HT (hydrochloride; Sigma) per 150 gm body weight, 20 min before
testing feeding latency. These injections increased levels of 5-HT in
the hemolymph within 5 min to 25.8 ± 4.9 nM, a 5-HT
level normally seen at night. Control animals received an equivalent
injection of seawater. All feeding experiments were performed blind.
Specifically, the investigator responsible for measuring feeding
latency was different from the investigator responsible for injection
of 5-HT.
Analysis of 5-HT from animals versus time spent in the laboratory was
performed with a paired, Student's t test. Time-dependent changes, after electrical stimulation and the effect of emetine on the
24 hr increase in 5-HT were analyzed via two-way ANOVA. Elevation of
5-HT in the hemolymph and rhythms in levels of 5-HT were analyzed for
significance using one-way ANOVA. The relationship between duration of
siphon withdrawal and the magnitude of increase in hemolymph 5-HT was
analyzed via product-moment correlation. Feeding latency at midday and
midnight, and after 5-HT injection was analyzed using a Wilcoxon
two-sample test. All post hoc analyses were performed using
the T-method of unplanned comparisons among means (Sokal and Rohlf,
1995). Statistical significance for all tests was set at
p  0.05.
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RESULTS |
The mean level of 5-HT in the hemolymph during the day taken from
Aplysia  3 d after their arrival to the laboratory was
18.0 ± 0.6 nM (SEM; n = 117), and is comparable to the mean level of 5-HT in the hemolymph
measured from animals in the field (16.7 ± 1.3 nM; n = 17). Interestingly, this
value is also close to the level of 5-HT in the hemolymph of another
mollusc, Helix (Cardot, 1971). This level of 5-HT in the
hemolymph of Aplysia is close to threshold values for a
number of physiological responses (Table 1).
Initially, animals were sampled immediately (15-30 min, between 10:00
A.M. and 12:00 P.M.) and 3 d after arrival into the laboratory, a
time at which animals are normally used in experiments. The levels of
5-HT in the hemolymph of animals that had just arrived were
significantly elevated relative to levels observed after animals had
been in the laboratory for 3 d (Fig.
1; t = 5.32; df = 7;
p < 0.001). Levels of 5-HT in the hemolymph did not
appear to decrease further after animals had been in the laboratory for >3 d (data not shown). The decrease in 5-HT with time in the
laboratory suggested that levels of 5-HT in the hemolymph may be
regulated by environmental stimuli and be indicative of the state of
the animal. Moreover, this observation indicates that animals should be
equilibrated in the laboratory before performing behavioral or cellular
studies. Several factors may have contributed to the increase in
hemolymph 5-HT during shipment of the animals. Animals arrived in the
laboratory after approximately one half day of travel, during which
they were maintained in cold seawater (~4°C) in dark, styrofoam
boxes. Some possible factors that might contribute to the elevated 5-HT
include stress caused by water temperature, handling, or being held in
darkness for a long period of time (see Fig. 3). Further experiments
are required to determine which of these factors, or others, might be
responsible for this change.
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Figure 1.
Levels of 5-HT in the hemolymph decrease with time
after arrival in the laboratory. Concentration of 5-HT in the hemolymph
is plotted as a function of time spent in the laboratory for individual
animals. Hemolymph was sampled immediately after arrival at the lab and
3 d afterward. There was a significant decrease in 5-HT during
this period.
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Diurnal rhythm of 5-HT
Aplysia have circadian locomotor rhythms, and 5-HT
plays an important role in regulation of ocular circadian pacemakers in Aplysia (Jacklet, 1969; Strumwasser, 1973; Kupfermann, 1974;
Lickey et al., 1977; Corrent and Eskin, 1982; Corrent et al., 1982;
Eskin and Maresh, 1982; Colwell, 1990). Therefore, we investigated
whether the concentration of 5-HT in the hemolymph varied with respect to time of day. Hemolymph was sampled from individual animals once at
various times throughout a complete photoperiod. Levels of 5-HT in the
hemolymph were significantly affected by light/dark cycles (Fig.
2; F = 3.54; df = 47; p < 0.01). The average concentration of 5-HT
(18.0 ± 1.2 nM; average of samples from
ZT1-ZT12) in the hemolymph during the day was significantly
(t = 4.53; df = 6; p < 0.0001)
lower than the average value at night (24.4 ± 0.7 nM; average of samples from ZT15-ZT24).
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Figure 2.
Concentration of 5-HT in the hemolymph is
regulated by light/dark cycles. Concentration of 5-HT in the hemolymph
is plotted as a function of time of day. Hemolymph was sampled one time
from each animal maintained in 12 hr LD cycle. Five to eight animals
were sampled at each time point. ZT0 is dawn, ZT12 is dusk. The
concentration of 5-HT shows significant variation as a function of time
of day. Error bars indicate SEM.
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To determine whether changes in the levels of 5-HT observed in a
light/dark cycle were under control of a circadian pacemaker, hemolymph
was sampled in another experiment from individual animals placed in
constant darkness. In these animals, no significant temporal variation
of 5-HT in the hemolymph was observed (Fig. 3; F = 0.38; df = 35; p > 0.9). The mean concentration of 5-HT in the
hemolymph from animals in DD (25.1 ± 0.7 nM) was similar to levels seen at night in the LD
experiment (24.4 ± 0.7 nM). This finding
indicates that the rhythm of 5-HT observed in LD is caused by light
decreasing levels of 5-HT in the hemolymph. Light could lower levels of
5-HT in the hemolymph by affecting levels of enzymes involved in 5-HT
biosynthesis. A similar mechanism explains the inhibition of melatonin
production by light in the mammalian pineal (Deguchi and Axelrod, 1972;
Klein and Weller, 1972; Moore and Klein, 1974). Alternatively, light
could modulate the activity of serotonergic neurons to decrease their
release of 5-HT, light could inhibit enzymes involved in 5-HT
degradation, or light could increase renal clearance of 5-HT.
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Figure 3.
Concentration of 5-HT in the hemolymph does not
change when animals are kept in constant darkness. Each animal was
sampled only once. No significant variation was seen in the
concentration of 5-HT in the hemolymph. In this experiment, 5-HT
remained near the night-time levels that were observed when animals
were maintained under a light/dark cycle (Fig. 2). Four animals were
sampled at each time point.
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Effect of electrical stimulation on 5-HT in the hemolymph
Sensitization in Aplysia appears to be mediated, at
least in part, via 5-HT. Application of 5-HT mimics many of the effects produced by electrical stimulation that produces sensitization (Brunelli et al., 1976; Bernier et al., 1982; Klein et al., 1982; Walters et al., 1983; Hammer et al., 1989; Schacher et al., 1990; Emptage and Carew, 1993; Trudeau and Castellucci, 1993; Emptage et al.,
1996; Zhang et al., 1997), and serotonergic neurons appear to be
necessary for sensitization (Glanzman et al., 1989). We investigated
whether electrical stimulation would release 5-HT and subsequently
increase levels of 5-HT in the hemolymph. Electrical stimulation
consisted of 4 blocks of 10 shocks to one side of the animal, with 0.5 hr between each block of shocks. Hemolymph was collected 0.5 hr before
stimulation and 1 min after each block of stimulation. Changes in
levels of 5-HT in the hemolymph are expressed relative to the level of
5-HT measured 0.5 hr before training. Electrical stimulation
significantly elevated levels of 5-HT in the hemolymph (Fig.
4; F = 21; df = 65;
p < 0.0001). Post hoc analysis indicated
that 5-HT was significantly elevated during training [Fig. 4; minimum
significant difference (MSD; Sokal and Rohlf, 1995) = 15%;
p < 0.05]. In addition, an increase in the level of
5-HT in the hemolymph was seen 1.5 and 24 hr after electrical
stimulation, however no significant increase of 5-HT in the hemolymph
was seen 48 hr after electrical stimulation (Fig. 4).
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Figure 4.
Electrical stimulation elevated 5-HT in the
hemolymph during, 1.5, and 24 hr after stimulation. Concentrations of
5-HT in the hemolymph of experimental animals sampled during, 1.5, 24, or 48 hr after the end of electrical stimulation are plotted relative
to the value of 5-HT for each animal 0.5 hr before initiation of
stimulation. Control animals were handled exactly as animals that were
trained, with the exception that control animals did not receive any
electrical stimulation. Four hemolymph samples were taken during
training; individual values were averaged to yield a concentration of
5-HT in the hemolymph during training. Individual hemolymph samples
were taken 1.5, 24, and 48 hr after training from individual animals
that had not been previously sampled. In this and subsequent
illustrations, an asterisk indicates a significant
(p 0.05) difference relative to control
values.
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If the increase in 5-HT in the hemolymph was related to the behavioral
sensitization caused by noxious stimulation of the body wall, then
increases in 5-HT in the hemolymph should occur in animals that were
sensitized by noxious stimulation. Therefore, siphon withdrawal and
changes in levels of 5-HT in the hemolymph were measured in the same
animal. Unilateral sensitization of the siphon withdrawal reflex was
observed in animals 24 hr after electrical stimulation. The side of the
animals that was trained showed a significant 152 ± 22%
(n = 8; SEM) increase in the duration of siphon
withdrawal compared to the control side, which showed no sensitization
(2 ± 3% increase; t = 6.96; df = 7;
p < 0.001). In these same animals, 5-HT in the
hemolymph significantly increased 20 ± 4% 24 hr after electrical
stimulation (t = 4.6; df = 7; p < 0.005). Furthermore, control animals that received no electrical stimulation showed no long-term behavioral sensitization as measured by
siphon withdrawal duration (9 ± 6% increase, n = 5) and no change in levels of 5-HT in the hemolymph (0 ± 7%).
The magnitude of the change in duration of siphon withdrawal was
significantly correlated with the magnitude of the change in 5-HT in
the hemolymph (Fig. 5; r = 0.66; df = 7; p = 0.05). This result indicates
that changes in the levels of 5-HT in the hemolymph are related to changes in the siphon-withdrawal reflex, and thus may be involved in
behavioral sensitization. In these experiments in which siphon withdrawal and 5-HT were both determined, it is unlikely that the
behavioral testing itself led to the increase in 5-HT in the hemolymph
since levels of 5-HT in the hemolymph did not increase in control
animals which received no electrical stimulation (Fig. 5), and
electrical stimulation alone led to a similar increase in 5-HT when no
behavioral measurements were made (Fig. 4).
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Figure 5.
Increases in the duration of siphon withdrawal
produced by electrical stimulation were correlated with increases in
5-HT in the hemolymph. Animals were electrically stimulated, and 24 hr
later, 5-HT in the hemolymph was measured after determination of the
duration of siphon withdrawal. Behavioral changes were significantly
correlated with changes in levels of 5-HT in the hemolymph. Measurement
of siphon withdrawal did not lead to the elevation of 5-HT in the
hemolymph because electrical stimulation alone without behavioral
measurement also led to elevation of 5-HT in the hemolymph (see Fig.
4).
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The increase we observed in 5-HT in the hemolymph 24 hr after training
could be caused by several factors. An initial increase of 5-HT could
lead to a long-lasting elevation if 5-HT in the hemolymph was stable
and the skin of the animal was impermeable to 5-HT. To ascertain
whether long-term increases in 5-HT could be caused by its persistence
in the hemolymph once it was elevated in sensitized animals, we
investigated how long an increase in 5-HT in the hemolymph would
persist. Sensitized animals were used in this experiment because the
turnover of 5-HT or its diffusion through the skin might be altered by
the training procedure. Animals were sensitized and then bathed in 250 µM 5-HT for 0.5 hr to elevate 5-HT in the hemolymph
(Alberini et al., 1994). Hemolymph was sampled from individual animals
before and at various times after exposure to 5-HT. Immediately after
this treatment, 5-HT in the hemolymph was ~1.4 ± 0.4 µM (Fig. 6;
F = 36.9; df = 16; p < 0.0001;
MSD = 188 µM; p < 0.05).
By 0.5 hr after the end of treatment, 5-HT had returned to baseline
values (Fig. 6). Similar results were obtained when this experiment was
performed in nonsensitized animals and when smaller changes in
hemolymph 5-HT were produced (data not shown). It is possible that the
clearance mechanisms used for micromolar concentrations of 5-HT differ
from those used at the nanomolar concentration. If this is so, there
should be a rapid decrease in levels of 5-HT in the hemolymph from
micromolar to nanomolar, followed by a slower decay back to baseline.
This pattern was not seen in our data. Levels of 5-HT returned to
baseline within 0.5 hr. This suggests that there is no "slow" decay
for 5-HT. Therefore, since increases in 5-HT in the hemolymph produced by in vivo exposure to 5-HT lasted relatively brief periods
of time, long-term sensitization training appears to lead to a
prolonged release of 5-HT from serotonergic neurons or other sources
that lasts at least for 24 hr.
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Figure 6.
Disappearance of 5-HT from the hemolymph.
Concentration of 5-HT in the hemolymph is plotted as a function of
different times before and after an in vivo exposure to
5-HT (Alberini et al., 1994). Hemolymph was sampled from sensitized
animals just before, immediately after, and then 0.5, 1, and 6 hr after
a 0.5 hr treatment of 250 µM 5-HT. The in
vivo treatment increased the concentration of 5-HT to 1.4 ± 0.4 µM. By 0.5 hr after the end of the treatment,
levels of 5-HT returned to baseline values. Three to four animals were
sampled at each time point. Animals were only sampled once. No
significant differences were observed for samples collected 0.5 hr
after treatment or later when compared to baseline values.
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The long-lasting increase in 5-HT in the hemolymph could be produced by
mechanisms similar to those responsible for producing long-term memory
in Aplysia and other organisms. New protein synthesis appears to be a general requirement during the induction phase of
long-term memory (Flexner et al., 1963; Agranoff, 1967;
Barondes, 1975; Agranoff et al., 1978; Davis and Squire,
1984; Castellucci et al., 1986; Goelet et al., 1986; Milner et al.,
1998). Therefore, we investigated whether the 24 hr increase in 5-HT in
the hemolymph was dependent on protein synthesis. The protein synthesis
inhibitor emetine, which has been used for studies both in
vivo and in vitro in Aplysia (Schwartz et
al., 1971; Castellucci et al., 1986; Montarolo et al., 1986; Hegde et
al., 1997; Martin et al., 1997), was injected into animals 0.5 hr
before electrical stimulation. Inhibition of protein synthesis does not
appear to have effects on the neurophysiological properties of
Aplysia neurons (Schwartz et al., 1971) or synaptic transmission (Castellucci et al., 1986; Montarolo et al., 1986). The
concentration of emetine in the hemolymph after injection (1 ml, 9 mM per 150 gm body weight) was estimated to be
~100 µM. The effect of emetine on protein
synthesis was assayed by measuring [3H]leucine incorporation into protein
in ganglia removed from Aplysia 0.5 hr after they were
injected with emetine (see Materials and Methods). Emetine injections
inhibited protein synthesis by 93% (± 2%; n = 3) in
the pleural-pedal ganglia and 91% (± 2%; n = 3) in
the abdominal ganglion. Emetine blocked the increase in 5-HT in the
hemolymph 24 hr after electrical stimulation (Fig. 7; F = 5.42; df = 20; p < 0.02; MSD = 24%; p < 0.05). Furthermore, emetine injection alone did not change levels of
5-HT in the hemolymph (Fig. 7). Although other neural effects cannot be
ruled out, the results suggest that blockage of the long-term increase
in hemolymph 5-HT by emetine was caused by inhibition of protein
synthesis.
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Figure 7.
Inhibition of protein synthesis blocks the 24 hr
increase in hemolymph 5-HT caused by sensitization training. The level
of 5-HT in the hemolymph was measured 24 hr after sensitization
training and compared to baseline values measured 1 hr before training.
Animals were injected with 1 ml, 9 mM emetine (in BFSW with
3% ethanol) per 140 gm body weight 0.5 hr before sensitization
training. The control group received an injection of vehicle (BFSW with
3% ethanol) 0.5 hr before sensitization training. There was a
significant increase in 5-HT in the hemolymph of the control group 24 hr after the end of sensitization training
(p < 0.05). However, animals that received
an injection of emetine before sensitization training did not have
increased levels of hemolymph 5-HT. Also, injection of emetine alone
(Not Trained group) did not alter levels of 5-HT 24 hr
later. The asterisk indicates that the control group was
significantly different from the two groups that received emetine. Each
group consisted of seven different animals.
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Influence of hemolymph 5-HT on feeding
There are many possible functional consequences of changes in the
levels of 5-HT in the hemolymph (for example, see Table 1). To begin
investigating functional consequences of elevations of 5-HT in the
hemolymph, we examined an aspect of appetitive feeding behavior, since
5-HT has been implicated in regulation of feeding behavior (Weiss et
al., 1975; Weiss et al., 1978; Kupfermann et al., 1979).
Aplysia were exposed to dried seaweed, and the time required
to elicit the feeding posture was measured (see Materials and Methods).
Confirming Kupfermann (1974), we found that Aplysia
expressed a diurnal rhythm of latency to assume feeding posture (Fig.
8A). Aplysia
responded much more quickly during the day (13.7 ± 1.9 sec) than
they did at night (97.0 ± 11.8 sec). This rhythm was specific for
food, because stimulation of animals with filter paper failed to elicit
the feeding posture at either time (n = 5; data not
shown). To investigate whether 5-HT in the hemolymph might be involved
in modulating feeding latency, 5-HT was increased in the hemolymph at a
time during the day (ZT6) when 5-HT was normally low. We predicted that
increasing 5-HT during the day would lead to an increase in the latency
to assume feeding posture, similar to what is normally observed at
night when 5-HT is elevated. Experimental animals received an injection of 5-HT during the day that increased levels of 5-HT in the hemolymph within 5 min to 25.8 ± 4.9 nM, a level
normally seen at night (see Materials and Methods; Fig. 2). As
predicted, animals that were injected with 5-HT had significantly
longer latencies to feed than animals injected with saline (Fig.
7B; U = 60; df = 16; p < 0.05).
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Figure 8.
5-HT in the hemolymph regulates feeding.
A, Latency to assumption of feeding posture expressed a
significant diurnal rhythm (Kupfermann, 1974). B,
Injection of 5-HT into the hemocoel of Aplysia in the
middle of the day (ZT6) significantly lengthened the latency to assume
feeding posture. Injections were designed to elevate 5-HT in the
hemolymph to levels normally seen at night. Asterisks
indicate a significant (p < 0.05)
difference relative to values at ZT6.
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DISCUSSION |
This study demonstrates that environmental events that affect the
state of the animal (LD cycles, sensitization training) also regulate
levels of 5-HT in the hemolymph. Furthermore, levels of 5-HT in the
hemolymph were significantly elevated after arrival of the animals in
the laboratory relative to levels measured 3 d later (Fig. 1). The
level of 5-HT in the hemolymph is close to threshold values reported
for several physiological responses (Table 1). Therefore, the changes
in 5-HT we observed, given their magnitudes and persistent nature, are
probably sufficient to produce significant effects on the properties of
neurons. In support of this idea, we found that elevating 5-HT in the
hemolymph from a "day" level to a "night" level led to an
increase in latency to assume feeding posture (Fig. 8). Our findings,
taken together with those of others showing effects of injections of
5-HT on behavior (Palovcik et al., 1982; Mackey and Carew, 1983;
Parsons and Pinsker, 1989), indicate that 5-HT in the hemolymph may
play a humoral role and modulate the behavior of
Aplysia.
Levels of 5-HT in the hemolymph could be regulated via several
processes. A number of possible sites of release of 5-HT into the
hemolymph exist. Electrical stimulation or training of the animal may
acutely release 5-HT from synapses in the CNS or peripheral nervous system and this 5-HT may "spill over" into the hemolymph (Peretz and Estes, 1974; Tritt et al., 1983; Goldstein et al., 1984;
Ono and McCaman, 1984; Kistler et al., 1985; Longley and Longley, 1986;
Rathouz and Kirk, 1988; Zhang et al., 1991; Hernádi et al.,
1992). Also, the eyes are surrounded by a basket of serotonergic fibers
originating from the accessory optic nerve, and the blood vessels of
Aplysia are highly innervated by serotonergic processes (Liebeswar et al., 1975; Takahashi et al., 1988; Alevizos et al., 1989;
Skelton and Koester, 1992). Finally, hemocytes may also release 5-HT
into the hemolymph (Stefano et al., 1989; Ottaviani and Cossarizza,
1990; Ottaviani et al., 1992; Levenson and Eskin, unpublished
observations). Clearance of 5-HT by the kidneys or hemocytes (Goldman
and Schwartz, 1977) may also contribute to the regulation of 5-HT in
the hemolymph.
The possible sites of action of 5-HT in the hemolymph are diverse. In
the nervous system, humoral 5-HT may exert its effects through synaptic
5-HT receptors, or perhaps through extrasynaptic 5-HT receptors located
on cell bodies (Drummond et al., 1980b; Li et al., 1995; Angers et al.,
1998). Humoral 5-HT would reach these sites through delivery via the
open circulatory system and by penetration of the connective tissue
sheath. Humoral 5-HT appears to have ready access to cell bodies, and
perhaps slightly less access to the neuropil (Brunelli et al., 1976;
Kaczmarek et al., 1978; Drummond et al., 1980a; Byrne et al., 1991;
Emptage et al., 1996; Bunge et al., 1997).
Diurnal variation of 5-HT in the hemolymph
The diurnal rhythm of 5-HT in the hemolymph may play a role in
regulating or modulating daily behaviors. In several invertebrates, 5-HT and other neurohormones have been shown to be important modulators of behavior or the neuronal circuitry underlying those behaviors (Poon,
1980; Livingstone et al., 1980; Willard, 1981; Schwarz et al., 1984;
Claassen and Kammer, 1985; Marder et al., 1986; Mulloney et al., 1987;
Tuersley and McCrohan, 1987; Nusbaum and Marder, 1988; Mangan et al.,
1994; Malyshev et al., 1997). Feeding behavior in Aplysia
undergoes daily changes (Kupfermann, 1974; Fig. 8A),
and we have found that those changes correlate with the level of 5-HT
in the hemolymph (Fig. 8A). Specifically, when levels
of 5-HT in the hemolymph were low, latency to feed was low. When levels
of 5-HT in the hemolymph were high, feeding latency was high. Moreover,
when levels of 5-HT in the hemolymph were elevated to nighttime levels
by injection of 5-HT during the day, latency to feed increased (Fig.
8B). These results suggest that one function of the
diurnal rhythm of 5-HT in the hemolymph may be to regulate feeding
behavior. Further tests of this specific idea will require examining
feeding latency when levels of 5-HT in the hemolymph are lowered or
5-HT receptors are blocked in vivo during the night.
Another daily behavior that could be regulated by 5-HT in the hemolymph
is locomotor activity. Injections of 5-HT increase locomotor activity
(Palovcik et al., 1982; Mackey and Carew, 1983; Parsons and Pinsker,
1989; see also Adamo and Chase, 1991), and the diurnal pattern of
locomotor activity peaks during the day (Lickey et al., 1977). However,
the levels of 5-HT in the hemolymph are lowest during the day, when
activity is highest. In the studies that examined the effects of
injection of 5-HT into Aplysia, behavior was measured no
more than 15 min after injection, and the effective concentration of
5-HT after injection was much higher than levels observed in this
study. Thus, lower levels of 5-HT might have opposite effects than high
doses. In addition, acute treatments may produce different effects than
longer term treatments. Indeed, modulation of pituitary lactotroph
prolactin secretion by endothelin-1 is either inhibitory or stimulatory
depending on whether the cells are exposed to an acute or chronic
treatment of dopamine, respectively (Kanyicska et al., 1995, 1997).
Elevation of 5-HT caused by electrical stimulation
A significant increase in levels of 5-HT in the hemolymph was
observed in our studies during electrical stimulation that leads to
sensitization (Fig. 4). This finding is the first evidence that release
of 5-HT actually accompanies sensitization training in
Aplysia and, as such, further suggests that 5-HT plays a
role in mediating the effects of sensitization training. Moreover, the
increase in siphon withdrawal duration was correlated with the
magnitude of the increase in 5-HT in the hemolymph (Fig. 5). This
further indicates a relationship between elevation of 5-HT in the
hemolymph and sensitization.
A significant increase in 5-HT in the hemolymph also occurred 1.5 and
24 hr after electrical stimulation (Fig. 4). The long-lasting increase
in 5-HT in the hemolymph appeared to be caused by a persistent release
of 5-HT from serotonergic neurons or other sources, because the
half-life of 5-HT in the hemolymph was relatively short (Fig. 6). These
results demonstrated that a single 1.5 hr block of electrical stimulation led to an increase in the levels of 5-HT in the hemolymph that persisted for 24 hr. Multiple blocks of such stimulation performed
over several (2-4) days leads to sensitization that persists for weeks
(Pinsker et al., 1973). It would be interesting to examine the effect
of multiple blocks of electrical stimulation on the duration of the
increase in 5-HT to determine whether the increase in 5-HT in the
hemolymph might persist even longer than a day.
Formation of long-term memory in Aplysia and many other
organisms requires protein synthesis during the induction phase of learning (Davis and Squire, 1984; Goelet et al., 1986; Milner et al.,
1998). Similarly, many of the long-lasting changes in the
Aplysia nervous system believed to be responsible for memory have been shown to be dependent on protein synthesis (Castellucci et
al., 1986; Montarolo et al., 1986; Manseau et al., 1998; Bailey et al.,
1992; O'Leary et al., 1995). Like these other processes, the 24 hr
increase in 5-HT in the hemolymph induced by sensitization training was
blocked via inhibition of protein synthesis (Fig. 7). The similarities
in time course and requirement of new protein synthesis between changes
in levels of 5-HT in the hemolymph and long-term changes responsible
for memory indicate that changes in 5-HT in the hemolymph might be one
of the constellation of factors that contribute to various forms of
long-term memory in Aplysia. Moreover, some of the same
molecular mechanisms (Abel et al., 1998) involved in the formation of
long-term memory produced by sensitization training may also be
involved in producing the long-lasting increase in 5-HT in the hemolymph.
What is the role of the elevated levels of 5-HT in the hemolymph or the
proposed persistent increase in activity of serotonergic neurons
produced by sensitization training? An increase in circulating 5-HT
produced by training may increase the sensitivity of peripheral mechanoreceptors and photoreceptors (Eskin and Maresh, 1982; Billy and
Walters, 1989). Elevations of 5-HT may also elevate heart rate and
increase vascular tone (Liebeswar et al., 1975; Krontiris-Litowitz et
al., 1989). Nonessential behaviors may be inhibited by increases in
circulating 5-HT. Humoral effects of 5-HT could contribute to, or even
cause in some cases the behavioral sensitization seen immediately after
sensitization training and 24 hr later. It is important to note that in
some instances, including the present study, electrical stimulation
leads to long-term unilateral sensitization (Scholz and Byrne, 1987;
Goldsmith and Byrne, 1993; Cleary et al., 1998). In these cases,
humoral 5-HT could not contribute to the induction of sensitization
unless the unilateral sensitization was produced by an associative
mechanism involving humoral 5-HT, and neural activity on the side of
the animal that was trained.
The increase in humoral 5-HT during training also could strengthen
memory formation by helping to induce facilitation of the sensorimotor
synapse and interneuronal connections (Bailey et al., 1981; Trudeau and
Castellucci, 1993; Xu et al., 1995). This possible role of an increase
in humoral 5-HT would be similar to the proposed enhancing role of
catecholamines in memory formation of mammals (for review, see McGaugh
et al., 1996). In the rat, elevation of epinephrine in the plasma
during aversive training enhances memory acquisition (McCarty and Gold,
1981; Galvez et al., 1996).
5-HT was also elevated in the hemolymph 1.5 and 24 hr after
sensitization training of Aplysia. The prolonged elevation
of 5-HT or the activation of neurons causing the prolonged elevation of
5-HT may aid in the induction of facilitation and participate in the
mechanism maintaining facilitation for long periods of time. Thus, the
prolonged release of 5-HT might be part of the "memory" for
long-term sensitization. Moreover, the prolonged release of 5-HT might
potentiate future training as epinephrine does in the rat, and be
relevant for additional training that lasts for several days.
Subsequent training sessions of Aplysia on following days
would be done in the presence of heightened activity of serotonergic
neurons or elevated 5-HT in the hemolymph. Prolonged increases in
neuronal activity and levels of humoral factors present a possible
mechanism for strengthening formation of memories when learning takes
place over a relatively long time scale, such as days or weeks.
Our findings raise the possibility that 5-HT may act as a
neuroendocrine molecule in the hemolymph to regulate the behavioral state of Aplysia. Humoral 5-HT may directly mediate some
events, whereas it may act as a modulator in other cases. The acute
changes in levels of 5-HT in the hemolymph may have different roles
than the chronic presence of 5-HT in the hemolymph (Kanyicska et al., 1995, 1997). It is also possible that 5-HT released at synapses because
of persistent neural activity induced by sensitization training may be
more or less important than humoral 5-HT. Ultimately, it will be
important to determine the role of 5-HT in the hemolymph and
distinguish its humoral role from its neurotransmitter role. It will
also be important to determine the site of release of 5-HT into the
hemolymph and the mechanisms responsible for persistence of 5-HT in the
hemolymph after sensitization training.
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FOOTNOTES |
Received Feb. 4, 1999; revised June 7, 1999; accepted June 29, 1999.
This work was supported by grants from the National Institutes of
Health (MH41979 and NS28462 to A.E. and NS19895 to J.H.B.). We thank R. Fernandez, S. Hattar, K. Herynk, G. Kelly (Marinus, Inc.), G. Patterson, and J. Selcher for technical assistance.
Correspondence should be addressed to Arnold Eskin, University of
Houston, Department of Biology and Biochemistry, 3201 Cullen Boulevard,
HSC 401, Houston, TX 77204-5513.
| |
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TOP
ABSTRACT
INTRODUCTION
