Abstract
The snail Calliostoma canaliculatum secretes a yellow mucous ('Yellow Stuff' or YS) in
response to attack from predatory starfish Pisaster giganteus and Pycnopodia helianthoides.
Previous studies have shown that YS inhibits the opening of voltage-gated potassium channels,
and the active component responsible for this action is proposed to be a serotonin dimer
derivative, 6-bromo-2-mercaptotryptamine. Because serotonin is a crucial neurotransmitter in
invertebrates, components of YS may also have an effect on the behaviour of neural networks
where serotonin plays a role. Several lines of experiments were carried out to identify any
possible relationships between the actions of these two compounds. Behavioural studies on the
brittle star Ophiopteris papillosa, showed a similar response to both these compounds -
immediate arm retraction. Results from these experiments established a biologically relevant
concentration of YS for further testing. Control experiments carried out in Shaker potassium
channels expressed in Xenopus oocytes demonstrated that at this concentration, YS did not lead
to a slowing of K-current activation. Additional experiments were performed using the buccal
ganglia of Aplysia californica. B cells in this ganglia contain serotonin receptors and many of
the cells and synaptic connections are modulated by serotonin. Single electrode recordings from
B cells after application of serotonin demonstrated a marked increase in spontaneous activity
(action potentials and post-synaptic potentials). Subsequent application of YS significantly
decreased this activity, and further application of serotonin after YS had been washed away,
renewed activity. This suggests that in the neural circuits involved in stimulation by serotonin,
YS may function as an antagonist, and therefore Yellow Stuff may be acting in invertebrates via
neuromodulatory pathways that involve serotonin.
2
Introduction
The gastropod Calliostoma canaliculatum which lives in the kelp forest of Monterey Bay
has an interesting and aggressive escape mechanism that distinguishes it from other snails of this
genus. In order to escape from its predators, the seastars Pycnopodia helianthoides and Pisaster
giganteus, C. canaliculatum, it will secrete a yellow mucous (Yellow Stuff or YS), from its
hypobranchial gland, that causes the seastar to retract its tube feet or to physically move away
from the exudate. Previous studies (Bryan et al, 1997) focused on the behavioural response of
the seastars to this exudate, but attempts at purifying and identifying this compound were
unsuccessful. It was only recently that an active component of Yellow Stuff was identified (Sack
et al) to be 6-bromo-2-mercaptotryptamine, a serotonin dimer derivative (fig 1). This compound
affects Shaker potassium channels expressed in Xenopus oocytes. The most prominent effects of
6BrMT (or raw Yellow Stuff) are a marked slowing of activation kinetics and a reduction of
peak potassium current amplitude. However, there have been no tests of either Yellow Stuff or
6BrMT on any serotonergic system despite the obvious structural similarity of serotonin and
6BTMT.
The goal of this investigation was to demonstrate a relationship between Yellow Stuff
and serotonin using a variety of different lines of experiments, and more specifically to
determine if Yellow Stuff was behaving in an agonistic or an antagonistic manner in relation to
serotonin.
Materials and Methods
Specimens:
Specimens of Calliostoma canaliculatum and Ophiopteris papillosa were collected from
Monterey Bay by Sealife Supply (Sand City, CA). Pycnopodia helianthoides was collected from
the Great Tide Pool in Pacific Grove, CA. Aplysia californica were from the National Resource
for Aplysia, University of Miami, FL.. Immediately after arrival all organisms were placed in
separate holding tanks with circulating natural seawater at 12°C.
Yellow Stuff Extraction:
In order to extract the hypobranchial gland, the snail was anaesthesised in a solution of
7.5% MgCl2 in dH2O and natural seawater (mixed in a 1:2 ratio) for 15 minutes. The shell of the
snail was then cracked with a vise, and the snail was pinned down in order to extract its gland.
The average mass of a gland was 0.040g. 0.8ml of natural seawater was then added and the
gland was homogenized. The supernatant was then removed and centrifuged at 12,000 RPM for
15 minutes. This solution of YS had a concentration of 0.05g/ml of NSW. This solution was
fürther diluted by 100 times with seawater for use in the experiments below. All solutions were
kept at 4°C until ready for use.
Seastar response to Yellow Stuff and serotonin:
Individual Ophiopteris papillosa were placed in a plastic container measuring 15cm by
20cm by 1Ocm containing 1800ml of natural seawater. Äfter each assay, the container was
rinsed and the seawater replaced. The seastars acclimated in the box for five minutes before any
tests were carried out A variety of compounds were tested: 1:10 and 1:100 YS, ImM 5-HT,
O.1mM 5-HT, 1mM octopamine, and 1mM 5-hydroxytryptophan.. Using a Pipetman, 5Oul of
the compound, was slowly ejected Icm away from the seastar’s arm. Care was taken to ensure
that the force of the ejected liquid did not cause a reaction from the seastar. Controls were
carried out by applying 50ul of natural seawater Icm away from the seastar’s arm. Äfter each
assay, the seastar was returned to its tank, and a different seastar used. The same seastar would
not be used more than once a day to avoid desensitization. Together there were 6 Ophiopteris
papillosa, and over a period of two weeks each seastar was used in an average of 12 assays.
Similar trials were performed on the single Pycnopodia helianthoides specimen.
Behavioural responses to application of the test solutions were classified using a scale of
0 to 3, where 0 = no reaction, 1 = arm tip recoil, 2 = whole arm recoil, and 3 = whole arm recoil
accompanied by movement of seastar away from application site. Data were analysed using the
chi-squared test.
Intracellular recordings from Aplysia buccal ganglion
Buccal ganglia from Aplysia californica were excised, and desheathed in order to expose
the B cells. Once excised and desheathed the cells were healthy for up to 36 hours if stored in
natural seawater at 4°C. The ganglia was pinned to a layer of Sylgard at the bottom of a
perfusion chamber. Recordings were performed in this chamber with volume 1.5ml. An input
tube for fresh seawater and output tube operated by a vacuum pump allowed the solution in the
chamber covering the ganglia to be replaced at anytime. Once the ganglia was desheathed, it
was hard to specifically identify particular cells, however it was unnecessary to pinpoint the
exact cells as it was an area of the ganglion that was of specific interest. This area was the
region with the Bl and B2 cells (bottom half of the ganglion) in which many cells are recognised
as having serotonin receptors (Gerschenfeld and Paupardin-Tritsch, 1974). These neurons were
impaled with a single micro-electrode that were filled with 3M KCl. Recordings were done at
room temperature.
Three different protocols were followed.
A baseline recording of the cell was taken for 3 minutes with the ganglia immersed in
fresh seawater. 50ul of 1:100 YS was then added to the bath using a Pipetman, and a
recording was taken immediately after this for 3 minutes.
2. A baseline recording of the cell was taken for 3 minutes. 5O0ul of 1mM 5-HT was then
added to the bath and a recording taken for 3 minutes. 50ul of 1:100 YS was then added
into the same bath without perfusing the previous solution out, and a recording was taken
for another 3 minutes.
3. A baseline recording of the cell was taken, 5Oul of 1mM 5-HT was then added, a
recording taken for 3 minutes, followed by addition of 50ul 1:100 YS and another
recording taken for 3 minutes. The ganglia was then perfused with natural seawater for
several minutes in order to wash away all the compounds previously added. 5Oul of 5-
HT was then added again to the bath, and a third recording was taken of the cell.
In addition, control recordings of 10 minutes were run to ensure that the effect of YS on the cell
was not confounded by the decrease in serotonin induced activity. Because desensitization of the
cells in the ganglia to both YS and 5-HT was a potential problem, only a few recordings could be
used from each freshly desheathed ganglia.
Voltage clamp:
In conjunction with Michael Hughes, voltage clamp was performed on Shaker A6-46 channels
expressed in Xenopus oocytes bathed in ND96. 2 glass microelectrodes with resistances between
0.7 and 2.0 M£2 were inserted just below the surface of the oocyte membrane and a holding
potential of -60mV was established using a conventional two-electrode voltage clamp. Ooctyes
were subjected to voltage pulses lasting 25 ms and stepped at 10 mV increments between -50
and 40 mV. 1:100 dilution of YS was added to the bath by perfusion. A family of Ig traces at
various voltages were recorded upon establishment of a successful voltage clamp; after a control
solution change to ND96 in a 1:100 dilution with seawater, and again after the addition of 1:100
YS. To wash out the YS, 3-4 ml of ND96 in 1:100 dilution of seawater was used (to control for
the effects of natural seawater). The solution of YS used was from the same stock of YS used
for the intracellular recordings on the buccal ganglia.
Results
Preliminary behavioural studies showed that Ophiopteris papillosa responded to Yellow
Stuff, although it is not a predator of C. canaliculatum. This class of echinoderms is unique in
its anatomy, in that it has giant axons present in the radial nerve cord which run the length of
each arm and mediate rapid escape responses (Yee et al, 1986). As a result this seastar
demonstrates a very visible and easily classifiable response to noxious chemicals. Responses to
application of various compounds are summarised in fig. 2A. Seawater (the negative control)
induced almost all level 0 responses, thus indicating that the force of ejection of the liquid from
the Pipetman was not large enough to induce a behavioural response from the seastar’s sensitive
arms. Application of both dilutions of Yellow Stuff resulted in a large percentage of level 2 and
a significant number of level 3 responses. A chi-squared test concluded that the differences in
frequency of each of the 4 levels was highly significant (p-value of «0.01). Application of YS at
dilutions over 1:100 did not result in any significant responses (data not shown). Application of
both concentrations of 5-HT also caused a high proportion of level 1 and level 2 reactions (p-
value of £ 0.01). To show that this behavioural effect was not common to all biological peptides
and amines, octopamine another common invertebrate neurotransmitter was applied. Over 60%
of the responses were level 0, similar to application of natural seawater. Application of 5-
hydroxytryptophan (the precursor to 5-hydroxytryptamine), resulted in an even spread of
reactions from level 0 to level 3, and did not show any statistically significant differences
between the frequency of the responses in the 3 levels.
Pisaster giganteus did not respond to either 5-HT or Yellow Stuff in a manner that was
easy to identfy in a reasonable period of time, therefore this species was not used.. As there was
only one Pycnopodia specimen, the results may not be as valid as that of the Ophiopteris, but the
response of this specimen did correspond with previous literature (Bryan et al, 1997). Again, as
can be seen in fig. 2B, natural seawater did not elicit a response in Pycnopodia, while both YS
and 5-HT resulted in a noticeably strong response. The Pycnopodia’s response to octopamine
was similar to the response seen in Ophiopteris.
Based on these results, it would appear that there is relationship between serotonin and
Yellow Stuff in these seastars as both compounds elicited escape responses that could not be
induced with octopamine or 5-hydroxytryptophan. These behavioural results give further
credence to the idea that 6BrMT may act biologically by interacting with serotonergic pathways
Intracellular recordings from the buccal ganglia of Aplysia californica
In order to further investigate any effects Yellow Stuff may have on serotonergic
pathways, a suitable assay based on the buccal ganglia of Aplysia was utilised. The buccal
ganglia (see fig. 3) contains a central pattern generator (CPG) that mediates rhythmic movements
of the buccal apparatus during feeding. The activity in the CPG is modulated by extrinsic
serotonergic inputs from the giant cerebral neurons (Evgeni et al, 2000) making these cells an
appropriate assay for comparison of YS and 5-HT.
In experiments where 1:100 YS was applied to the bath without the presence of 5-HT,
there is a clear trend - the application of YS did not change the baseline activity of the cell (see
figs. 4A and B). The firing activity of the cell continued after application of YS, and although
throughout a 3 minute recording the frequency of the action potentials in a cell does not usually
remain constant (bursts of activity are seen followed by periods of relative inactivity), there was
no discernable effect of YS on the activity of the cell, e.g. no PSPs were produced after
application of YS, and the shape of the action potential remained the same.
The B cells are primarily pacemaker cells firing regular action potentials but there were
some that had a quiet baseline (see fig. 5A). In the cells that did not display any spontaneous
activity (either in the form of post-synaptic potentials or action potentials) application of 5-HT to
the bath resulted in the appearance of regular action potentials (fig. 5B) indicating 5-HT had an
excitatory effect on the ganglia. Subsequent application of YS in the same chamber resulted in
an immediate silencing of the cell - no further action potentials were seen (fig. 5C).
In order to determine whether the effect of YS was reversible, the chamber and ganglia
were perfused with natural seawater after the application of 5-HT and YS. After the application
of ImM 5-HT following perfusion with seawater, the excitatory effect of serotonin was seen
again to a similar extent of that seen after the first application of 5-HT. Fig. 6B shows an EPS.
with an increase in the firing frequency of the action potentials situated over the small
depolarising wave. This is not seen after application of YS (fig. 6C), but reappears after
application of 5-HT (fig. 6D). The traces shown in these figures are representative of each of the
3 minute recordings.
Effect of 1:100 YS on Shaker potassium channels
Previous work (Sack et al) determined that application of 6BrMT at uM concentrations to
Xenopus oocytes expressing Shaker potassium channels resulted in a marked slowing of
activation kinetics and a reduction in the peak amplitude of Ig (fig. 7B). These effects were also
seen when raw exudate was used (Sack, personal communication). However 1:100 dilution of
YS used in the present study only resulted in a decrease in peak current amplitude, and no
significant decrease in activation rate (Fig. 7A). The implications of this will be discussed in
greater detail below.
Discussion
The results from the three different lines of experiments in this study indicate that Yellow
Stuff is behaving via some serotonergic pathway. The behavioural studies provided a base for
the latter electrophysiological experiements, by demonstrating a relationship between YS and
serotonin that was not seen with other biological substances. These behavioural results thus
provided a justification for further comparison of the two substances, and more importantly
determined the threshold concentration beyond which no visible response from the seastars was
observed. This dilution of YS was then used in all subsequent electrophysiology experiments
The voltage clamp experiment demonstrated that at 1:100 dilution, YS was not affecting the
activation kinetics of Shaker potassium channels, and the intracellular recordings from the
Aplysia buccal ganglia clearly demonstrated the effect of YS on serotonergic activity.
In 1974 Gerschenfeld and Paupardin-Tritsch first convincingly demonstrated that 5-HT
functioned in Aplysia californica as a neurotransmitter by showing that of the 13 cells they
studied in the buccal ganglia, nine had an excitatory response and four had an inhibitory response
due to specific serotonin receptors. Using iontophoretic methods of application, they
demonstrated that 5-HT resulted in pronounced EPSPs in buccal ganglia cells. The results
obtained in this present study are therefore in accordance with previous literature, showing
serotonin behaving in an excitatory fashion, either generating pronounced excitatory post-
10
synaptic potentials or in a few cases initiating action potentials. The application of YS after 5-
HT resulted in a significant decrease of serotonin-induced activity. Thus, YS at a 1:100 dilution
appears to be functioning as an antagonist of 5-HT.
The buccal ganglia is responsible for generating rhythmic chewing and swallowing
motions in the buccal apparatus through the central pattern generator (CPG) which produces
buccal motor programmes, BMPs. The CPG receives extrinsic serotonergic inputs, e.g. from the
serotonergic metacerebral cell (MCC) which synthesises 5-HT. Perfusion of a semi-intact head
with 5-HT induced swallowing-like movements, seen in intracellular recordings as an increase in
excitability, thus demonstrating that 5-HT is an important neuromodulator in the buccal ganglia
system (Evgeni et al, 2000). As can be seen from fig. 5B and 6B, the application of 5-HI
induced a large excitatory effect seen in most of the recordings as an increase in PSPs (as most of
the B cells tested were pacemakers), and in the cells that were not pacemakers, this excitation
was in the form of action potentials. Application of 1:100 YS after 5-HT resulted in a significant
decrease in the cell’s activity indicating that YS functions as a serotonin antagonist. In addition.
the application of 5-HT after perfusion of the ganglia with natural seawater (fig. 6D) resulted in
the return of excitatory activity, therefore suggesting YS could be a reversible antagonist.
Because the recordings were on intact ganglia where the cells were still a part of a large
neural network, there is not enough evidence to determine whether YS is behaving as an
antagonist on a cellular level or on the whole serotonergic system, although the structural
similarities between 5-HT and 6BrMT provides strong indication that it might be antagonising at
the cellular level. To determine whether YS is an antagonist of 5-HT at the receptor level would
require isolation of the B cell and many more tests to be performed.
11
The lack of effect of 1:100 Yellow Stuff on Shaker potassium channels is consistent with
the argument that YS is behaving antagonistically to serotonin. 6BrMT has a well documented
effect on Shaker potassium channels - a reduction of peak potassium current amplitude and a
reduced activation rate (Sack et al). With 1:100 YS, only one of these effects was visible (fig.
7A), therefore at this dilution YS has a much smaller effect on Shaker potassiuam channel
kinetics. Since 1:100 YS has a pronounced effect on the serotonergic system of the buccal
ganglia it is likely that at this dilution, YS is interacting via serotonergic pathways and not
predominantly on Shaker potassium channels. It is very likely that this antagonistic effect is
primarily due to the presence of 6BrMT, a serotonin analogue.
Conclusions
6BrMT has a well documented effect on Shaker potassium channels, but the fact that a
low concentration of raw Yellow Stuff suppresses serotonin induced activity in the buccal
ganglia of Aplysia, provides a good indication that YS has multiple pathways through which it
functions, one of which being through a serotonin pathway. Although this study primarily
concentrated on the effect of YS on Aplysia, an organism which C. canaliculatum would not
normally encounter in its ecological niche, the conclusion that YS functions antagonistically to
5-HT could help determine the mechanism of how YS elicits the escape response in its predators.
This study also raises many more questions about the relationship of 6BrMT and YS to
serotonin, not only in seastars, but in humans, where a serotonin antagonist has many
pharmacological uses.
12
Acknowledgements
1 wish to thank everyone at Hopkins who has helped me during these last two months,
especially the Fisher’ people (Anna, Ryan, Jane, Rachel) for keeping me company during the
long days of electrophysiology, Mike for all the voltage-clamp work he did for me, the helpful
people in Gilly’s lab, my housemate Tori for her continued support, Brett for all his rides home
at 12am and Charles for making sure I ate dinner at lam..
Gilly - thank you for making sure I thought everything through before jumping in head
first, for being a natural skeptic, for your love of paperclips, and for all the time and invaluable
encouragement you supplied.
Stuart - thank you for your amazing enthusiasm and energy you put into this project, and
for helping me make sense of the complicated world of Aplysia.
13
References
Bryan, P.J., J.B. McClintock, and M. Hamann (1997). Behavioural and Chemical Defenses of
Marine Prosobranch Gastropod Calliostoma canaliculatum in Response to Sympatric
Seastars. J. Chem Ecol 23: 645-658
Evgeni A. Kabotyanski, Douglas A. Baxter, Susan J. Cushman, and John H. Byrne (2000).
Modulation of Fictive Feeding by Dopamine and Serotonin. J. Neurophysiol. 83: 374-
392.
Gerschenfeld, H.M. and D. Paupardin-Tritsch (1974). Ionic Mechanisms and Receptor Properties
Underlying the Responses of Molluscan Neurones to 5-Hydroxytryptamine. J. Physiol.
243: 427-456.
Gerschenfeld, H.M. and D. Paupardin-Tritsch (1974). On The Transmitter Function of 5-
Hydroxytryptamine At Excitatory and Inhibitory Monosynaptic Junctions. J. Physiol.
243: 457-481.
Sack, J., W.P. Kelley, A.M. Wolters, R.A. Jockusch, J. Jurchen, E.R. Williams, J.V. Sweedler
and W.F. Gilly. Slowing the Activation Kinetics of K-channels with BrMT, Novel
Pharmacological Agent from a Marine Snail. Publication pending.
Yee, A., J. Burkhardt and W.F. Gilly (1986). Mobilisation of a Coordinated Escape Response by
Giant Axons in the Ophiuroid, Ophiopteris papillosa. J. Exp Biol 128: 287-305.
Figure Legends
The chemical structures of 5-HT and 6BrMT (a serotonin dimer derivative)
Fig.1.
Fig. 24
Behavioural response of Ophiopteris papillosa to various chemical stimuli. A
total of 6 Ophiopteris were used in, each for 12 assays.
Fig. 2B
Behavioural response of Pycnopodia helianthoides to the same chemical stimuli.
One specimen was used for the study.
Fig. 3
Map of the B cells in the buccal ganglia of Aplysia californica
Fig. 44
Baseline of a pacemaker cell in the buccal ganglia.
Fig. 4B
After application of 1:100YS to the bath. Trace is 5000ms and is representative
of the whole recording.
Fig. 5A
Baseline of a non-pacemaker cell (no action potentials)
Fig. 5B
After application of 50 ul 1mM 5-HT to the bath. Regular action potentials
generated.
Fig. 5C
Addition of 1:100 YS into the same chamber 3 min later. Action potentials no
longer seen.
Fig. 6A
Baseline of a pacemaker cell
Fig. 6B
After application of 5-HT to the bath. EPSPs generated.
Fig. 60
Subsequent application of 1:100 YS to the same bath. EPSPs no longer seen
Fig. 6D
After perfusion with seawater, 5-HT added again. EPSPs generated.
Shaker sensitivity to control and 1:100 YS
Fig. 7A
Fig. 7B
Shaker sensitivity to control and 6BrMT (Sack et al)
Figures
Fig 1 Serotonin
NH.
Ho.


5-H
6-bromo-2-mercaptotryptamine
N.
N
-S—S-
E

B
Fig 24
Fig. 2B
Response of Ophiopteris to various compounds
Elevel 0
Blevel 1
Dlevel 2
Dlevel 3
L



9

0
00

Compounds
Response of Pycnopodia to various compounds
Elevel
Elevel
Dlevel
—evet
II








Compounds
Fig. 3
B3
862
B11
B5
864/
8b
766
88a
B4
661
B7
863

T
S2834
S1
B1 8314
Fe

832-

S2


B2


from http:/www.biu. ac. il/LS/stafffsusswein/susswein2.html
CBC
1683

BN-2
BN-1
mV
mV
Fig 44
ee e e  e ehne e eae ethen e   n   en    enanet

Mtn,
Time (5000ms)
Fig. 4B
ene gehenene e nen e ane ae n enen ene aeane ne aee aean aeee
Tn.—
Time (5000ms)
19
e
mV
mV
Fig. 54


TEGeenens    e  onenann e

Tn
Time (5000ms)
Fig. 5B
.

120
ae
2

etetesetoe e et t ose e e s o

P

e
60000
Tme (ma)
Time (5000ms)
Fig. 5C
1 2
Fonchegea heh ehanhehengen eof ena enen h a ehgnedoshs e  aoe er  een o un oe  e  a n o
20
Time (5000ms)
20
Fig. 64

—t
..
1..
Time (5000ms)
Fig. 6B
1
tt
„1
Heetttttrtettreeetr

—
4.
Time (5000ms)
Fig. 60
tt



Time (5000ms)
Fig. 6D

t
11 Knettrttfrttttet
Hettrt

T
Time (5000ms)
Fig. 74
—
Hughes (2001)
Fig. 7B
Sack et al


10 ms
—

400 ms
5 UA
20 UA
22