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The Journal of Neuroscience, June 15, 2001, 21(12):4523-4529
Dual and Opposing Modulatory Effects of Serotonin on Crayfish
Lateral Giant Escape Command Neurons
Terri
Teshiba1,
Ashkan
Shamsian1,
Bahram
Yashar1,
Shih-Rung
Yeh2,
Donald H.
Edwards3, and
Franklin B.
Krasne1
1 Department of Psychology, University of California,
Los Angeles, Los Angeles, California 90095, 2 Institute of
Life Sciences, National Tsing Hua University, 30013 Taiwan, Republic of
China, and 3 Department of Biology, Georgia State
University, Atlanta, Georgia 30302-4010
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ABSTRACT |
Serotonin modulates afferent synaptic transmission to the lateral
giant neurons of crayfish, which are command neurons for escape
behavior. Low concentrations, or high concentrations reached gradually,
are facilitatory, whereas high concentrations reached rapidly are
inhibitory. The modulatory effects rapidly reverse after brief periods
of application, whereas longer periods of application are followed by
facilitation that persists for hours. These effects of serotonin can be
reproduced by models that involve multiple interacting intracellular
signaling systems that are each stimulated by serotonin. The dependence
of the neuromodulatory effect on dose, rate, and duration of modulator
application may be relevant to understanding the effects of natural
neuromodulation on behavior and cognition and to the design of drug therapies.
Key words:
neuromodulation; crayfish; lateral giant; serotonin; facilitation; inhibition; escape behavior
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INTRODUCTION |
Serotonin, octopamine, and GABA are
all known to be present in crayfish and/or lobster nerve cord (Kravitz
et al., 1976
; Livingstone et al., 1980; Beltz and Kravitz, 1983;
Glanzman and Krasne, 1986; Mulloney and Hall, 1990; Schneider et al.,
1993) and can modulate the response of the crayfish lateral giant
escape command neurons (LGs) to excitatory synaptic input. As a result,
they regulate the excitability of escape (Glanzman and Krasne, 1983; Vu
and Krasne, 1993; Yeh et al., 1996, 1997). As far as is known, the effect of GABA is always to inhibit and that of octopamine is to
facilitate. However, the effect of 5-HT appears not to be fixed but to
vary as a function of the animals' past social experience. When 5-HT
is applied to the nerve cord of crayfish that have lived for several
weeks as social subordinates, its effect is to inhibit EPSPs produced
in the LGs by sensory activity, whereas 5-HT exposure to the cord of
social dominants usually causes a facilitation of EPSP amplitude (Yeh
et al., 1996, 1997). In social isolates, the picture is less clear,
with different laboratories obtaining opposite effects (Glanzman and
Krasne, 1983; Vu and Krasne, 1993; Yeh et al., 1996, 1997).
We report here that the LGs of social isolates have machinery for both
serotonergic facilitation and inhibition and that the modulatory effect
produced depends in complex ways on the dose and schedule of 5-HT
exposure. These dependencies account for the opposing effects of
serotonin obtained in different laboratories. We show that these
complex dependencies could result from serotonergic activation of
parallel and possibly interacting intracellular signaling cascades.
However, whatever their mechanisms, variations in the qualitative form
of aminergic neuromodulation as a function of modulator dose, onset
characteristics, and exposure duration may have important
consequences for pharmacological therapies and ultimately be relevant
to comprehending the ways in which neuromodulators are used naturally.
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MATERIALS AND METHODS |
Animals. Experiments were performed on either adult
Procambarus clarkii measuring 8-9 cm rostrum to telson or
on juveniles 2-3 cm in length. Crayfish were obtained from Atchalafaya
Biological Supply (Raceland, LA) and kept isolated in 14 × 20 cm polyethylene dishes for at least 2 weeks before experimentation.
Adults and juveniles gave similar results, and their data were pooled.
We avoided use of animals kept in the lab for many months because we
believe that they have diminished responsiveness to applied 5-HT.
Experimental procedures. Animals were cooled gradually to
5°C, the abdomen was pinned on Sylgard in crayfish saline, and the nerve cord was exposed dorsally as in Krasne (1969). Thereafter, experiments were at ~20°C. KCl-filled (3 M), ~10 M
recording electrodes were
placed in proximal dendrites of LGs, usually in the sixth abdominal
ganglion but in the third or fourth in a few experiments, and
platinum wire stimulating electrodes were placed on roots 2-4
(root 2 in third and fourth ganglion experiments). Test EPSPs were
evoked every 2 min by a 0.2 msec voltage pulse at stimulating
electrodes. Exposure to 5-HT was begun when the amplitude of evoked
EPSPs, which often drifted up or down after dissection and penetration,
stabilized (usually ~30 min). Electrical activity was stored
digitally for later analysis.
5-HT application. 5-HT was applied by superfusion in
juveniles and by arterial perfusion (as in Mulloney et al., 1987) in adults. LOW concentration was 10
8
M (perfusion) or 5 × 106 M
(superfusion), and HIGH concentration was 5 × 105 M (perfusion)
or 104 M
(superfusion). Exposure periods were either SHORT (10 min) or LONG (45 min or 40-60 min in initial experiments). For FAST applications, 5-HT was stepped to full concentration as rapidly as
possible (within a few minutes). For SLOW applications, it was dripped
so that full concentration was reached in ~20-30 min (assessed by
replacing 5-HT solutions with salt solutions and measuring
conductance). 5-HT creatinine sulfate crystals (Research Biochemicals,
Natick, MA) were stored with desiccant in a freezer (for not
more than a few months) and dissolved just before use in 4 mM HEPES-buffered crayfish Ringer's solution, pH
7.2.
Data analysis. The EPSPs evoked by stimulation of ganglionic
roots generally begin with an early depolarizing peak (
) caused by
arrival of monosynaptic input and a slightly later peak (
) produced
by disynaptic input (Fig.
1A,B).
Subsequent portions of the response reflect a mixture of polysynaptic
excitatory and inhibitory inputs that follow the stimulus. In analyzing
our data, we measured membrane potential at the times at which and
peaks were located at the beginning of the experiment, even when
these peaks subsequently shifted somewhat. In some cases, the peak was not unimodal. We then usually measured the amplitude of the earlier
peak. For graphing, EPSP amplitudes were normalized to their value on
the last control EPSP before introduction of 5-HT. Although we
attempted to set initial EPSP levels so that LG spikes, which
interfered with measurement of EPSP amplitudes, would be unlikely,
sometimes serotonin increased EPSPs enough to cause spikes. In these
cases, we entered into analyses an EPSP amplitude 20% higher than the
largest EPSP amplitude of the experiment that was not contaminated by a
spike. Mean EPSP amplitudes were calculated for the last 5 pre-serotonin trials, the last 5 serotonin trials (the last three in
the case of the SHORT condition), and wash trials 15-19 (30 min after
the start of serotonin washout). Statistical analyses were on
differences between the mean EPSP amplitude at the time point of
interest and the pre-serotonin mean. Statistical tests were two-tailed
t tests.
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Figure 1.
Effect of 5-HT on synaptic potentials
evoked in LG by 0.2 msec root shocks. A, Schematic of
afferent portion of LG circuit. Chemical synapses (Chem
excite) of sensory neurons on interneurons and rectifying
electrical synapses (Rect elect) of sensory neurons and
interneurons on LGs are indicated, as are the recording site at the LG
axon initial segment and the soma of the unipolar LG. The monosynaptic
innervation of LG causes an initial () depolarization, followed by
another depolarization () attributable to the disynaptic pathway;
action potentials can be triggered by or sometimes but not
later parts of the EPSP, which reflects a complex mix of excitation and
depolarizing inhibition. B, Time plots of normalized (open circles) and (filled
circles) component amplitudes for illustrative experiments
under each of the indicated conditions of 5-HT delivery. 5-HT exposure
as indicated by solid bars, with the width of the
bar approximately indicating 5-HT concentration. Test
stimuli were at 2 min intervals. To the right of each
graph are sample synaptic potentials recorded before 5-HT application
(thin continuous lines), at the end of 5-HT application
(solid bold lines), and after 30 min of 5-HT washout
(dashed bold lines).
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RESULTS |
The LG receives converging synaptic input from primary
mechanosensory afferents and mechanosensory interneurons (Fig.
1A) that produce monosynaptic () and disynaptic
() EPSPs, respectively (Fig. 1B) (Krasne, 1969);
LG spikes and reflexive tail flips are usually produced only when the
peak exceeds firing level (Krasne, 1969; Olson and Krasne, 1981),
although under some circumstances the component can also become
large enough to trigger spikes and behavior (Edwards et al., 1994).
The effect of rapid application of 5-HT (maximum level reached within
1-2 min) for 10 min or less at a high concentration (the
FAST-SHORT-HIGH condition; see Materials and Methods) is shown in
Figure 1B1 for an illustrative
preparation. Both the and components of the LG EPSP declined
during the 5-HT but were restored to their baseline values after
several minutes of 5-HT washout. This same pattern can be seen to hold
for the averaged normalized component in Figure
2, which plots the time course of 5-HT
effects for all conditions, superimposed to facilitate comparison.
Figure 3A shows mean EPSP
amplitudes at the end of 5-HT exposure and after 30 min of washout. The
decline of the component is statistically reliable at the end of
the 5-HT exposure (p < 0.05) but not
significantly different from baseline after 30 min of wash. As in
several conditions of this study, the effects of 5-HT on the component paralleled those on the component but were not
statistically reliable. However, in two previous studies in which 5-HT
exposures comparable with those of the FAST-SHORT-HIGH conditions
were examined (Glanzman and Krasne, 1983; Vu and Krasne 1993), the
decline of the component amplitude, although generally smaller than
that of the component, was reliable. Moreover, if the data
from the FAST-SHORT-HIGH condition are combined with the identically
obtained data from the first 10 min of 5-HT exposure in the
FAST-LONG-HIGH condition, the mean percentage decline of the component at the end of 10 min 5-HT exposure does become statistically
significant (p < 0.05).
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Figure 2.
Group mean time courses of component EPSP
amplitude during and after 5-HT exposure. component amplitudes were
normalized by their values on the last stimulation before the start of
5-HT exposure. Exposure began at the dashed vertical
line. The short vertical line segments at ~45
min indicate omission of a few time points for those preparations that
were exposed to 5-HT for >40 min. The EPSP amplitude on the last trial
in 5-HT is taken as the 0 time point for the washout graphs.
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Figure 3.
Group means and SEs of the percentage
change in EPSP amplitudes at the end of 5-HT exposure and after 30 min
of wash for each regimen of 5-HT application. Significance levels are
indicated by asterisks: *p < 0.05;
**p < 0.01. Numbers of cases in each condition
(n) are shown near the
x-axis.
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Very different results were obtained when the same concentration of
serotonin was applied much more slowly, so that the modulator reached
its maximum level only after 20-30 min (SLOW-LONG-HIGH condition).
Under this condition, facilitation rather than inhibition of both LG
EPSP components developed, and the facilitation became persistent, long
outlasting removal of the serotonin (Figs.
1B2, 2, 3). In both Figures 1 and 2, the component amplitude appears to fall during the latter half of the
period of 5-HT exposure and to rise again during wash. This was seen in
half of the preparations, but it was not statistically reliable across
the full population. The component changes, which were in the same
direction as the statistically reliable component changes, were not
statistically reliable (Fig. 3B). However, because increases
in the component in comparable previous work (Yeh et al., 1996,
1997) were statistically reliable, we believe that the increases,
although insignificant here, are real.
These findings for the FAST-SHORT-HIGH and SLOW-LONG-HIGH
conditions duplicate the opposing results of previous studies (Glanzman and Krasne, 1983; Vu and Krasne, 1993; Yeh et al., 1996, 1997) and
define the circumstances under which they are obtained.
With slow 5-HT application, EPSP facilitation began when the
gradually rising concentration of 5-HT was still quite low
(consistent with Yeh et al., 1997). This suggested the possibility that
the concentration threshold of facilitation was lower than that needed to cause EPSP reduction. We thus examined the effects of rapid application of a low concentration of 5-HT that was maintained for
45-60 min (FAST-LONG-LOW condition; see Materials and Methods). This
procedure produced prompt facilitation of the LG EPSP instead of
inhibition, and the facilitation persisted after washout of 5-HT, as in
the response to the SLOW-LONG-HIGH application (Figs. 1B4, 2, 3). The increases of both
the and components at the end of 5-HT application and after 30 min of wash were statistically significant (Fig. 3D).
Because persistent facilitation was seen during washout in both the
SLOW-LONG-HIGH and the FAST-LONG-LOW
conditions, but not in the FAST-SHORT-HIGH condition, we
examined the effect of FAST-LONG-HIGH exposures.
Consistent with the findings for the FAST-SHORT-HIGH condition, the initial effect of the 5-HT was inhibitory. This inhibition persisted throughout the ~45 min period of 5-HT exposure (Figs. 1B3, 2, 3C; both
and component reductions statistically significant). However,
when the 5-HT was washed out, the inhibition gave way to a persistent
facilitation (Figs. 1B3, 2). This
facilitation was statistically reliable for the but not the component (Fig. 3C). Thus, persistent facilitation occurred
in the aftermath of all LONG exposures, regardless of the initial
effect of serotonin (Figs.
1B2-B4; 2, WASH curves; 3B-D, open bars).
In many of the present experiments, facilitation persisted for >1 hr
without decline, and in a previous report using slow application (Yeh et al., 1997), facilitation of both and were routinely seen to
persist throughout 1 hr of wash and in some cases seen to persist unabated during 5 hr of wash.
The LGs became depolarized during exposure to 5-HT in all conditions
and all experiments and by approximately the same amount in each
condition (mean ± SD, 2.7 ± 1.3). Depolarization
also occurred during facilitation in Yeh et al. (1997) and during
inhibition produced by exposures, comparable with our FAST, HIGH
conditions (Vu and Krasne, 1993).
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DISCUSSION |
These experiments have uncovered two distinct forms of
serotonergic modulation affecting the LGs in socially isolated
crayfish, a facilitation that is produced by low concentrations of 5-HT ("low-threshold facilitation") and an inhibition that is produced by high 5-HT concentrations ("high-threshold inhibition"), both of
which are associated with depolarization of the LGs.
The fact that 5-HT effects were generally more pronounced for the component than for the monosynaptic component might indicate that
the effects of 5-HT on the component are in part attributable to modulation of the first-order synapse of the
disynaptic pathway. However, inhibition of the component was
statistically significant at both 10 and 45-60 min after rapid
introduction of a high concentration of 5-HT, and facilitation of the
monosynaptic response was statistically reliable during exposure to low
concentrations of 5-HT. Reliable facilitation and inhibition of
monosynaptic () input to the LGs have also been seen in previous
studies (Glanzman and Krasne, 1983; Vu and Krasne, 1993; Yeh et
al., 1996, 1997). Additionally, the fact that 5-HT altered the membrane
potential of the LGs in both this and previous studies and that LG
input conductance increased in association with the inhibition (Vu and Krasne, 1993) and decreased in association with the facilitation (Yeh
et al., 1997) supports the view that both facilitatory and inhibitory
effects of 5-HT in isolates are attributable at least in part to
modulation directly at the level of the LG and/or synapses directly on it.
The same SLOW-LONG-HIGH application of serotonin that here and in
previous studies (Yeh et al., 1996, 1997) produced facilitation in
social isolates produces inhibition of the response of the LG in
social subordinates (Yeh et al., 1997). Because the inhibition studied
in subordinates, unlike the high-threshold inhibition studied here, is
associated with LG hyperpolarization, it appears to be a distinct type
of serotonergic modulation of LG, different from either of those
observed here.
The present experiments also add to the complexity of serotonergic
modulation of LG in that directly opposite effects of serotonin are
obtained not only in animals with different social histories but also
in animals of a given history when different patterns of serotonin
application are used. It remains to be determined whether the effect of
serotonin on the LGs in subordinates is also sensitive to the pattern
of application.
Possible mechanisms
The cellular mechanisms that cause the modulatory effects of
serotonin to depend on the concentration, application rate, and duration of exposure are unknown, but some preliminary conclusions may
be drawn. The results shown here suggest that 5-HT exposure can
activate two parallel intracellular signaling pathways, a lower-threshold facilitatory pathway, and a higher-threshold inhibitory pathway. These pathways might be activated by either different 5-HT
receptors or different levels of a common initial second messenger (as
in Artola and Singer, 1993; Lisman, 1994; Bear, 1995).
The production of inhibition when higher concentrations of 5-HT are
introduced rapidly but facilitation when the same concentrations are
reached slowly suggests that intracellular signals generating one form
of modulation may interfere with early steps involved in producing the
other, as shown in Figure
4A1.
If such mutual suppression operated, then whichever modulatory pathway
became active first would prevent development of the other form of
modulation and, thus, would dominate. With gradual application of 5-HT,
the lower-threshold facilitatory pathway would dominate because it would be engaged first. On the other hand, if the high-threshold inhibitory pathway had a faster response time than the facilitatory signaling pathway, then sudden increases of 5-HT concentration to high
levels would cause inhibition rather than facilitation. This is by no
means the only possible explanation of the application rate sensitivity
we observed, but it was the most plausible one we could think of.
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Figure 4.
Illustrative explanations of key data features.
Here and in Figure 5, boxes indicate activated
receptors, activated G-proteins, second messengers, or activated
enzymes such as kinases. Lines with
arrowheads indicate stimulation of production of the
targeted factor, possibly via a number of unspecified steps.
Filled circles indicate processes that cause breakdown
or inactivation of a factor. Open circles indicate
inhibition of a process. Open arrows indicate
usage-produced decline in the efficacy of a process. A,
Possible mechanism for dependence on rate of 5-HT application (see
Discussion).  LO, Low-threshold
facilitatory pathway; HI, high-threshold
inhibitory pathway (see Discussion). B, Possible
mechanism for 5-HT duration-dependent persistence of facilitation (see
Discussion).
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Serotonergic facilitation that lasts for hours after prolonged but not
after brief 5-HT exposures is reminiscent of a similar effect at
sensorimotor synapses of Aplysia. There, hours long facilitation can result from either translation-dependent (but transcription-independent) processes (Ghirardi et al., 1995;
Mauelshagen et al., 1996, 1998) or entirely post-translational
processes (Ghirardi et al., 1995). The exact mechanisms operating
either in Aplysia or here are unknown, and there are many
possibilities. For example, such properties can in principle arise from
mutual facilitation between signaling pathways, as
illustrated by modeling of the interactive properties of
mitogen-activated protein (MAP) kinase and protein kinase C
(Bhalla and Iyengar, 1999). Another possibility is that the
process that normally destroys facilitatory precursors gradually
inactivates and allows the remaining precursors and resultant
facilitation to persist, as shown in Figure 4B.
The mechanisms by which G-protein-coupled receptors adapt
(Ferguson and Caron, 1998; Lefkowitz, 1998) provides a possible
mechanism, although at a shorter time scale, for such inactivation.
Ability of mechanisms to account for findings
To show that signaling pathway interactions really could cause the
phenomena we observed, we constructed a specific computational model
(Fig. 5A) that combined the
mutual suppression and the inactivation processes just discussed; the
quantitative details of the model are given in Appendix. The model
consists of interacting facilitatory (F) and
inhibitory (I) pathways activated by serotonin
(S). Each pathway has three reaction steps in which
an initial product (Fo or
Io) increases as a sigmoidal function
of log S and is converted to secondary and tertiary products
(F1,
F2, or
I1,
I2). Products increase at a rate
proportional to the amount of precursor (Fig. 5A,
processes 3, 7, 10, 11) and
are broken down at a rate proportional to their own amount (Fig.
5A, processes 5, 9, 13,
14), and where necessary, feedback inhibition was
used to avoid run-away growth of products (Fig. 5A,
processes 4, 8). Whichever form of modulation becomes established first, be it facilitation or inhibition, dominates because product F2 suppresses the
production of I2 and conversely (Fig.
5A, process 12; compare with Fig.
4A). Facilitation persists after extended serotonin
exposure because breakdown of F1 (Fig. 5A, process 5) inactivates (Fig. 5A,
process 6; compare with Fig. 4B). The
final products (F2 and
I2) determine multiplicative factors that adjust EPSP amplitude; the facilitatory multiplier was constructed to increase the EPSP by a percentage proportional to the amount of
facilitatory product and correspondingly for the inhibitory multiplier
and the final inhibitory product. EPSP amplitudes predicted by the
model under the four different conditions of 5-HT application are shown
in Figure 5B.
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Figure 5.
A model that predicts the observed results.
A, The model; Discussion contains a qualitative
explanation, and Appendix contains full quantitative characterization.
Symbols are defined in the legend of Figure 4.
F0-F2 and
I1-I2 are key
intermediaries in the conjectured facilitatory and inhibitory pathways,
respectively; processes are numbered for reference and
correspond to equation numbers in Appendix.
B1-B4,
EPSP amplitudes predicted by the model under the four different
conditions of 5-HT application. The width of the
dark bars approximately indicate concentration of
5-HT.
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The FAST-SHORT-HIGH 5-HT stimulus (Fig.
5B1) is inhibitory because the early,
rapid response of the inhibitory pathway suppresses production of
F2. Facilitation is not activated long
enough for process 5 (Fig. 5A) to inactivate; therefore,
F1 does not become self-sustaining
and consequently facilitation fails to develop during wash.
The gradual increase in 5-HT concentration during the SLOW-LONG-HIGH
condition (Fig. 5B2) promotes facilitation
because the low-threshold facilitatory response
(F2) develops first and
F2 laterally inhibits the production
of I2.
F1 becomes self-sustaining because its
breakdown inactivates; thus, facilitation persists during wash. The
trend in the biological data for facilitation to diminish after
extended exposure to 5-HT and to rise further during wash was generated
by introducing feedforward inhibition of the effects of
F2 by
I1 (Fig. 5A, process
15).
The FAST-LONG-HIGH stimulus (Fig. 5B3)
causes inhibition for the same reasons as does the FAST-SHORT-HIGH
stimulus. F1 becomes self-sustaining
because F1 is elevated long enough for
the breakdown reaction (Fig. 5A, process 5) to
inactivate. Therefore, F2 is produced
once inhibition of its production by
I2 (Fig. 5A, process 12) ceases, with the consequence that facilitation becomes
manifest during washout of 5-HT.
The FAST-LONG-LOW stimulus (Fig. 5B4) activates
the lower-threshold facilitatory pathway for long enough to result in
self-sustaining elevation of F1, but
it fails to activate the higher-threshold inhibitory pathway. Thus,
persistent facilitation is produced.
The signaling pathways mediating the effects of serotonin on the LG are
not yet known. Thus, attempts to suggest specific signaling pathways
and interactions that could implement this model, which is itself only
one of a number that could reproduce our findings, would be premature.
However, it should be pointed out that the model is not unrealistic in
terms of types of mechanisms that are known to operate in cellular
signaling pathways. Thus, F1 (or
I1) in the model might plausibly
correspond to a second messenger, such as cAMP or
diacylglycerol, and F2 (or
I2) to the activated form of the
associated protein kinase of the second messenger. The activated
kinases might then alter membrane conductances by directly
phosphorylating channel proteins, the decreased conductance and
depolarization associated with low-threshold facilitation being
consistent with closure of a K+ channel
and the increased conductance and depolarization associated with
high-threshold inhibition being consistent with opening of a
Cl+ channel. The suppression of
F2 production by
I2 would then result if the activated
kinase corresponding to I2 were to
inhibit the activation of the kinase corresponding to
F2 by phosphorylating suitable sites
on its regulatory region and conversely (as in inhibition of MAP kinase
and CaM kinase kinase activation by PKA) (D'Angelo et al., 1997;
Wayman et al., 1997; Soderling, 1999). The
F1 persistence postulated in the model
could be attributable to inactivation of an enzymatic reaction breaking
down the second messenger (see above) or to some other mechanism
leading to prolonged elevation of second-messenger levels such as those
responsible for prolonged elevation of DAG in some systems
(Nishizuka, 1995). Given this set of proposals, feedforward
inhibition (Fig. 5A, process 15) would have to be
explained by postulating that the second messenger corresponding to
I1 causes a reduction in the extent to
which the phosphorylation caused by facilitatory kinase closes the
K+ channel. Because some second messengers
can directly gate ion channels (Yau, 1994), this effect might be direct.
Alternatively, F1 and
I1 could correspond to two different
activated protein kinases and F2 and
I2 to the sets of phosphorylated proteins that are the targets of the kinases. Then, if the activated kinase corresponding to I1 were to
inhibit the catalytic action of the activated kinase corresponding to
F2 by phosphorylating it, and
conversely, the mutual inhibition postulated by the model would be
accomplished. The persistence of F1
would here be equivalent to the sustained activation of the kinase to
which it corresponds; sustained activation of PKA in fact occurs in
Aplysia after suitably extended periods of serotonin
application (Muller and Carew, 1998). Under this set of assumptions,
feedforward inhibition would have to be explained by assuming that the
activated kinase corresponding to I1
reduces the extent to which the phosphorylation of the
K+ channel catalyzed by the facilitatory
kinase effectively closes the K+ channel;
the I1 kinase could do this by
directly phosphorylating the K+ channel at
a suitable regulatory site.
More specific and more complete hypotheses must await identification of
the signaling pathways recruited in the LGs by 5-HT. However, whatever
the actual mechanisms, our simulations show that the dependence of the
effect of 5-HT on dose, rate, and duration of 5-HT application may in
principle be understood in terms of complex interactions between second
messengers and enzymes in intracellular signaling systems. Interactions
of this kind provide a capability for logical operations, memory, and
temporal pattern generation based on intracellular biochemical
networks rather than on multicellular neural networks (Katz,
1999; Weng et al., 1999).
Functional importance
These different application regimens may mimic the different ways
that naturally released 5-HT can reach the LG neuron. A serotonergic
neuron has been found that projects caudally along the dorsal aspect of
the LGs and has terminal varicosities in apparent contact (at the light
microscopic level) with the initial dendritic segment of the LG in each
abdominal ganglion (Yeh et al., 1997). Activation of this serotonergic
neuron could cause the concentration of serotonin in the vicinity of LG
to rise quickly to high levels, mimicking the FAST-HIGH application
regimen. Serotonin is also a hormone in the blood of crayfish and
lobsters (Beltz and Kravitz, 1983); the lack of a blood-brain barrier
allows serotonin in the blood to reach the LG, but it presumably does
so more slowly and at lower concentrations than with synaptic release,
perhaps mimicking the SLOW and LOW application regimens of this study.
The functional significance of serotonergic modulation of LG excitation
is not yet clear. However, because injected serotonin seems to have
effects on social behavior (Huber et al., 1997) and because its effects
on the LGs are social experience dependent, it seems likely that the
modulation is important during agonistic interactions. There also is
evidence for both increases in medial giant-mediated escape (Herberholz
et al., 2001) and decreases of lateral giant-mediated escape (Krasne et
al., 1997) during social interactions of freely behaving crayfish,
although a role for serotonin in such modulation has not been established.
The factors determining 5-HT exposure pattern in crayfish are not
unique. Cortical targets located close to the release sites of
serotonergic neurons projecting from the raphe nuclei might be expected
to experience potentially large, rapidly changing levels of 5-HT
exposure, whereas more distant targets would experience smaller, slower
changes (Stamford et al., 2000). If similar to crayfish, these proximal
and distal targets might be modulated very differently. Understanding
how intracellular signaling systems are tailored to respond to
different aspects of the 5-HT signal may guide both the study of the
cognitive and behavioral responses to natural neuromodulation and the
design of drug therapies.
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FOOTNOTES |
Received Oct. 26, 2000; revised April 5, 2001; accepted April 5, 2001.
This work was supported by National Institutes of Health Grants NS8108
(to F.B.K.) and NS26547 (to D.H.E.). We thank Ronald Harris-Warrick for
helpful discussions and Paul Katz for suggestions on an early version
of this manuscript.
Correspondence should be addressed to Frank Krasne, Department of
Psychology, University of California, 1285 Franz Hall, Box 951963, Los
Angeles, CA 90095-1563. E-mail: krasne{at}psych.ucla.edu.
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APPENDIX |
This appendix provides a mathematical characterization of the
model described in Discussion and diagramed in Figure 5A.
Variable names correspond to those of the figure, and equation numbers correspond to the numbered processes of the figure.
Serotonin at concentration S produces initial intracellular
facilitatory and inhibitory factors,
F0 and
I0, that are sigmoidal functions of
log S. Specifically,
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(1)
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where A = 2, log
S0 = 
8, and
z0 is chosen so that
F0 = 0 when log S = 10,
and
|
(2)
|
where A = 2, log
S0 = 5, and
z0 is chosen so that
I0 = 0 when log S = 10.
F1 is produced at a rate proportional
to F0 but is attenuated by a factor,
repiF, because of endpoint
inhibition, which is constructed to keep
F1 from exceeding unity even when breakdown of F1 is totally inactivated
(see below). Thus,
|
(3)
|
where
|
(4)
|
F1 breaks down at a rate
proportional to its concentration, but the rate of degradation is
attenuated by an inactivation factor,
ri, that declines as breakdown occurs.
This inactivation of F1 breakdown is
responsible for the persistence of F1
when 5-HT is removed after long exposures. Thus,
|
(5)
|
where
|
(6)
|
Taking into account both production and breakdown,
Production and breakdown of I1
are computed similarly exceptthat breakdown of
I1 does not inactivate. Thus,
|
(7)
|
where endpoint inhibition is given by
|
(8)
|
which prevents I1 from exceeding
unity, and
|
(9)
|
Taking into account both production and breakdown,
F2 and I2 are each
produced at rates proportional to their precursors, but production of
each intermediary is inhibited by the other (mutual inhibition,
rmi). Thus,
|
(10)
|
and
|
(11)
|
where the mutual inhibition attenuation factor is given by
|
(12)
|
with the argument, x, being either
I2 or
F2.
As above, breakdown of F2 and
I2 are given by
|
(13)
|
|
(14)
|
Hence,
Finally, EPSP amplitudes are assumed to be changed by factors
proportional to final facilitatory and inhibitory products. Thus,
However, in approximately half of our experiments under the
SLOW-LONG-HIGH condition and in the mean curves of Figure 2, the
elevated component of the EPSP fell somewhat as 5-HT concentration increased and then grew again as 5-HT was washed out. Although this
effect was not statistically reliable, it was sufficiently common (and
clear in some individual experiments) that we wanted our model to be
able to emulate it. It could not be explained by production of small
amounts of I2 as the slowly rising
5-HT concentration grew, because mutual inhibition necessarily drives I2 to zero when 5-HT concentration
rises slowly. Therefore, to account for this feature of the data, we
assumed that I1 attenuates F2-caused facilitation by a factor,
rffi (feedforward inhibition) via the
dashed pathway of Figure 5. Thus,
where
|
(15)
|
Simulations were done with this feedforward inhibition
operative. When it is omitted, the prewash dip and postwash rise of EPSP amplitude seen in Figure 5B2 do not occur.
For computations, 4000 updates were taken to correspond to 30 min of
real time or 0.45 sec per update. For HIGH conditions, log S
was set to 3, and for LOW to 7. FAST applications were simulated by
steps of log S from 10 to final concentration, whereas for
SLOW application log S was started at 10 and altered over time according to 
log S/t = 0.008 (log
Sfinal log S). LONG was
equal to 4000 iterations and SHORT to 1000, corresponding nominally to
30 and 7.5 min of 5-HT application, respectively. Parameters used in
simulations were as follows:
| |
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TOP
ABSTRACT
INTRODUCTION
![&Dgr;F<SUB>2</SUB>/&Dgr;t=&agr;<SUB>2</SUB>[r<SUB><UP>mi</UP></SUB>(I<SUB>2</SUB>)F<SUB>1</SUB>−F<SUB>2</SUB>],](/content/vol21/issue12/fulltext/4523/img017.gif)
![&Dgr;I<SUB>2</SUB>/&Dgr;t=&agr;<SUB>2</SUB>[r<SUB><UP>mi</UP></SUB>(F<SUB>2</SUB>)I<SUB>1</SUB>−I<SUB>2</SUB>].](/content/vol21/issue12/fulltext/4523/img018.gif)
![<UP>EPSP</UP><SUB><UP>normalized</UP></SUB>=[1+&sfgr;<SUB>F</SUB>F<SUB>2</SUB>][1−&sfgr;<SUB>I</SUB>I<SUB>2</SUB>].](/content/vol21/issue12/fulltext/4523/img019.gif)
![<UP>EPSP</UP><SUB><UP>normalized</UP></SUB>=[1+&sfgr;<SUB>F</SUB>r<SUB>ffi</SUB>F<SUB>2</SUB>][1−&sfgr;<SUB>I</SUB>I<SUB>2</SUB>]](/content/vol21/issue12/fulltext/4523/img020.gif)
