Abstract
The shapes and frequencies of action potentials of Doriopsilla
albopunctata neuronal soma were studied in artificial sea water (ASW),
and ASW solutions that were Na -free, Caz-free with Co¬, or with
tetraethylammonium jons (TEA). Action potentials were also measured in
ASW solutions containing 67 micromolar forskolin, 1 micromolar
chlorophenylthiol CAMP (CPT CAMP), or 50 nanomolar
tetradecanoyl-phorbol-acetate (TPA).
The Nat-free, Ca2-free Co2*, and TEA solutions were used to study
action potentials missing Na’, Caz*, and K' currents respectively.
Na’-free solution caused action potentials to decrease in amplitude and
increase in width. Action potentials became slight undulations or sine
wave like. Ca2'-free solution removed repetitive firing. Potentials also
lost amplitude and gained width. TEA removed the undershoot of
hyperpolarization as well as increased the width of action potentials. he
addition of both TEA and Ca2-free Co2 solutions to cells produced and
two stage effect where the voltage fluctuated between a resting potential
and a potential 40 mV above rest.
Forskolin and CPT CAMP were used to observe the putative effects of
CAMP on action potentials. Forskolin increased action potential width and
seemed to decrease threshold voltage. CPT CAMP had no observable
effects. TPA was used to determine the putative effects of diazoglycerol.
It seemed to decrease threshold voltage in Na free solution.
These results suggest that action potential shape is determined by
varjous current components and that second messengers may affect action
potential shape by blocking or enhancing certain currents.
Introduction
It has been hupothesized that second messengers plag a role in
changing rates of ion currents in neurons thus changing action potential
shape resulting in different amounts of transmitter released from
sunaptic terminals. Klein and Kandel have suggested a model where CAMP
levels may control sensitization in Aplysia gill withdrawal response.
Accordinq to this model, serotonin binding to receptors activates
adenulate cuclase leading to an increase in cAMP levels in the presynaptic
terminal of the sensoru neuron. CAMP activates a cAMP-dependent protein
kinase which phosphorglates a voltage sensitive K* channel, inactivating
it. The subsequent decrease in K' current prolongs the time of
depolarization during action potentials, allowing more calcium jons to
enter with the result that more transmitter is released from the terminal
(1). Castellucci et al. (2) and Kaczmarek et al. (3) have injected the
catalutic subunit of bovine cAMP-dependent protein kinase into Aplusia
sensory neurons and bag cells respectivelg. Action potential height and
width were incressed. Results seem to indicate an increase in Ca¬
current and a decrease in K' current were responsible for this effect.
TPA (12-0-tetradecanoul-phorbol-13 -acetate), a tumor promoting
phorbol ester, has also been reported to increase action potential height in
Aplusia bag cells. Presumably, it acts by mimicing the effects of
diszoglucerol which activates protein kinase C. Injection of purified
protein kinase C also increases action potential height by increasing Ca¬
current (4). Ca channels may be phosphorglated by this kinase thus
bringing more channels from an inactive to an active state.
In this paper, the effects of forskolin, chlorophenglthiol cAMP, and
TPA on action potential shape in the mollusc Doriopsilla albopunctata
were studied. Forskolin is a membrane soluble diterpene which stimulates
the increase of cAMP levels by activating adenglate cyclase. It also seems
to have the effect of increasing the slow inward Na' current in molluscan
neurons which is not medisted by CAMP (5). CPT CAMP is a nonhgdolyzable
membrane permeable analog of cAMP. To try to determine what types of
currents were affected bu each of the chemicals, action potentials were
observed in solutions where Na ions were removed, Ca ions were removed,
or tetraethulammonium ions (TEA) was added. This allowed the
observation of action potentials with certain currents blocked.
Materials and Hethods
Doriopsilla albopunctata were obtained from Tom Otis who dove foi
them in the Monterey Bay area. They were kept in a tank through which
filtered sea water was continuouslg run through. The sea water was put
through a degassifier which consisted of a tube filled with plastic beads
in order to prevent gas from coming out of solution inside the animals due
to the difference in temperature between the sea and the tank.
Preparation of Brains
Doriopsilla brains were dissected by making two incisions from the
gill to each rhinophore using scissors. The flap of skin was pulled up
exposing the brain which could be easilg identified because of its orange
color, two black eues that were attached to it, and because of its position
between and slightlu posterior to the rhinophores. The brain and
surrounding muscle tissue were removed using fine scissors and pinned to
a petri dish with a silgard base. They were then rinsed in an artificial sea
water solution (ASW) (see Table 1). Excess tissue such as salivarg gland
tissue was removed using fine forceps. Next, the brain was treated with a
small amount of dispase, covered with a small piece of kimwipe to
preserve moisture and incubated for 1 hour at 4 degrees centigrade. he
dispsse was rinsed off and the brain was incubsted for at lesst 2 hours in
artificial sea water also at 4 degrees centigrade. This procedure loosened
the epineurial sheath surrounding the ganglions. The sheath was later torn
off using fine forceps.
Intracellular Recordings
Recordings were done on either the three large cells of the pedal
ganglions or the large posterior cells in the pleural ganglions. Iwo
electrodes filled with 3 molar KCI with resistances of between 2 and 15
megachms were inserted into the cells using manipulators. The electodes
were connected to amplifiers. One electrode was used to regulate current,
while the second was used to record voltage. Current and voltage were
monitored using an oscilloscope, and a computer was used to program
current pulses. During the recording procedure, the brain was pinned in a
perfusion dish on a cooling plate at about 15 degrees centigrade. The
perfusion dish was designed so that the bathing solution could be changed
while keeping the brain totally immersed with minimal damage to the
cells. The insertion of the electrodes was done under a light microscope
at a magnification of 500 times.
Bathing Solutions
Na'-free, Ca-free Co-*, and TEA solutions are listed in Table 1.
Half Ca2-free Co*, half TEA solution was made by mixing equal volumes
of Ca2-free Co* and TEA solutions. TPA was made in a 50 micromolar
stock solution in dimethul sulfoxide (DHSO) and added to the cells so that
the final concentration was 50 nanomolar. Forskolin was stored as a 10
millimolar stock in DHSO and used at a final concentration of 67
micromolar. Stock CPT CAMP was 1 millimolar in distilled water. It was
used at a final concentration of 1 micromolar. Solutions were added to the
cells by perfusion. Chemicals were added directly into the bathing
solution and wash out by perfusion.
Results
Figure 1. shows control action potentials in normal artificial sea
water. Cells had resting potentials between -20 and -50 mV. The peaks of
action potentials were between 130 and 150 mV. Cells fired repetitivelu
when stimulated by long current injections.
In Na -free solution, the resting potentials consistently became
more negative to approximately -70 mV. During the replacement of ASW
with Na -free, spike frequency decreased, spike height decreased, and
spike width incressed. The rate of voltage increase of the rising phase of
the action potential decreased and the rate of voltage decrease of the
falling phase decreased. When perfusion was completed, repetitive firing
was either abolished after the first action potential or verg slight
undulations followed the first action potential. Figure 2. shows action
potentjals in Na’-free solution. The upper traces show an action potential
during perfusion, and the lower traces show an action potential after
perfusion was complete. These effects were all reversible by washing
with ASW.
The effects of Ca+-free Co* solution is shown in Figure 3. Spike
height was decreased and spike width increased although not as much as in
Na'-free solution. Repetitive firing was decreased with a long current
pulse causing two spikes, the second much shorter and wider than the
first, and then a flat line.
TEA caused a major incresse in the width of action potentials.
Action potential width at half maximal height changed from control values
of approximatelu 12 mS to approximately 180 mS. Resting potential
became more positive by approximately 5 mV, and peak action potential
height decressed slightlg. The huperpolarization at the end of a spike was
abolished (see Figure 4.).
When the TEA solution and Ca--free Co* solution were mixed, an
interesting effect was observed in besting cells, shown in Figure 5.
Following a spike, a cell never huperpolarized back to resting potential but
huperpolarized to a voltage approximately 40 mV obove rest. A verg weck

ad f.
dimdfld
second spike was obser veu 10n0weu ug à sever di seconu fiat intei vai
during which the voltage was slowlg but consistently decreasing. After
reaching a certain voltage, slight action potential like spikes began
appearing growing in size (both action potential peak voltage was rising
and lowest voltage was becoming more negative). Äfter these potentials
resched a certain height, voltage returned to resting potential. This
process was repeated in a beater like fashion. At resting potential, a
definite membrane resistance could be measured but during the time that
the cell was at the 40 mV above the resting stage, membrane resistance,
as messured by injecting a huperpolarizing current, decreased to the
extent that a 1 nA injected current had an immeasurable effect on voltage.
The addition of ASW to the TEA/Ca-free Co
2 solution decreased the
amount of time spent in the higher voltage state. It could also abolish the
higher voltage state totally and insteed, make the cell fire repetitivelg for
a short interval followed bu an interval where the cell did not fire
spontaneously similar to a bursting pacemaker cell.
Forskolin increased resting potential (ie. from -22 to -12.9 mV) and
in some cases may have increased action potential frequency. Action
potential width at half meximal height was increased approximatelg 300
percent. Observations were made where low current pulses (ie. 1 nA) did
not change voltage in control (ASW) cells while higher current pulses (ie. 5
né) produced action potentials and repetitive firing if maintained. Atter
cells were treated with forskolin, the lower voltage produced action
potentials as well as repetitive firing if maintained while the higher
current pulses produced a weaker action potential followed by inhibition
of firing. This effect was observed in all four trials but the results mag
be misleadinq since in all cases the same current pulses were used
regardless of resting potential height. Since forskolin raised action
potential levels, injecting equivalent currents mag not have resulted in
same absolute voltages compared to controls. Only in one case was the
cell huperpolarized to its previous resting potential before being tested.
This cell acted in the same way as the others.
Forskolin consistently increased the width at half maximal height of
action potentials: from 10 mS to 30 mS. It consistently caused a décrease
in height of action potentials: from 85 mY from peak to bottom of
huperpolarization to 65 mV, and in one out of four trials, it noticeably
increased action potential frequency during a stimulus.
CPI CAMP was added in concentrations of 1 micromolar. At this
concentration, no effects were observed measuring action potential
frequencu, height, or width. Resistance of the cell also did not change.
Four experiments were done with CPT CAMP. All gielded the same results.
Adding TPA at a concentration of 50 nH to cells in ASW produced no
noticeable effect on action potential frequency and no consisted etfects on
action potential height or width. Adding TPA to cells in Na'-free sea
water stimulated action potential production over control levels at low
voltages. Cells in Na’-free sea water had resting potentials of
approximatelu -70 mV. At this potential, pulses of up to 5 nA produced no
action potentials in control cells. Cells treated with TPA however,
produced low frequency firing as a result of these pulses. When cells were
depolarized to higher resting potentials, ie. the resting potential of the
cell before addition of Na’-free solution (appoximatelg -50 mV), control
cells and TPA treated cells both produced similar numbers and frequencies
of action potentials (see figure 8.).
Discussion
It has been observed that in molluscan cell soma, the major currents
involved during action potentials are carried by Na“, Ca2*, and K* ions (6).
Hat and Ca2t currents are necessarg for the rising phase as Na“ and Laz-
jons enter the cell and Kt ions leaving the cell account for the subsequent
huperpolarization during the potential. The experiments in which Nar¬
free. Ca2t-free, and TEA sea water were used to observe action potentials
in the selective absence of these ions seems to verify this. In all cases,
action potential shape was changed drastically. In Na“-free solution, the
sine wave like undulations indicate that Ca2* current has a slower
activation and a slower deactivation than Hat current. The repetitions of
the undulations seems to indicate that in these cells, Ca¬* current may be
necessary for repetitive firing. In contrast, with the Cazt free solution,
Nat current turned on rapidly and deactivated rapidlg. The difference in
the time it takes from the peak of the action potential to the
repolarization is different. Since K* current should be the same in both
cases, this difference can be accounted for by the rapid deectivation of the
Nat channels. In Ca2t-free solution, there are one or two sharp action
potentials followed by a flat line. This seems to indicate that Ha“ current
bu itself cannot maintain repetitive firing. A verg simplified model of an
action potential would be that the Na* current plays a major role in the
quick upward and downward slopes of the spikes while Caz* current plags
a major role in the repetition of the spikes.
The summation of the voltages during a spike in Na“-free and a spike
in Ca2t-free solutions does not result in the shape of a "normal“ action
potential in ASW. This indicates that another factor is necessarg for
achieving action potential shape. This factor mag be the negative or
positive feedback that the currents experience as a result of the change in
potential. As the voltage increases, the probability or a Cazt or Na
channel being open also increases. Ädding the spikes observed in Na“-free
and Ca2t-free solutions increases the voltage which in turn changes Na
and Ca2t currents. To obtain a normal action potential shape by the
addition of Nat and Ca2* currents, these currents should be measured at
voltages ranging from resting potential to maximum spike height. As the
Nat-free curves and the Ca2t-free curves are added to form a higher
voltage, the Nat and Ca2* curves for the new voltage should be used to
continue
Tetraethul ammonium ions block K* current. The addition of IEA
decreased the rate of hyperpolarization during an action potential as
would be expected if K* current was hindered. The slowed
huperpolarization that remained was probebly caused by the inactivation
of Nat and Ca2t current. The undershoot at the end of an action potential
was abolished. This also was expected since this is caused by increased
Ktcurrent. The slight lowering of the peak of the action potential was not
expected and does not seem to be a result of the direct action of the K*
ions. It can be explained indirectly by the result that adding TEA reduces
some resting Kt current resulting in an increased resting potential (a 5
my increase in resting potential was observed when cells were in TEA
solution). This increased resting potential mag prevent some of the Na
channels from going to the closed state from the inactive state (Na¬
channels need a huperpolarized voltage to go the the closed state from the
inactive state). Having more Nat channels in the inactive state would
prevent an action potential from acheiving maximum height. Also, the
absence of the huperpolarization mag have the same etfect.
The addition of the combination of TEA solution and Ca2*-frée Coz*
solution produced an interesting effect in beating cells. The cell seems to
be in an unstable equilibrium between two states: the resting state and a
state approximatelu 40 mV above the resting state. The resting state can
be explained bu the permeability of the membrane to potassium ions.
Although TEA blocks some Kt channels, mang K* channels necessarg for the
maintenance of the resting potential are not effected by TEA. Also, in the
combination solution used in this case, the TEA has been diluted bg Lazr¬
free and ASW solutions. The state in which the cell is at a potential 40
my above resting can be explained by the opening of Ca2* channels. This is
unexpected since the presence of Ca2t-free Co would be expected to block
Ca2t channels. However, the fact that the resistance of the cell at the 40
my above resting state is verg low compared to the resistance at resting
shows that this effect cannot be caused solely by a decrease in K
conductance. An increase in Nat conductance would explain this
phenomenon except that voltage gated Nat channels would be expected to
inactivate instead of staging open so long. Therefore, this second state
seems to be explained best by a state achieved by the equilibrium of the
conductances of both Kt channels and Ca2* channels, both of which have
been decreased by TEA and Ca2+-free Co.
Since the cell originally was a beater, resting potential is inherentig
unstable. Slow inward current or B current probablg depolarizes the cell
past threshold voltage. The first two spikes can be attributed to Na“
current and inactivation. However, since voltage dependent K* current is
inhibited to an extent by TEA, the voltage does not return to resting
potential but reaches this second state where the equilibrium is dependent
on a balance between Ca2t and Kt current. At this point, Na“ current is
blocked since the Nat channels are trapped in the inactive state by the
depolarization. This second state is not stable since the voltage has been
observed to decrease steadily. This decrease can be explained by factors
such as the Nat/Kt pump, slow Ca2* current inactivation, and an increase
in Ca2t dependent K* current as Ca2* concentration rises in the cells. As
voltage decreases, more and more Na“ channels are transferred from the
inactive to the closed state. Since voltage is above threshold at this
stage, these Nat channels open and close causing fluctuations that
increase in amplitude as voltage decreases. During this time, Cazt current
continues to inactivate until Kt current overwhelms it and brings it back
to resting potential. B current brings the voltage back to threshold again
causing the cycle to repeat.
The time spent in the second state is longer than the 300 ms that the
cell is above restinq potential when it is in a TEA solution. This indicates
that some other factor besides the decrease in K* current and the
inactivation of Ca2t current is involved in the cell remaining in the
second state. It may be that Co2t has an effect of leaving some Ca¬
channels open longer or blocking a different subset of K* channels than
TEA does, although both of these hypotheses are speculation.
Forskolin was expected to behave in a wag similar to TEA. It was
expected to lead to increases in cAMP dependent protein kinase which
would phosphorglate voltage dependent K* channels thus blocking them,
leading to a result similar to that observed when TEA is added. The
increase in resting potential observed when forskolin was added is similar
to the effect observed in TEA solution and mag be the result of decreased
Kt conductance. The increase in action potential width also agrees with
the possibilitu that K* channels were blocked. The decrease in injected
current needed to produce cell spiking mag imply that forskolin lowers the
threshold voltage of cells. However, it mag also be an artifact resulting
from the incresse in resting potential of the cell when exposed to the drug.
Hore experiments must be done to confirm which of the two is correct. In
the literature, there is no mention of forskolin affecting threshold
voltages of cells.
Since forskolin has been observed to have effects other than those
mediated bu CAMP, such as changes in A current or transient K* current,
chlorophenulthiol CAMP, a membrane permeable cAMP analog, was used to
tru to duplicate its effects and separate which effects were CAMP
mediated and which were not. 1 micromolar concentrations were used and
no changes in spike height, width, or frequency were observed. It is
possible that changing CAMP levels has no effect on action potential shape
or frequency in Doriopsilla although this seems verg unlikelg, since cAHF
is such a ubiquitous second messenger and has been shown to affect spike
shape in Aplusia neurons. The more likelg possibilitg is that 1 nH
concentrations of CPT CAMP are too low to cause an effect. Higher
concentrations should be used for further experimentation.
14
The effects of TPA in Nat-free sea water indicate that TPA lowers
the threshold voltage of a cell. Spiking occurs in the presence of TPA at
voltages where in its absence, it does not occur. However, at voltages
where spiking occurs in both, frequency remains unchanged. In ASW,
observations were only made at voltages where spiking occurred in both
cases. Threshold was not measured so further experiments will have to be
done to see if the lowering of threshold voltages occurs in ASW as well as
Nat-free sea water. An incresse in spike height and width such as has
been observed in Aplusia neurons was not observed. A possible explanation
for the decreased threshold voltage observed in Nat-free solution with
IPA is that TPA activates protein kinase C which phosphorglates Caz
channels bringing them from the inactive to closed state. This increases
the number of Ca2t channels that may be opened. Although the probability
of ang one channel being open at a particular voltage mag not change, since
there are more channels available, more of them will be open at a
particular voltage thus lowering threshold, similar to the idea that the
threshold voltage is lower at the spike initiating zone because there is a
higher concentration of Nat channels there.
Conclusions
Although the results of this experiment are far from conclusive, it
seems likelu, from the current literature, and from some results here, that
second messengers do have an effect on action potential shape, frequencg,
or threshold. Neurons do not just passivelg transmit action potentials. Il
this were the case, learning and memorg would not occur. Neurons must
integrate and modifu the information that is transmitted to them. Second
messengers, among other things, seem to plag a role in this moditication.
Using second messengers, models for simple forms of learning, such as the
Klein and Kandel model for Aplusia sensitization, can be made. Therefore,
the effects that second messengers have on neurons should be fürther
studied.
Acknowledgements
would like to thank Dr. Stuart Thompson for his helpful advice and
ideas and for teaching me the techniques involved in Doriopsilla dissection
and intracellular recording. Also, I would like to thank Drs Baxter, Epel,
Swezeu, and Dennu, the postdoctoral fellows and graduate student in the
Thompson lab, and Tiffanu Tom for their advice and concern, and Tom Utis
for the Dorids.
Figure Legends
Figure 1. Action potentials in ASW. Upper trace is injected current; lower
trace is voltage.
Figure 2. Action potentials in Na free sea water. The upper two traces are
input current and voltage measured during perfusion to replace ASW
with Na free sea water. The lower traces are input current and
voltage measured after perfusion was complete.
Figure 3. Effects of Ca free Co sea water on action potentials. The upper
two sets of current and voltage traces show input current and action
potentials in ASW. The lower sets show input current and action
potentials in Ca free Co solution.
Figure 4. Action potential in TEA containing sea water. Upper trace is
current; lower trace is voltage.
Figure 5. Action potentials in a mixture of Ca free Co sea water and sea
water containing TEA. The upper two voltage traces show the two
different states that the voltage fluctuated between: resting voltage,
and 40 mV above resting. The lowest voltage trace shows how
diluting the Ca free Co and TEA mixture with ASW results in a burster
like activity of the cell.
Figure 6. Effect of forskolin on action potentials. The upper two traces
are current and voltage in ASW. The lower two are current and
voltage in ASW with forskolin added.
Figure 7. The effect of TPA in Na free sea water with resting voltage at -
70 mV. The upper two traces are current and voltage in Na free
solution without TPA. The lower two traces are current and voltage
in Na free solution with TPA.
Figure 8. The effect of TPA in Na free sea water with resting voltage
depolarized to -50 mV. Upper two traces are in Na free solution -
TPA; lower two are in Na free solution +TPA.
18
gure 1.
19
Figure 2.
20
Figure 3.
Figure 4.
Figure 5.
23
Figure 6.
24
Figure 7.
Figure 8.
26
Nacl
KCI
Caci
rigc12
Hepes
Tris
CoCl2
TEA-CI
Table 1. Composition of bathing solutions in millimolars
A5W
Na free
La free C0
TEA
470
370
470
10
10
10
10
50
50
10
470
100
References
1. Kandel, E. R., Schwartz, J. H., Principles of Neuroscience Elsevier Second
Edition (1985).
2. Castellucci, V. F., Kandel, E. R., Schwartz, J. H., Wilson, F. D., Nairn, A. C.,
and Greengard, P., Proc. Natl. Acad. Sci. USA. 77, 7492-7496 (1980).
3. Kaczmarek, L. K., Jennings, K. R., Strumwasser, F., Nairn, A. C., Walter, U.,
Wilson, F. D., and Greengard, P., Proc. Natl. Acad. Sci. USA 77, 7487¬
7491 (1980).
4. DeRiemer, S. A., Strong, J. A., Albert, K. A., Greengard, P., and Kaczmarek,
L.K., Nature 313, 313-316 (1985).
5. Coombs, J., Thompson, S. H., J. Neurosci. 7, 443-452 (1987).
6. Adams, D. J., Smith, S. J., Thompson, S. H., Ann. Rev. Neurosci. 3, 141-
167 (1980).