ABSTRACT
The pleural ganglia of Doriopsilla albopunctata were mapped,
allowing for exploration into the effects of various neuroactive
substances on particular types of molluscan cells. Using a current
clamp, we studied those cells that demonstrated autorhythmic firing
characteristics - either beating or bursting. Two amines, Dopamine
and 5-Hydroxytryptamine, and one peptide, FMRF-amide, were applied
onto the ganglia, and the resulting gross voltage changes were recorded.
FMRF-amide and 5-HT were applied in combination, producing effects
unlike those when the chemicals were applied singly. Observed effects
range from membrane depolarization and hyperpolarization to changes in
synaptic input and input resistance. To decipher possible second
messenger systems underlying these changes, membrane permeable
CAMP analog (CPT-CAMP) and an adenylate cyclase activator, forskolin,
were employed. We found that CAMP did not mimic the cellular
responses to these chemicals, nor did the forskolin produce the same
effect as the CAMP analog; clearly other second messenger systems are
being employed in both cases. Some voltage clamping was performed,
although many of the conclusions drawn concerning currents underlying
these changes are based on analogy with other molluscan systems.
INTRODUCTION
Many studies aimed to model the electrophysiological properties of
neuroactive substances have been performed on molluscan nervous
systems. While much work has been performed on its larger relative
Aplysia californica here we present the feasibility of using the dorid
nudibranch, Doriopsilla albopunctata, for long-term
electrophysiological investigations.
Some of the cells in the D.albopunctata ganglia demonstrate
auforhythmic firing characteristics - either beating or bursting.
Beating cells are identified by repetitive firing at a constant regular
interval. Bursting, meanwhile, appears as a complex pattern involving
spike trains of increasing then decreasing frequency, and long
inter-burst hyperpolarizations. Indicative of the burst is the
depolarizing afterpotential, appearing as a bump after the last spike in
the burst. Bursting cells would often be observed in a beating phase,
when they would resemble a beater; we could differentiate between the
two, however, with knowledge of ganglia topography enabling individual
cell recognition, as well as by the symptomatic spike frequency
changes, and the appearance of slight bumps after the spikes typical of
a burster.
Application of various neuroactive peptides and amines on the cells
often resulted in gross, observable changes in firing pattern under
current clamping situations. Two amines, 5-Hydroxytryptamine and
Dopamine, and one peptide, FMRF-amide, were applied and voltage
changes were recorded. To decipher possible second messenger systems
underlying the effects observed, membrane permeable CAMP as well as
an adenylate cyclase activator were employed. Dramatic effects such
as depolarization and hyperpolarization of the membrane, or subtle
changes like increased synaptic input or input resistance changes, were
among the types of responses seen.
MATERIALS AND METHODS
Doriopsilla albopunctata were collected subtidally from Monterey
Bay, CA., and stored in flowing natural sea water tanks of approximately
13°0. They were not fed, and were typically used within two weeks of
collection. Ganglia were dissected out and treated with neutral protease
(Dispase; Calbiochem.) for one hour, followed by a minimum two hour
rinse in ASW to facilitate the surgical 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 =
3-10M22) filled with 3M KCI solution. Basic current clamping protocol
was performed, in which one microelectrode injected a square current
pulse while the other measured the resulting voltage change,
Administration of long hyperpolarizing current pulses enabled us to
measure Tau and input resistance.
Several 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 the initial gross effects of the applied
substances were observed. Perfusion continued at a rate of 1-10 cc per
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. From the holding
voltage, voltage was stepped from -90mV to -10mV in steps of +10mV.
each lasting 100ms with a 5sec interval between steps. The other
microelectrode measured the underlying current changes that resulted
from these steps.
RESULTS
Identification of Autorhythmic Cells
We were able to map the dorsal side of the asymmetrical pleural
ganglia using morphological and electrophysiological means of
identification. Our map represents a composite of all the individual
ganglion observed (Figure 1). Each side of the ganglia yielded one 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. RP1, meanwhile, proves more
difficult to locate as there are many large cells in the right ganglia,
with seemingly greater variability amongst individual D.albopunctata
The most reliable characteristic of RP1 is its whitish glow (under light
microscope), 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 variability, RP3 was difficult to map, although it is
usually a medium sized cell found in the vicinity of the base of the
nerve root.
Phe-Met-Arg-Phe-NH2 (FMRF-amide)
FMRF-amide induced an instantaneous depolarizing effect in the LP1
burster (Figure 2). An input resistance drop from a control of 6.5MQ to
1.5M52 accompanied the observed tonic spiking condition. In the beating
LP2 cell, on the other hand, we observed complete inhibition of firing
upon the peptide application.
Disruption of beating pattern was observed in the rapidly firing RP3
beater; a constant control spike interval of 800ms fluctuated after
FMRF-amide application from 680ms to 960ms. From the appearance of
synaptic input during these intervals, we infer that cells presynaptic to
RP3 exhibit the FMRF-amide response consequently effecting RP3. AIl
FMRF-amide effects were reversible upon perfusion.
5-Hydroxytryptamine(5-HT)
Serotonin produced a delayed response in cell RP2, inhibiting its
beating pattern approximately 1.5min after application (Figure 3A). The
original firing pattern did eventually resume. Meanwhile, 5-HT caused a
depolarizing effect in the LP1 burster and the LP2 beater, exciting both
to a high rate of repetitive firing (Figure 3B). Voltage clamping of the
beater demonstrated an insignificant change in inward rectification,
although the spike interval decreased from 2.75-3.Osec to 1.36sec.
Clamping of the burster when exposed to a 5-HT bath concentration of
1OUM, however, revealed a marked increase in inward rectification of
1.43nA at -90mV to .89nA at -60mV (Figure 4).
In the beating cell RP3, 5-HT application also induced a higher rate
of repetitive firing that attenuated with each successive application.
Spike interval was measured 60 seconds after administration of 5-HT in
three separate applications, perfusing between each (Figure 5).
Following the first application spike interval showed a dramatic drop
from a control of 3.5sec to 0.5sec, and thereafter a progressive increase
to 2.9sec and 3.25sec in applications two and three, respectively. Input
resistance measurements also showed a gradual increase from 12MQ to
21.3MO and 22.0MQ in applications one, two and three, respectively.
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 15.4MQ to 14.4MO, the input resistance dropped
to 10.2M2 with the combination. 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.
Evidence for synaptic input effecting this cell's behavior was
observed after the application of this peptide-amine combination. Its
manifestation is in the form of instantaneous, excitatory spurts, which
increase spike frequency whether the cell be in a bursting or beating
state (Figure 6). Interburst hyperpolarizations were not smooth, rather
irregular with EPSP's, while sharp rises to spikes instead of gradual
depolarizations were observed.
Forskolin and Chlorophenylthio-CAMP (CPT-CAMP)
1o 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 the diterpene forskolin, a known stimulator of CAMP
production in a variety of cells, including molluscan neurons (Coombs &
Thompson, 1987).
In cell LP1, forskolin inhibited bursting activity and initiated an
erratic beating pattern indicating much synaptic input (Figure 7). In
addition, doublet, triplets and trains of up to five spikes replaced the
single spikes in the original bursting condition. The effects of forskolin
were long lasting, being visible over an hour after application, even
though perfusing at a rate of 10cc per min. Input resistance
measurements reflect a drop from 7.OMQ to 5.25M0 upon application of
the diterpene. Voltage clamp was performed on the burster RP1 in the
opposite ganglion. Under these conditions, inward rectification was
found to decrease by a maximum of 39.3% at -60mV (Figure 8).
CPT-CAMP was then applied to LP1 without perfusion. Even after
30min, 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 seem to reproduce
the effects of forskolin.
Dopamine (DA)
Dopamine instantaneously inhibited all firing upon application to the
beating cell LP2 (Figure 10). The beating pattern did eventually recover.
initially firing at a slower rate and gradually returning to its original
condition.
The LP1 burster depolarized in response to DA, transforming its
bursting into a fast rate of repetitive firing. Tonic spiking activity
gradually slowed with an eventual resumption of the bursting pattern.
DISCUSSION
Individual neuropeptides and amines often exhibit varied effects on
different autorhythmic cells, even cells that display the same firing
pattern. Elucidation of the mechanisms of each particular observation
is difficult without appropriate voltage clamping experiments, but
general trends in other molluscan systems may prove analogous.
In the Aplysia bursting neuron L2, FMRF-amide activates inward
rectification (Thompson, 1988). If this were the case, one would expect
hyperpolarization of the membrane - thus, a probable explanation of
the observed inhibition in the beating LP2 cell. But we also observed a
depolarizing effect characterized by tonic spiking activity in both a
beater and a burster, RP3 and LP1. One possible explanation is a
decrease in inward rectification be responsible, or, more generally, the
opening of inward cation channels.
FMRF-amide's effect on cation channels in L4 and L6 bursting neurons
of Aplysia reveals its neurotransmitter/hormonal properties; it vields
a biphasic response in which it first activates an inward cation current
carried by Nat, then , after a longer latency activates an outward
current predominantly carried by Kt (Ruben, et al.,1986). The short
latency first observed between application and activation suggests that
the neurotransmitter binds directly to the receptor that gates the Nat
channels. The longer latency of the second response, meanwhile.
indicates that the compatible receptors are away from the site of
activation, implying a second messenger system. The particular
messenger which initiates this Kt response is uncertain; while
FMRF-amide is found to increase cAMP production in Aplysia gill tissue
(Weiss, et al., 1984), forskolin, an adenylate cyclase activator, causes
no change in the FMRF-amide response. It seems that a different second
messenger phosphorylates, thus activates, the channels; which
messenger is not clear. But perhaps the unexplained depolarizing
effects relate to the first phase of this biphasic response. If, in these
dorids, the Nat channels continued to be activated, tonic firing would be
expected. Observations of hyperpolarizing and depolarizing effects not
only amongst different autorhythmic cells, but also within individual
cells, illuminate the neurotransmitter-like properties of this peptide.
thus warranting further investigation into its properties and
mechanisms.
Serotonin produced the varied effects not uncommon of a known
neurotransmitter - both hyperpolarizing and depolarizing, depending on
the cell investigated. Depolarization observations such as those in LP1
and LP2 could be explained by noting that high concentrations of
5-HT(S1OuM) act via increasing CAMP production to increase
subthreshold Catt currents near resting potential in Aplysia R15
(Levitan & Levitan, 1988). If this were the case, we would expect that
application of a membrane permeable CAMP analog(CPT-CAMP) would
produce a similar response; after a 30min application, we observed no
effect whatsoever. So it seems that 5-HT acts via another mechanism to
open Catt or other cation channels. Levitan and Levitan continue by
stating that lower concentrations of 5-HT, such as those in the range of
10uM, tend to enhance the amplitude of the interburst hyperpolarization
in the R15 burster, acting via cAMP to increase inward rectification
that they conclude is carried by Kt. The resulting increase in K+
conductance makes K+ currents the predominant determinants of cell
activity, and pushes the resting potential down toward the K+ reversal
potential; this would hyperpolarize the membrane. Lower
concentrations were applied under voltage clamping conditions, and
indeed an increase in inward rectification was observed. No voltage
clamping of the cell after application of CPT-CAMP was performed, but
it would be appropriate to see if cAMP is responsible for the increase of
inward rectification. Finally, in the RP3 beater, we witnessed that the
depolarizing effect was reduced with successive 5-HT applications, the
cell apparently desensitizing itself to 5-HT. An increase in input
resistance accompanied the inactivation of the channels that caused the
initial gross changes; it seems each application decreased the number of
inward cation channels available to depolarize the cell. The observation
is not only interesting but also critical to experimental technique. It
demonstrates that although the gross effects of the substance may be
washed away and control conditions apparently returned, the subtle
effects underlying the observations may still affect data collection.
reinforcing the known complexity of mechanisms and receptor
properties that are responsible for our observations.
Of particular interest in studying the pharmacology of the
autorhythmic cells is the appearance of synaptic input, as was observed
after the co-application of 5-HT and FMRF-amide. The excitatory spurts
of activity could either be due to this cell's individual response to the
substances, to the cell presynaptic to it, or even a combination of the
two; elucidation of which it is is critical to our analysis for if bursting
behavior is not entirely endogenous to the cell, data collection will be
more complicated than expected. To test for synaptic input,
hyperpolarizing current could be injected which would allow EPSP's to
be observed.
Application of forskolin also resulted in a great increase in synaptic
input in the LP1 burster in addition to a tonic spiking condition. One
effect of forskolin is its ability to depolarize the cell by closing
transient Kf currents, 1 (Coombs & Thompson, 1987). If this cell has
multiple axons each with its own spike initiating zone, the resulting
lower resting potential from the accumulation of internal Kt cations
may exceed the threshold of these zones, thus accounting for the
doublets, triplets, and trains of spikes observed. Voltage clamping
revealed a decrease in inward rectification, not surprising if carried by
KTions. As la is active at subthreshold voltages, its main effect is on
spike shape and rate of firing (Connor & Stevens, 1971; Byrne, 1980), as
observed. To complete this analysis, CPT-CAMP was applied under
voltage clamping conditions, and exhibited no change in inward
rectification. Thus it seems forskolin has its effect by a different
mechanism than cAMP, contrary to some findings (see Coombs &
Thompson, 1987).
Finally, dopamine exhibited the varied effects typical of a classic
neurotransmitter. In Aplysia R15, DA decreases subthreshold Catt
currents (Lewis et al, 1984), thus hyperpolarizing the cell. This
hyperpolarization stems from the fact that in nudibranchs, both Catt and
Naf constitute the upstroke of the action potential. As Catt currents
operate mainly at subthreshold voltages, determining if the cell reaches
threshold, if the Catt current were adequately reduced, the voltage
dependent, strong Nat channels will never be able to be activated, thus
inhibiting spikes. The observed depolarizing effects in the LP1 burster
can only be partially explained by suggesting that DA probably acts via
another cation channel to depolarize the cell, for it does decrease Catt
conductance. As with serotonin, DA's possible mechanisms are as
varied as are its effects.
Clearly, this pharmacological analysis of the D. albopunctata ganglia
is far from complete (Figure 11). It is, however, evidence for this
dorid's potential for electrophysiological study. Further elaboration
into these effects and unknown mechanisms of neuroactive substances
on this organism, using both current and voltage clamping techniques,
will open a new and exciting realm in neurological study that can only
fortify our understanding of cellular physiology.
FIGURE LEGEND
Figure 1. Map of the dorsal pleural ganglia of Doriopsilla albopunctata:
Figure 2. Various effects of FMRF-amide on two autorhythmic cells. A,
FMRF-amide causes inhibition of beating in LP2 . B, FMRF-amide induces
tonic spiking in LP1 burster.
Figure 3. Various cellular responses to 5-HT. A, 5-HT inhibits beating
activity in RP2. B, 5-HT depolarizes LP2 leading to increased spike
frequency.
Figure 4. 5-HT increases inward rectification in LP1 burster.
Figure 5. Depolarizing effects of 5-HT on cell RP4: Spike interval
measurements during three successive 5-HT applications. Dotted arrows
represent 60sec following application of 5-HT. Control spike interval is
3.5sec. #1, The first 5-HT application results in 0.5 sec spike interval.
42, The second application results in a 2.9 sec interval. #3, The third
and final application results in a 3.25 sec spike interval. Note: Different
time scales used in each measurement.
Figure 6. Evidence for synaptic input in LP1 beater after application of
1:1 FMRF-amide and 5-HT. A,B, Bursts of increased frequency are
apparent during beating phase. CIrregular spike frequency within bursts
and sharp rises to spikes suggest synaptic input.
Figure 7. Multiple effects of forskolin on LP1. Forskolin induced
doublets, triplets, and quintuplets, with erratic firing pattern
characterized by much synaptic input.
Figure 8. Forskolin's effect on inward rectification.
Figure 9. CPT-CAMP shows negligible effects on inward rectification.
Unlike the plot of forskolin, this shows basically no change in inward
rectification.
Figure 10. Various cellular responses to DA. A, DA transforms RP1
bursting activity into a high rate of repetitive firing. B, DA inhibits
firing in LP2
Figure 11. Summary of autorhythmic neuron responses to applied
chemical substances.
FIGURE 1
Sarz

6
M.
Kei
2
Doriopsilla albopunctata
Pleural ganglia, dorsal view
FIGURE 2


FMRF
FMRF
FIGURE 3
Mneee
5-HT
20 V
10.
FIGURE 4

-4
-99

-89

CONTROL
QO 3-HT
k


2
-59
-79
-69
-49
—
39
FIGURE 5
5-HT
10-
4
rrtrtrtttttittrn
20 V
FIGURE 6
n

neen



+
FIGURE 7


25mV
108
FIGURE 8
—
CONTROL
O FORSKOLIN


1

8


-
-79
-69
-99
89
-59
-49
FIGURE 9
9
-2
9
-99
—
8

-89
CONTROL
OCPT-CAMP
kaa

-79
-69

-50
-39
FIGURE 10
nn
FIGURE 11
RP3
LPI
RPI
RP2
LP2
70
VAS
++
FNRE
+
——
Au
——
++
DA
ESK
++
507
0
O
AME
—
+
+
SAT
+
FNAFT
54T

indicates membrane hyperpolarization
indicates membrane depolarization
indicates no apparent response
blank implies experiment not performed
224
REFERENCES
Byrne, J.H. (1980) Analysis of ionic conductance mechanisms in motor
cells mediating inking behavior in Aplysia californica. J. Neurophysiol.
43, 630-650.
Connor, J.A. and C.F. Stevens (1971) Voltage clamp studies of transient
outward membrane current in gastropod neural somata. J.Physiol. 213.
21-30.
Coombs, J. and S.H. Thompson (1987) Forskolin's effect on transient K
current in nudibranch neurons is not reproduced by CAMP. J.Neurosci.
7(2);443-452.
Levitan, E.S. and I.B. Levitan (1988) Serotonin acting via CAMP enhances
both the hyperpolarizing and depolarizing phases of bursting pacemaker
activity in the Aplysia neuron R15. J.Neurosci. 8(4);1152-1161.
Lewis, D.V., G.B. Evans, W.A. Wilson (1984) Dopamine reduces slow
outward current and calcium influx in burst firing neuron R15 of
Aplysia. J.Neurosci 4, 3014-3020.
Ruben, P., J.W. Johnson, and S.H. Thompson (1984) Biphasic response of a
bursting neuron to FMRF-amide. Soc.Neurosci.Abstr. 10, 1116.
Ruben, P., J.W. Johnson, and S.H. Thompson (1986) Analysis of
FMRF-amide effects on Aplysia bursting neurons. J.Neurosci,
6(1);252-259.
Thompson, S.H. and P. Ruben (1988) Inward rectification in response to
FMRF-amide in Aplysia neuron L2: Interaction with transient K current,
(Unpublished).
Thompson, S.H. and S.J.Smith(1976) Depolarizing afterpotential and
burst production in molluscan pacemaker neurons. J.Neurophys. 39(1),
153-161.
Weiss, S., J.I. Goldberg, K.S. Chohan, W.K. Stell, G.I.Drummond, and K.
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Acknowledgments
Many thanks to Stuart Thompson for his guidance and patience in
directing my arduous metamorphosis toward neurobiological competence,
Invaluable, too, has been the insight and assistance of Tony Morielli and
Brett Premack, and the moral support of dorid reaper Tom Otis. To my
partner Maggie, cheers. 1 wish you good moods and the very best of luck
always.