ABSTRACT
The modulation of the central pattern generator for swimming
in Melibe leonina by serotonin is the focus of this paper. Changes in
behavior and neuronal firing patterns were monitored during the
application of serotonin in order to elucidate how a neural circuit can
be modified by a chemical transmitter. Behavioral observations
indicate that micromolar concentrations of serotonin cause an
increase in swimming frequency immediately after application in a
whole animal preparation. Recordings from Interneuron 1 in the
swimming circuit show that serotonin causes a decrease in burst
duration and an increase in both instantaneous frequency and
spikes/second/burst immediately upon application. These three
parameters are all consistent with a transient acceleration and
tightening of the burst firing pattern of Interneuron 1. Äfter
washout, a second application of serotonin to the same ganglia
results in no significant changes in the firing pattern of Interneuron
1. Similar results were seen in a whole animal preparation; after 1-3
applications of serotonin no changes in swimming frequency occurred
upon additional application. Two possible mechanisms are given to
explain the effects of serotonin. One explanation is that serotonin
works as hormones affecting all the cells within the swimming neural
circuit. The other explanation is that serotonin works as a
neurotransmitter at specific synapses between the cells of the
circuit.
INTRODUCTION
The concept of a central pattern generator, or CPG, has played a
key role in the understanding of how rhythmic motor patterns are
generated. It is theorized that discrete neural circuits present in the
central nervous system, operating in the absence of proprioceptive
feedback, control the stereotypical motor patterns seen in many
behaviors (Harris-Warrick et al., 1989). Studies of systems like that
of the crustacean digestive system and stomatogastric ganglion have
provided insight into, not only the control of a rhythmic motor pattern
by a CPG, but also the modulation of a CPG by neuroactive substances
like serotonin and dopamine, and the peptide hormones proctolin and
red pigment concentrating hormone (RPCH) (Harris-Warrick et al.,
1989). For the studies of chemical modulation of rhythmic behavior,
alternative systems can give new insight into understanding CPG
control. One such system consists of the swimming motor pattern of
Melibe leonina.
Melibe swimming behavior has been characterized by Stuart
Thompson (unpublished observations) and by Anne Herst (1968). The
swimming motor pattern consists of alternating contractions of the
body wall when the foot is dislodged from its substrate. These
alternating contractions cause the animal to bend from side to side
(Figure 1). Although Melibe exhibit several other behaviors including
feeding, crumple, and shrug responses (Diane Scott, personal
communication), it is the swimming behavior which has been a
primary focus of previous studies.
The neural circuit, or CPG, underlying the control of swimming
was studied and modeled by Thompson (personal communication). His
model centers on two pairs of interneurons. One cell of each pair,
Interneuron 1, is located on the dorsal surface of the pleural ganglia,
and the other cell, Interneuron 2, is located on the dorsal surface of
the pedal ganglia (Figure 2). The two pairs of interneurons synapse on
motoneurons in the pedal ganglia, which innervate the body wall of
the animal and cause the swimming contractions. Interneuron 1 and
Interneuron 2 form mutually inhibitory synapses with the
interneurons of the other pair while electrically synapsing on each
other (Figure 3). The pattern of connections between the interneurons
of this CPG results in an oscillating burst firing pattern in which one
pair fires a burst while inhibiting the other pair and vice versa.
Modulation of the swimming CPG by serotonin (5-HT) is the
focus of this paper. Changes in behavior and neuronal firing patterns
were monitored during application of 5-HT in an effort to elucidate
how the output of one neural circuit can be modified by a chemical
transmitter that is widely found in molluscan nervous systems.
MATERIALS AND METHODS
Melibe leonina were collected from Monterey Bay using SCUBA.
They were maintained at Hopkins Marine Station in flowing filtered
sea water at a temperature of 10-15° C. As a precautionary measure,
the sea water was degassed by filtering it through a column of
marbles to quard against bubble disease. The tanks were sheltered
from bright sunlight and were filled with kelp to provide the Melibe
with a somewhat natural habitat. The animals were fed mysids/brine
shrimp every other day. The mysids were collected by dip-net in kelp
beds.
For the first few weeks, the Melibe exhibited normal behavior
(swimming, crumple, feeding, shrug, etc.), and during this time,
experimental animals were selected at random from the individuals in
the tanks. After 3-5 weeks, the Melibe began to exhibit abnormal
behavior such as lethargic swimming and no feeding. From this point
onward, experimental animals were selected based on their ability to
exhibit normal, vigorous behavior.
Preparation of Animals
lwo types of experimental preparations were used. Behavioral
observations were made using a whole animal preparation developed
by Diane Scott and Stuart Thompson. An incision was made on the
dorsal surface of the animal so that the head ganglia were exposed.
An additional incision was made just to the right of the head ganglia
and esophagus. A metal stage was inserted through this second
incision so that it rested directly ventral to both the esophagus and
head ganglia. The head ganglia and esophagus were then pinned to this
metal stage so that the ganglia were stabilized. Once the ganglia
were stabilized, the animal hung suspended in a tank of cold (13° C)
running sea water. While suspended, Melibe exhibit normal swimming,
crumple, and shrug behaviors. This preparation allowed behavioral
observations to be made while applying solutions to the head ganglia.
A second preparation was used for extracellular suction
electrode recording. The head ganglia and a portion of esophagus were
surgically removed from the animal by cutting all nerve roots to and
from the ganglia and by cutting through the esophagus just anterior
and posterior to the head ganglia. Particular care was taken to make
sure the circumesophageal connectives between the right and left
pedal ganglia were not damaged.
Once removed from the animal, the ganglia and esophagus were
bathed in a cold (14° C) artificial sea water solution (ASW)
containing 470 mM Nacl, 10mM KCI, 50mM MgCl», 10mM Cacl», and
10mM Hepes (pH 7.8). The ganglia were placed in a perfusion chamber
and pinned to a Sylgard base in the chamber. The perfusion chamber
was kept at approximately 14° C by a cold plate.
Drug Application
The effect of serotonin (5-HT) was studied in whole animal
preparations and isolated ganglia preparations. In the whole animal
preparation, 10 ml of 10°2 M 5-HT was pipetted directly onto the
stabilized head ganglia of the animal. This concentration was chosen
so that some of the drug would reach the neuronal synapses in the
ganglia without eliciting non-specific responses. Solutions of 5-HT
creatinine sulfate were made in ASW + 0.1% ascorbic acid (pH 7.5).
Ascorbic acid was needed to preserve the stability of the drugs in
solution. Even with the addition of ascorbic acid, 5-HT solutions
were stable for only a few hours.
10 ml of 10-9 M 5-HT solution was applied directly to the
isolated ganglia preparation. Solutions of 5-HT were again made up in
ASW + 0.1% ascorbic acid (pH 7.5). Drug application was followed
immediately by a 10 ml washout with ASW.
Extracellular Recordings
Recordings were made directly over Interneuron 1, which is
located in the pleural ganglia (Figure 2). Recordings from Interneuron
1 were only done in the isolated ganglia preparation. Interneuron 1
was located by its topographical location in the pleural ganglia and by
its burst firing pattern (Thompson, unpublished data; Wesseling,
personal communication). The isolated ganglia preparation was
chosen for recording because of its convenience and because isolation
of the ganglia has been shown to have no effect on the burst firing
pattern of Interneuron 1 (Thompson, unpublished data).
Extracellular suction electrode recording allowed quick and
easy location of Interneuron 1 by monitoring its firing pattern.
Electrodes consisted of glass micropipettes with fire-polished tips.
Neuronal firing activity was monitored on a digital oscilloscope
(Nicolet) and recorded on a chart recorder (Gould/Brush).
RESULTS
Behavioral Observations
Live animals were observed in tanks of cold sea water devoid
of substrate so that their natural swimming behavior could be studied
and compared to the swimming behavior seen in whole animal
preparations. When dislodged from substrate with a tactile stimulus,
the Melibe were observed to crumple and slowly sink before initiating
swimming. Once swimming was initiated, the animals were observed
to swim without pausing until their foot touched substrate. A pause
was defined as a momentary stop at either midline or to one side
during a swim cycle. Observed swimming frequencies were fairly
constant with a mean of 15.5 + 2.6 cycles/minute (n=19; all values
given with s.d.) (Figure 4). Swimming frequency was defined as the
number of full alternating left and right bending cycles per minute.
Observations using the whole animal preparation were made to
assess pre-serotonin swimming behavior and frequency. Swimming
was only observed when the animal was suspended without a
substrate touching its foot. When a substrate was touching the foot,
the animal crawled on its surface. Dislodging of the foot from
substrate by a tactile stimulus resulted in crumple behavior which
was usually followed by swimming. Swimming frequencies ranged
from 4 to 22 cycles/minute (Figure 5) with an average of 12.7 + 5.3
cycles /minute (n=25). Maximum swimming frequencies resulted from
vigorous left and right contractions without pauses. Minimum
swimming frequencies resulted from pauses between and within
cycles. Öften, an animal was observed to swim for a few cycles, to
pause at midline or to one side, and then resume swimming for a few
more cycles before pausing again. The resumption of swimming from
midline was observed to occur both in-phase (left, pause, right) and
out-of-phase (left, pause, left) without apparent pattern.
Behavioral Changes After 5-HT Application
5-HT application to the stabilized ganglia of a swimming
Melibe in the whole animal preparation resulted in an increase in
swimming frequency immediately upon application in 5 of 7 animals
(Figure 6). There was an average increase in swimming frequency of
181% upon 5-HT application. Swimming contractions were more
vigorous as defined by tight right and left bends of the body wall so
that the cerata touched the oral hood (Figure 1), after the application
of serotonin compared to those before 5-HT.
5-HT application to the stabilized ganglia of a Melibe standing
on substrate resulted in writhing-consisting of alternating left and
right contractions of the body wall reminiscent of swimming. When
the foot was dislodged from the substrate using a tactile stimulus,
vigorous swimming ensued. If only the posterior portion of the foot
were attached to substrate, the animal would writhe until it
dislodged itself from the substrate. Äfter successive 5-HT
applications to the same animal, no noticeable behavior changes were
seen, i.e. contractions were no longer produced while the animal was
standing on substrate, and swimming frequency was no longer
increased.
Applications of 10 ml sea water alone or 10 ml ASW + 0.1%
ascorbic acid (pH 7.5) to the stabilized head ganglia of a swimming
Melibe were also done to control for movement or vehicle artifact. In
7 of 10 trials, neither sea water nor ASW + 0.1% ascorbic acid
resulted in an increase in swimming frequency. Only 3 of 10 control
applications resulted in an increase in swimming frequency with an
average increase of 169%. Application of control solutions to Melibe
standing on substrate also resulted in no noticeable behavioral
changes.
Effects of 5-HT Application in the Isolated Ganglia
Three parameters of the burst firing pattern of Interneuron 1
(Int 1) were analyzed. Burst duration is defined as the time interval
from the first spike of a burst to the last spike of a burst.
Instantaneous frequency is defined as 1/period where the period is
the time interval from the first spike of a burst to the first spike of
the next burst. Spikes/second/burst is used as a way of analyzing
how many spikes were occurring in a burst independent of burst
duration.
Application of 5-HT to Interneuron 1 in an isolated ganglia
resulted in an immediate statistically significant decrease in burst
duration and increase in instantaneous frequency. The most dramatic
example of this is seen in Figures 7, 8a, and 8b where serotonin
application results in a decrease in average burst duration from 10.8
+1.4 sec. to 3.1 + 0.6 sec. (Pc 0.001, df=19; df defined as degrees of
freedom) and an increase in instantaneous frequency from 3.1
cycles/minute to 9.0 cycles/minute (P« 0.001, df=18). Once the burst
duration dropped, it began to slowly increase; and once the
instantaneous frequency increased, it began to steadily decrease.
Although both trends were significant, neither parameter returned to
its pre-serotonin value.
Serotonin also had a significant effect on the third parameter,
spikes/second/burst (sp/s/b). In one recording, 5-HT application
resulted in an increase in the number of spikes/second/burst from
14.0 + 1.8 sp/s/b to a maximum of 16.9 + 0.8 sp/s/b (P« 0.01, df=28).
From this maximum value, the number of spis/b decreased to a
pre-serotonin level.
Consecutive applications of serotonin to the same isolated
ganglia preparation resulted in no significant changes in the three
parameters after a second application. As seen in Figures 9a, b, and c.
no significant increases or decreases were seen in burst duration,
instantaneous frequency, or spikes/second/burst upon the second
application of 5-HT.
A control was also done using ASW + 0.1% ascorbic acid as an
applied solution. The control solution was observed to have an
opposite effect on burst duration as 5-HT. Burst duration increased
from 2.1 +0.2 sec. to 2.7 + 0.1 sec. (P« 0.001, df=38). The control
solution was also observed to increase spikes/second/burst from 11.6
+1.7 sp/s/b to 15.2 + 0.4 sp/s/b (P« 0.001, df=18). Although this
effect is analogous to that seen with 5-HT, the time courses of the
two effects are different. Serotonin caused an increase in spis/b for
about 20 cycles while the control solution of ASW + 0.1% ascorbic
acid resulted in an increase for 5 cycles. Serotonin effects on sp/s/b
were seen immediately upon application of 5-HT while the effects of
the control solution were not seen until it had already been washed
out with ASW.
DISCUSSION
The aim of this study was to explore the possibility that
serotonin had a modulatory effect on the central pattern generator for
swimming in Melibe leonina. Both behavioral observations and
extracellular recordings during the application of 5-HT showed this to
be true.
In the whole animal preparation, serotonin increased swimming
frequency immediately after application with an average increase of
181%. Serotonin was also observed to initiate alternating left and
right body wall contractions if the animal was standing on substrate,
a state in which swimming is normally inhibited.
These observations suggest that serotonin can cause the
initiation of swimming behavior. This indicates that serotonin may
activate the CPG for swimming from a quiescent state. This type of
excitatory activity has precedent in the crustacean stomatogastric
ganglion. In this system, proctolin and red pigment concentrating
hormone have been shown to strongly activate the pyloric system of C.
borealis in quiescent preparations (Marder, 1989).
Extracellular recordings from Interneuron 1 have shown
serotonin to have a modulatory effect on swimming CPG firing
patterns. In the isolated ganglia preparation, serotonin had an
excitatory effect on Int. 1. Of the three parameters studied, serotonin
affected all in an excitatory way. Burst duration was shown to
decrease while instantaneous frequency and spikes/second/burst
were both shown to increase. Thus, serotonin excites the swimming
CPG by tightening the bursting pattern. Similar modulation of a CPG
is seen in the crustacean stomatogastric ganglion (STG) upon the
application of proctolin. In this system, Marder has shown that
proctolin can produce increases in cycling frequency in slow cycling
preparations in addition to increases in the number of action
potentials/burst from the LP neuron, an integral neuron in the pyloric
neural circuit (1987).
Based on behavioral observations and extracellular recording,
serotonin seems to have a state-dependent effect on the swimming
CPG. Modulation of the CPG activity depends on its state of activity
when serotonin is applied. If the CPG is quiescent, serotonin
activates it. If the CPG is exhibiting a burst firing pattern, serotonin
tightens the bursts and increases the frequency.
There are two ways in which serotonin could be acting as a
transmitter on the swimming neural circuit. One explanation is that
serotonin has a hormonal, or neuromodulatory, effect on the CPG.
Serotonin may be released locally in the pedal ganglia neuropil where
processes from all the neurons in the neural circuit are present. Once
released, the chemical could locally diffuse and affect all the cells.
This type of local release system and hormonal action is analogous to
that proposed to explain the role of 5-HT in the crustacean STG
(Marder, 1987) In this system, serotonin has been shown to influence
the STG of all species. In P.interruptus, Beltz has theorized that
serotonin plays a hormonal role (reviewed by Marder, 1987).
An alternative explanation for serotonin's actions on the
swimming CPG is that 5-HT acts on specific synapses within the
circuit. Serotonin may act as a neurotransmitter at a synapse
between Interneuron 1 and another interneuron resulting in a change
in the firing properties of Int. 1 and the firing pattern of the circuit.
In either case (neuromodulator or neurotransmitter), serotonin
may change the synaptic efficacy within the circuit. 5-HT could
increase the strength of synapses within the circuit, thereby
tightening the bursts and causing the excitatory effects seen in this
study. In analogy, the monoamines serotonin, dopamine, octopamine,
and histamine, have been shown to change the relative strength of a
specific chemical or electrical synapse within crustacean neural
circuits (Harris-Warrick, 1989).
In addition to the excitatory effects of 5-HT on the swimming
CPG, the results given here suggest that the serotonin receptors
within the circuit can be desensitized. This hypothesis is supported
by the fact that after 1-3 applications of 5-HT to the whole animal
preparation, no noticeable behavioral changes are seen in response to
later applications. Additional evidence for this hypothesis of
receptor desensitization is that a second perfusion of 5-HT on the
same isolated ganglia results in no significant change in the three
parameters of burst firing pattern studied.
The mechanisms underlying the serotonergic modulation of the
swimming CPG of Melibe are complex and leave ample room for
fürther experimentation. Additional studies will elucidate the
specific location and mechanism by which serotonin acts on this CPG.
Further experimentation on the effects of additional neuroactive
substances and peptides on the swimming CPG will also lend insight
into how neural circuits can be modulated to cause changes in
behavior and rhythmic motor patterns.
LITERATURE CITED
Harris-Warrick RM, Johnson BR (1989): Motor pattern networks:
Flexible foundation for rhythmic pattern production. In T.J.
Carew and D.B. Kelley (eds): Perspectives in Neural Systems and
Behavior. New York: Alan R. Liss, Inc., pp 51-71.
Hurst A (1968): The feeding mechanism and behaviour of the
opisthobranch Melibe leonina. Symp. zool. Soc. Lond.
22:151-166.
Marder E (1987): Neurotransmitters and neuromodulators. In A.I.
Selverston and M. Moulins (eds): The Crustacean Stomatogastric
System. Berlin: Springer-Verlag, pp 263-300.
Marder E, Nusbaum MP (1989): Peptidergic modulation of the motor
pattern generators in the stomatogastric ganglion. In T.J.
Carew and D.B. Kelley (eds): Perspectives in Neural Systems and
Behavior. New York: Alan R. Liss, Inc., pp 73-91.
FIGURE LEGEND
Figure 1. Dorsal view of vigorous swimming in Melibe.
Figure 2. Melibe head ganglia (dorsal view). Interneuron 1,
Interneuron 2, and specific ganglia are labeled.
Figure 3. Circuit model of the neuronal pattern generator for
swimming in Melibe. The model centers around 4
interneurons (L&R Int. 1's and L&R Int. 2's) which form
chemical synapses on antagonistic (Ant.) and synergistic
(Syn.) motorneurons. Circles indicate inhibitory
synapses; arrows indicate excitatory synapses. The
direct lines between ipsilateral Int. 1's and Int. 2's
indicate electrical connections.
Figure 4. Swimming frequencies of intact, free-swimming Melibe.
Average swimming frequency observed = 15.5 +2.6
cycles/minute.
Figure 5. Swimming frequencies of Melibe in the whole animal
preparation before serotonin application. Average
swimming frequency observed = 12.7 + 5.3
cycles/minute.
Figure 6. The effects of serotonin on the swimming frequency of
Melibe. Data were taken in the whole animal preparation.
The graph shows data from one representative animal.
Figure 7. The effects of serotonin on the burst firing pattern of
Interneuron 1. The recording was made in an isolated
ganglia. A 10-2 M solution of 5-HT was applied at the
arrow.
Figure 8a. The effects of serotonin on burst duration. Data were
taken by extracellular recording from Interneuron 1 in
an isolated ganglia. A solution of 10°9 M 5-HT was
applied at the arrow. Average burst duration before
5-HT application was 10.8 + 1.4 seconds. Average burst
duration immediately after application was 3.1 +0.6
seconds. Data shown are from a representative animal.
Figure 8b. The effects of serotonin on instantaneous frequency.
Data were taken by extracellular recording from
Interneuron 1 in an isolated ganglia. A solution of
10•° M 5-HT was applied at the arrow. Average
instantaneous frequency before 5-HT application
was 3.1 cycles/minute. Average instantaneous
frequency immediately after application was 9.0
cycles/minute. Data shown are from a representative
animal.
Figure 9a. The effects of two applications of serotonin on the
same ganglia with respect to burst duration. Data were
taken by extracellular recording from Interneuron 1 in
an isolated ganglia. A solution of 10°2 M 5-HT was
applied at the arrow. Data shown are from a
representative animal.
Figure 9b. The effects of two applications of serotonin on the
same ganglia with respect to instantaneous frequency.
Data were taken by extracellular recording from
Interneuron 1 in an isolated ganglia. A solution of
10° M 5-HT was applied at the arrow. Data shown are
from a representative animal.
Figure 9c. The effects of two applications of serotonin on the
same ganglia with respect to spikes/second/burst.
Data were taken by extracellular recording from
Interneuron 1 in an isolated ganglia. A solution of
10°9 M 5-HT was applied at the arrow. Data shown are
from a representative animal.
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