ABSTRACT
In order to illustrate the usefulness of the dorid
nudibranch Doriopsilla albopunctata for
electrophysiological studies, maps of the pleural ganglia
were made and gross cellular responses of autorhythmic
neurons to applied neuroactive peptides and amines were
observed.
Dopamine, 5-HT and FMRF-amide all induced both
depolarizations and hyperpolarizations in different
autorhythmic cells of D. albopunctata. Responses ranged
from complete inhibition of firing, to increased rate of
repetetive firing in a beating cell, and complete
transformation of bursting activity into beating activity.
In addition, 5-HT induced in a beating cell a
depolarization that attenuated with repeated
applications. 5-HT applied in combination with
FMRF-amide produced varied responses that were not
seen with applications of 5-HT and FMRF-amide
separately.
CPT-CAMP and forskolin, which both serve to enhance
CAMP production, were applied to selected cells in order
to observe if any of the previous cellular responses were
mediated by an increase in cAMP levels. Application of
CPT-CAMP, constituting an increase in CAMP levels only,
produced no response. Forskolin produced many effects,
but they did not resemble any responses induced by the
previously applied neuroactive peptides and
amines. It seems then, that the applied neuroactive
peptides and amines do not mediate their actions through
CAMP levels. In addition, although forskolin is a CAMF
analog, from the differing responses to CPT-CAMP and
forskolin, forskolin seems to be inducing its cellular
responses via a mechanism other than a rise in CAMF
concentration.
INTRODUCTION
Little work has been done on the nudibranch
Doriopsilla albopunctata although it is an ideal organism
for studying electrophysiology. Easy accessibility and
preparation, great viability and large easily identified
neurons are all good reasons why D. albopunctata should
be taken advantage of and studied further.
In our study of the pharmacology of various D.
albopunctata bursting and beating cells located in the
pleural ganglia, we observed both depolarizing and
hyperpolarizing responses to the applied neuroactive
peptides and amines, with none having a dominant effect
on all of the cells observed. We conclude from our
studies that the autorhythmic neurons of D. albopunctata
have various, differing mechanisms by which they
respond to a particular substance, depending on
individual cellular properties. These differences may lie
at the level of receptors, second messengers, ion channel
properties, etc. Comparing our results with known
cellular responses to the same neuroactive peptides and
amines on similar Aplysia neurons, we then determined
if they could also possibly account for our observations.
In some D. albopunctata cells, the mechanisms
operating in Aplysia could feasibly underly our observed
responses.
But in other cells, we saw unexpected results,
indicating the presence of new, unknown mechanisms.
Further studies to elucidate the mechanisms behind
these observed unique responses could add tremendously
to our existing knowledge concerning these neuroactive
peptides and amines.
MATERIALS AND METHODS
Doriopsilla albopunctata were collected subtidally
from Montery Bay, CA., and stored in flowing natural sea
water tanks of approximately 13°C. They were not fed.
Ganglia were surgically removed and treated with
dispase, a neutral protease, for one hour followed by a
minimum of a two hour rinse in ASW to facilitate
removal of the epineural sheath.
Experiments were performed on the pleural ganglia
pinned in a Sylgard dish of approximately 5ml bath
volume and perfused with ASW of the following
composition: 470mM Nacl, 10mM KCI, 10mM Cacl, 50mM
MgCl, and 10mM Hepes (pH 7.8). Bath temperature during
experiments was approximately 17’0.
Individual cells were impaled with two glass
microelectrodes (R = 3-10 MÖhms) filled with 3M KCI
solution. One microelectrode injected a square current
pulse while the other measured the resulting voltage
change. Long hyperpolarizing current pulses were
administered in order to measure Tau and input
resistance.
Various neuroactive peptides and amines were applied
directly onto the ganglia in dosages that, unless
otherwise stated, resulted in a final bath concentration
of 2uM. Except where otherwise noted, perfusion was
not operating until after observation of the initial gross
effects of the applied substances. Perfusion continued at
a rate of 1-10cc/min. until the control condition was
resumed.
Selected cells were then voltage clamped using two
microelectrodes similar to those used in the current
clamp. One electrode maintained the cell at a constant
holding voltage of -40mV, while the other measured the
underlying currents resulting from voltage steps from
the holding voltage.
RESULTS
Identification of Autorhythmic Cells
We were able to map the dorsal side of the
asymmetrical pleural ganglia using morphologhical and
electophysiological means of identification. Our map
represents a composite of all the individual ganglion
observed (Figure 1). Each side of the ganglia yielded a
cell with a characteristic bursting pattern. These cells
were consistently large and whitish in appearance. LP1
is one of two large cells in the left ganglia located
furthest anteriorly. RP2, meanwhile, proves more
difficult to locate as there are many large cells in the
right ganglia, with seemingly greater variablity amongst
individual D. albopunctata. The most reliable
characteristic of RP1 is its whitish glow, clearly visible
even through the epineural sheath. The beating cell LP2,
is typically the largest and most posterior cell in this
view of the left pleural ganglia. RP2 resides in a cluster
of three cells located most anteriorly in this ganglia,
each of which is about half the diameter of the numerous
large cells below. Due to the aforementioned ganglia
topographic variablity, RP3 was difficult to map,
although it is usually a medium sized cell found in the
vicinity of the base of the nerve root.
FMRF-amide
FMRF-amide (Phe-Met-Arg-Phe-NH2) instantaneously
depolarized the LP1 burster, converting all bursting
activity into tonic spiking activity. (Figure 2B) An input
resistance drop from a control of 6.5 MÖhms to 1.5
Möhms was also observed. In the beating LP2 cell, on the
other hand, we observed complete inhibition of firing
upon peptide application. (Figure 24)
Disruption of beating pattern was observed in the
rapidly firing RP3 beater. After FMRF-amide application,
the constant control spike interval of 800ms fluctuated
from 680ms to 960ms. From the appearance of synaptic
input during these intervals, we hypothesize that cells
presynaptic to RP3 exhibit responses to FMRF-amide that
consequently affect RP3. AII FMRF-amide effects on all
cells studied were reversible upon perfusion.
FMRF-amide and 5-HT
A 1:1 ratio of 5-HT and FMRF-amide applied during a
5-HT induced tonic spiking condition in the LP1 burster
resulted in an input resistance drop greater than that of
5-HT alone. While 5-HT lowered input resistance from
15.4 MÖhms to 14.4 MÖhms, the input resistance dropped
to 10.2 MÖhms with the combination of 5-HT and
FMRF-amide. Tau also decreased from a control of
360.6ms to 354.3ms with 5-HT alone, then to a final
value of 285.7ms when combined with FMRF-amide.
Of particular interest in studying this cell's
pharmacology is the evidence for synaptic input after the
application of 5-HT amd FMRF-amide. It manifested
itself in the form of instantaneous, excitatory bursts,
which increased spike frequency whether the cell was in
a bursting or beating state. In addition, interburst
hyperpolarizations were not smooth, and spikes rose
sharply rather than gradually depolarizing.
5-HT
Serotonin produced a depolarization in the LPI
burster (Figure 3A,B) and the LP2 beater (Figure 4B),
exciting both to a high rate of repetetive firing. Voltage
clamping of the LP2 beater revealed an insignificant
change in inward rectification, although the spike
interval decreased from 2.75-3.0s to 1.36s. Clamping of
the burster LP1 when exposed to a 5-HT bath
concetration of 10 uM, however, revealed a marked
decrease in inward rectification of 1.43 nA at -90mV to
89 nA at -60mV. (Figure 8)
In the beating cell RP3, 5-HT application also induced
a higher rate of repetetive firing, but the response
attenuated with successive applications. Spike interval
was measured 60s after administration of 5-HT in three
separate dosages, with perfusion between each.
Following the first application, spike interval dropped
dramatically from a control of 3.5s to 0.5s, and
thereafter increased progressively to 2.9s and 3.25s in
the second and third applications, respectively. (Figure
5-1,2,3) Input resistance measurements showed a
gradual increase from 12 MOhms to 21.3 MOhms and 22.0
MÖhms in applications one, two and three, respectively.
5-HT produced a delayed response in cell RP2 (Figure
4A), inhibiting its beating pattern approximately 1.5 min.
after application. The original firing pattern did
eventually resume.
Forskolin and Chlorophenylthio-CAMP (CPT-CAMP)
To observe whether any of the applied neuroactive
peptides or amines exerted their effects via CAMP, we
increased the intracellular levels of CAMP by applying
CPT-CAMP, a membrane permeable cyclic AMP analog, and
forskolin, a known stimulator of CAMP production.
In cell LP1, forskolin inhibited bursting activity and
initiated an erratic beating pattern characterized by
much synaptic input. (Figure 6) In addition, doublets,
triplets and trains of up to five spikes replaced the
single spikes in the original bursting condition. The
10
effects of forskolin were long lasting, being visible over
an hour after application even though perfusion was at a
rate of1Occ/min. Input resistance measurements
reflected an increase from 5.25 MÖhms to 7.0 MÖhms
upon application of forskolin. Voltage clamp was
performed on the burster RP1 in the opposite ganglion.
Voltage was stepped from -90mV to -10mV in steps of
+10mV, lasting 100ms each. Under these conditions,
inward rectification was found to decrease by a
maximum of 39.3 at -60mV.
CPT-CAMP was then applied to LPI without perfusion.
Even after 30 minutes, no effect on bursting activity or
input resistance under current clamping conditions was
observed. Voltage clamping under conditions identical to
that of forskolin, resulted in a negligible change in
inward rectification. (Figure 9) CPT-CAMP, then, does
not affect LP1, and more importantly does not seem to
reproduce the effects of forskolin.
Dopamine
Dopamine instantaneously inhibited all firing upon
application to the beater LP2. (Figure 7B) The beating
pattern did eventually recover after perfusion, initially
firing at a slower rate and gradually returning to its
original condition.
The LP1 burster depolarized in reponse to DA,
transforming its bursting activity into a fast rate of
repetetive firing. (Figure 7A) With perfusion, tonic
spiking activity gradually slowed, with an eventual
resumption of the bursting pattern.
DISCUSSION
The responses of the studied autorhythmic cells to
each neuroactive peptide and amine were varied, showing
no dominant pattern. It seems then, that there are a
number of different mechanisms by which individual D.
albopunctata cells respond to a substance. This
discussion focuses on some of the possible mechanisms
by which these cells may operate.
Past studies on FMRF-amide have shown that it
elucidates a wide spectrum of responses in many
different cells. Effects on Aplysia neurons range from a
biphasic excitation-inhibition (Ruben, Johnson and
Thompson, 1984), to complete inhibition. (Stone and
Mayeri, 1981) Furthermore, the giant serotoninergic
neuron of the land snail Helix responds to FMRF-amide
by first increasing a K+ -current near resting potential,
and then reducing a Ca++ and voltage dependent
K+-current at more depolarized voltages. (Cottrell, 1982:
Cottrell et al., 1984) The LP1 burster and LP2 and RP3
beaters of D. albopunctata that we studied proved to be
equally variable in their responses to FMRF-amide. The
LP2 beater's hyperpolarizing response can possibly be
explained by Ruben, Johnson and Thompson's (1984)
reports of an FMRF-amide induced biphasic response in
Aplysia burster neurons L4 and L6. In these cells, an
inward current increase carried by Na+ ions starts
approximately 100-200ms after FMRF-amide application,
followed by an outward current increase carried by K+
ions that begins approximately 2-5s after peptide
application. Due to the rapid onset of the voltage change
induced by the first depolarizing phase of the
FMRF-amide response, it could have easily been obscured
using our current clamping methodology. But the latter,
delayed, hyperpolarizing phase of the peptide response
which is easily seen, could feasibly account for the
inhibition of firing that was observed in LP2. An increase
in outward K+-current could act to keep LP2 in a
hyperpolarized, nonfiring state, as well as to counteract
any depolarizing stimuli it might have received.
Ruben, Johnson and Thompson (1984) also postulate
that the presence of two separate ion channels could
account for the observed immediate and delayed
responses to FMRF-amide. The inward Na--channels that
are instantaneously opened possibly involve one set of
receptors located at or near the site of peptide
application. The outward K+-channels, however, open
with a delay time that could be accounted for by the
existence of a separate set of receptors located further
away from the site of peptide application. The lag time,
then, represents the time taken for the FMRF-amide to
15
diffuse from application site to receptor site.
Alternatively, the rapidly acting Na--channels could
interact indirectly with FMRF-amide, via an intermediate
mechanism requiring only a few FMRF-amide molecule
interactions to initiate an amplified effect. Here then,
many channels could open in a short period of time. The
slowly activating K+-channels, though, would interact
directly with the FMRF-amide, thereby explaining the lag
time. If each individual K+-channel had to interact
directly with an FMRF-amide molecule, it would take a
reasonable amount of time before enough K+-channels
could be opened to produce an observable response. If the
LP1 burster were to have only the receptors responsible
for the rapidly acting Na--channels, its sole depolarizing
response to FMRF-amide could easily be explained.
Tentative support for this mechanism lies in the
decrease in input resistance observed, which could
correspond to an increase in number of open
Na--channels.
The absence of either of these two possible
FMRF-amide receptors on RP3 could account for a lack of
a gross depolarizing or hyperpolarizing responses.
However, the presence of the FMRF-amide induced erratic
beating pattern and synaptic input could feasibly
represent the depolarizing or hyperpolarizing responses
16
of cells to FMRF-amide presynaptic to RP3, depending on
which of the two posssible FMRF-amide receptors these
cells had.
An interesting point to notice is the unique effect
that the combination of 5-HT and FMRF-amide had on the
burster LP1. Applied by themselves, neither induced any
synaptic input onto LP1. But together, synaptic input that
had not been seen previously , ocurred. What possible
mechanism could account for this? Because FMRF-amide
and 5-HT both induce depolarizing responses in LP1, they
may possibly be acting via the same mechanism, such as
the opening of the same inward cation channels.
FMRF-amide and 5-HT may not be able to produce a
depolarization by themselves in cells presynaptic to LPI
that is sufficient to exceed the threshold of these cells,
resulting in no transmitter release and therefore no
synaptic input. But both FMRF-amide and 5-HT applied
together may have the dual effect of depolarizing the
presynaptic cells more than either is capable of doing
alone, resultingly exceeding threshold and producing the
synaptic input seen.
5-HT, like FMRF-amide, also produced various
responses in D. albopunctata. The burster LP1, and the
beaters LP2 and RP3 exhibited a depolarizing response
that is possibly explained by Levitan and Levitan's (1988)
proposed mechanism of 5-HT. Levitan and Levitan (1988)
found that in the Aplysia burster R15, a 5-HT
concentration of 500uM (Tremblay et al., 1976) could act
via an increase in CAMP levels to increase the
subthreshold calcium current, causing an enhancement of
the depolarized phase of the bursting cycle. Although the
5-HT concentrations applied to the cells in our
experiments only had a final bath concentration of 2uM,
application method was such that 5-HT concentrations
exceeding 500uM could have been possible. To test this
CAMP mediated increase in LACPT-CAMP was applied to
LP2, with no observable response. Therefore, although
the depolarizing effects caused by 5-HT in LP2 may still
be mediated by an increase in L,,5-HT is not acting via
CAMP to produce this response. However, a CAMP
mediated depolarization has not yet been ruled out for
LPI and RP3, as CPT-CAMP was not applied to these
cells.
The beating cell RP3 also exhibited a depolarizing
response to 5-HT, but differed from LPI and LP2 in that
the excitatory response desensitized. The progressive
increase in spike interval from the first to the third
5-HT application exhibits this attenuation well. A
possible mechanism can be deduced from the observation
of a gradually increasing input resistance with each
5-HT application. This is consistent with a gradual
inactivation of channels responsible for causing the
initial large, depolarizing response. These inactivated
channels remained in this state during the second and
third 5-HT applications, and so were not available for
opening and resultingly depolarizing the cell. Each
successive 5-HT application then, represents a
progressively decreasing population of inward cation
channels available to depolarize RP3. However, another
possible mechanism that could account for the
desensitzation to 5-HT could lie in downregulation of
5-HT receptors on RP3. No experiments were performed
to test either of these hypotheses, and therefore
represent only speculations at best. Further work to
elucidate the exact mechanism of the 5-HT
desensitization observed in RP3 is needed.
5-HT induced a unique response in the beater RP2.
After a delay time of approximately one and a half
minutes, the cell hyperpolarized and all firing activity
was inhibited. A possible mechanism for this response
lies in Levitan and Levitan's (1988) finding that in
Aplysia R15, 5-HT concentrations at or below 1OuM can
act via increasing CAMP levels to increase the inwardly
rectifying K+-current. With the resulting increase in K+
conductance, the cell resting potential would be driven
19
to hyperpolarizing voltages near the reversal potential
for K+ (around -70my), resulting in an inhibition of
cellular activity. The lag time observed before the
response, however, could indicate another possible
mechanism involving either Cl- channels or a different
set of K+-channels located far away from the site of
5-HT application, which do not operate via CAMP. These
are only suggested mechanisms, as we did not perform
any voltage clamp or CAMP analog experiments, and
therefore cannot be sure that 5-HT does actually act
through CAMP to affect the inwardly rectifying
K+-current.
The results of the CPT-CAMP and forskolin
applications indicated that none of the applied
neuroactive peptides or amines operate using cAMP as an
intermediate. In addition, it appears that although
forskolin is a stimulator of CAMP production, the effects
resulting from its application are due to mechanisms
other than increased levels of CAMP.
In LP1, forskolin caused a number of effects that did
not resemble any of the responses of the other applied
neuroactive peptides and amines, giving the first
indication that these substances did not operate via a
CAMP intermediate. Evidence for a possible mechanism
underlying the various responses to forskolin lies in the
20
observation of an increase in input resistance following
application. This corresponds well with Coombs and
Thompson's (1987) finding that forskolin acts on certain
D. albopunctata neurons to close transient K+-channels.
Closure of K+-channels would depolarize the cell, and
could resultingly contribute to the creation of a new
resting potential that exceeds the thresholds of the
multiple spike initiating zones located at various points
along the axon; or possibly the single spike initiating
zones on multiple axons extending from the soma. If this
depolarization effect were to occur, trains of spikes
would result that were in fact observed upon application
of forskolin. The decrease in inward rectification seen
under voltage clamp could add to the decreased K+
conductance, assuming that this current was carried
mostly by K+ ions. This in turn, would act to depolarize
LP1 still further, thus providing even more feasibility
for the multiple spikes.
The decrease in the transient K+- current that Coombs
and Thompson (1987) described could also provide a
mechanism by which a number of other effects of
forskolin acted. Because  is active at subthreshold
voltages, it exerts its main effects not on changing spike
shape, but on slowing the rate of repetetive firing and
controlling spike interval. Therefore, it is reasonable to
21
conclude that if 1is blocked, the effects will be
manifested in an increase in repetetive firing as well as
erratic spike intervals, which are in fact the effects
forskolin induced.
Finally, forskolin has been found to cause an increase
in an inward cation current near resting potential in a
number of nudibranchs.(Coombs and Thompson, 19871
Although this response may contribute to the
depolarization of the resting potential and the multiple
spikes mentioned earlier, it could also be responsible for
the great deal of synaptic input that was observed in
LP1. This synaptic input might be the effects of forskolin
activating the inward cation current in cells presynaptic
to LPI.
Because forskolin is a known activator of adenylate
cyclase, an increase in cAMP levels is one of the most
feasible mechanisms by which it could exert its effects
on LP1. To test whether CAMP did serve as an
intermediate, we applied CPT-CAMP, a membrane
permeable analog of cAMP that would produce effects in
LP1 that could be the result of a sole increase in cAMF
concentration. Under current and voltage clamping
conditions, no effects of CPT-CAMP were observed
giving strong evidence that forskolin does not act via
CAMP levels, but instead via changes in 1, as suggested
22
earlier.
Dopamine proved to exhibit responses that were as
variable as those that our other amine, 5-HT, had
produced. The inhibitory response shown by LP2 can
possibly be explained by the finding that DA has been
shown to cause a decrease in the subthreshold Ca++
current in Aplysia R15. (Lewis, et al., 1984) A decrease
in Ca++ -current could greatly hinder LP2's ability to fire
a spike, as in molluscan neurons, the upstroke of the
action potential consists of a combination of Ca+ and
Na-- currents. If the Ca+--current is eliminated, opening
of Na--channels only may not produce sufficient
depolarization to produce a spike. Even more significant
is that the Ca++- current operates mainly at
subthreshold voltages, and consequently has great power
in determining if a cell will reach threshold. If the lgis
decreased enough, the cell will never depolarize to the
point where enough Na--channels are activated to
initiate an action potential. As a result all firing
activity will be inhibited, which is in fact what the LP2
response was.
The LP1 burster responded to DA by shifting from its
bursting pattern to a high frequency tonic firing pattern.
In Aplysia R15, the same decrease in L just mentioned
acts to increase the interburst hyperpolarization, and at
23
times to stop all bursting activity altogether. (Äscher,
1972) As this reported response is directly contrary to
the depolarizing response observed, it seems safe to say
that DA is not exerting its effects here via 1-Although
DA may be acting to increase an inward cation
conductance in LP1, it is hard to say anything about the
underlying mechanisms, as the change in firing pattern is
all the data that could be collected in this experiment.
Doriopsilla albopunctata neurons show a wide
spectrum of responses to the various neuroactive
peptides and amines applied to them. Although we have
just barely scratched the surface of the pharmacology of
these various cells, we hope that we have sparked an
interest in this useful animal for further studies. As
some of the observed responses were unexplainable by
traditional mechanisms, elucidation of the exact
mechanisms by which the cells responded will serve to
enhance knowledge of neuropharmacology immensely.
FIGURE LEGEND
Fig.1. Map of the dorsal pleural ganglia of
Doriopsilla albopunctata.
Fig.2. A. FMRF-amide causes inhibition of beating
activity in LP2.
FMRF-amide transforms LPI bursting pattern
into a beating pattern.
Bursting activity
Fig.3. A,B. 5-HT depolarizes LPI.
converted into a high rate of repetetive
firing.
Fig.4. A. 5-HT has a delayed response on RP2, eventually
inhibiting its beating pattern.
B. 5-HT excites LP2, increasing its rate of firing.
Fig.5. 5-HT induces a higher rate of repetetive firing
in RP3, The response attenuates with repeated
applications. Arrows represent spike interval
measurements 60s after 5-HT application in trials
1,2, and 3.
1. spike interval.5s
2. spike interval 2.9s
3. spike interval 3.25s
Effects of forskolin on LPl. Note multiple spikes,
Fig.6.
erratic beating pattern and synaptic input.
Fig.7. A. DA transforms LPI bursting activity into a
high rate of repetetive firing.
B. DA inhibits firing in LP2.
Fig.8. 5-HT causes an increase in inward rectification
in LPI.
Fig.9. CPT-cAMP causes no significant change in inward
rectification in LPl.
Fig.10. Summary of autorhythmic neuron responses to
applied chemical substances.
Fig.11. The varied bursting cycles of RPl and LP1.
FIGURE 1
JRP2

S2
Doglees,aalata
ew
ALPORNCTAT
8
o
8
A






NA
o

NA
FIGURE 10
VAS
FMRE
A
DA
t
c07
CAMI


SAT
—

FNRF+
54T
LPI
+
O
+
RPI
70
+++
+
++
++
O
++

LP2
——
——
+
RP2
—
RP3
+
P
C
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ACKNOVLEDGEMENTS
I would like to thank my advisor, Stuart Thompson,
as this project vould not have been possible without his
support and quidance. In addition, I would like to thank
Ion Ctis, Tony Morielli and Brett Premack for their heln
and advice, Finally, areat thanks go to my partner,
Scott Forrison, for being a great research covorker, d
super buddy, and a wonderfully tolerant person who was
always willing to put un with my alleded moodiness.