Department of Integrative Biology, Pharmacology and Physiology, University of Texas-Houston Medical School, Houston, Texas 77030
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ABSTRACT |
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Liao, Xiaogang, Christine G. Brou, and Edgar T. Walters. Limited Contributions of Serotonin to Long-Term Hyperexcitability of Aplysia Sensory Neurons. J. Neurophysiol. 82: 3223-3235, 1999. Serotonin (5-HT) has provided a useful tool to study plasticity of nociceptive sensory neurons in Aplysia. Because noxious stimulation causes release of 5-HT and long-term hyperexcitability (LTH) of sensory neuron somata and because 5-HT treatment can induce long-term synaptic facilitation of sensory neuron synapses, a plausible hypothesis is that 5-HT also induces LTH of the sensory neuron soma. Prolonged or repeated exposure of excised ganglia to 5-HT produced immediate hyperexcitability of sensory neurons that showed little desensitization, but the hyperexcitability decayed within minutes of washing out the 5-HT. Prolonged or repeated treatment of either excised ganglia or dissociated sensory neurons with various concentrations of 5-HT failed to induce significant LTH even when long-term synaptic facilitation was induced in the same preparations. Use of a high-divalent cation solution to reduce interneuron activity during 5-HT treatment failed to enable the induction of LTH in excised ganglia. Pairing 5-HT application with nerve shock failed to enhance LTH produced by nerve shock or to reveal covert LTH produced by 5-HT. The induction of LTH by nerve stimulation was enhanced rather than inhibited by treatment with methiothepin, a 5-HT antagonist reported to block various 5-HT receptors and 5-HT-induced adenylyl cyclase activation. This suggests that endogenous 5-HT may have inhibitory effects on the induction of LTH by noxious stimulation. Methiothepin blocked immediate hyperexcitability produced by exogenous 5-HT and also inhibited the expression of LTH induced by nerve stimulation when applied during testing 1 day afterward. At higher concentrations, methiothepin reduced basal excitability of sensory neurons by mechanisms that may be independent of its antagonism of 5-HT receptors. Several observations suggest that early release of 5-HT and consequent cAMP synthesis in sensory neurons is not important for the induction of LTH by noxious stimulation, whereas later release of 5-HT from persistently activated modulatory neurons, with consequent elevation of cAMP synthesis, may contribute to the maintenance of LTH.
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INTRODUCTION |
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Sensory neurons in Aplysia
(Castellucci et al. 1970; Walters et al.
1983a
) have played a prominent role in revealing potentially general mechanisms for the acquisition and storage of memory (for review, see Byrne et al. 1993
; Krasne and
Glanzman 1995
; Walters 1994
). This work has
taken advantage of the fact that a single neuromodulator, serotonin
(5-HT), can induce long-term facilitation (LTF) and growth of sensory
neuron synapses (Bailey et al. 1992
; Clark and
Kandel 1993
; Emptage and Carew 1993
;
Montarolo et al. 1986
; Zhang et al. 1997
)
that closely resemble effects expressed by these cells during long-term
sensitization of defensive behavior (Bailey and Chen 1983
,
1988
; Cleary et al. 1998
; Frost et al. 1985
; Walters 1987a
,b
). This similarity, and the
observation that a 5-HT-immunoreactive interneuron that facilitates
synaptic connections from Aplysia sensory neurons is
strongly activated by noxious stimulation (Mackey et al.
1989
), suggested that 5-HT is a major signal for inducing
long-term sensory alterations after noxious stimulation. 5-HT thus has
been used to mimic sensitizing stimulation in many studies of sensory
neurons in excised ganglia and in cell culture preparations.
An additional effect of intense or prolonged noxious stimulation is
long-term hyperexcitability (LTH) of the sensory neuron soma
(Cleary et al. 1998; Miller and Walters
1998
; Scholz and Byrne 1987
; Walters
1987b
) and peripheral receptive field (Billy and Walters
1989a
). Hyperexcitability of the soma can lead to soma
afterdischarge during peripheral stimulation, amplifying the synaptic
output of the sensory neuron (Clatworthy and Walters 1993
). The role of 5-HT in LTH has received little attention, although there is one report of LTH induction by 5-HT in dissociated sensory neurons (Dale et al. 1987
). A contribution of
5-HT to LTH induction in Aplysia also is suggested by
additional observations: 1) noxious peripheral stimuli like
those that induce LTH (Cleary et al. 1998
; Scholz
and Byrne 1987
; Walters 1987b
) appear to release 5-HT (Glanzman et al. 1989
; Levenson et al.
1999
; Mackey et al. 1989
), 2)
exogenous 5-HT stimulates both cAMP synthesis (Abrams et al.
1984
; Bernier et al. 1982
; Jarrard et al.
1993
; Ocorr and Byrne 1985
; Ocorr et al.
1985
) and protein kinase A (PKA) activity (Muller and
Carew 1998
) in sensory neurons, and 3) cAMP
injections can induce LTH of sensory neurons (Lewin and Walters
1999
; Scholz and Byrne 1988
). However, it
recently has been shown that cAMP synthesis and PKA are not required
for LTH induced by noxious stimulation of the body surface or by
axotomy (Lewin and Walters 1999
; Liao et al.
1999
). At least two other contributions of 5-HT to LTH are
possible. First, by rapidly increasing peripheral and central
excitability, 5-HT may enhance the discharge of action potentials
during prolonged or repeated noxious stimulation of a sensory neuron's
receptive field, thereby amplifying activity-dependent signals for the
induction of LTH (Billy and Walters 1989a
;
Clatworthy and Walters 1993
). Second, 5-HT might be
released persistently long after peripheral trauma (Levenson et
al. 1999
), thereby producing LTH that is mediated continuously
by short-term mechanisms in the sensory neuron.
We have found that prolonged 5-HT treatment induces little or no LTH of sensory neurons in either excised ganglia or dissociated cell culture. 5-HT does not potentiate the induction of weak LTH by tetanic nerve stimulation, nor does a potent 5-HT antagonist prevent induction of LTH by nerve stimulation. We have also tested the ability of this 5-HT antagonist to reduce excitability when applied 1 day after noxious stimulation.
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METHODS |
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General
Aplysia californica (70-200 g) were supplied by the NIH-Aplysia Resource Facility (Miami, FL) and Alacrity Marine Biological Services (Redondo Beach, CA). Animals were housed in artificial seawater (ASW; Instant Ocean, Burlington, NC) at 15-17°C. Constant body weight was maintained on a diet of Gracilaria seaweed. Animals were dissected after injection of isotonic MgCl2 (equivalent to ~50% of body volume) and each of the two pedal-pleural ganglia complexes (see Fig. 1B) was excised separately, put into its own dish, and desheathed in a 1:1 solution of isotonic MgCl2 and ASW. Thus no communication from other ganglia was possible during treatment or testing. In experiments in which immediate effects of 5-HT were tested in excised ganglia, the ganglia were treated with 0.5% glutaraldehyde in a 1:1 solution of ASW and isotonic MgCl2 for 30 s to immobilize the contractile sheath.
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Drugs and solutions
5-HT (Sigma) was applied in the bath to the final concentrations
indicated. Methiothepin (ICN) first was dissolved in DMSO (at 10-20
mM) and then slowly mixed with ASW to the indicated final
concentrations. The pH was adjusted to 7.4-7.5, and the final
concentration of DMSO was 0.5%. At pH > 7.6, the solubility of
methiothepin was reduced greatly. Excitability testing usually was
conducted in ASW containing (in mM) 460 NaCl, 11 CaCl2, 10 KCl, 30 MgCl2,
25 MgSO4, and 10 Tris buffer (pH 7.6). To
reduce activation of interneurons, some tests and training procedures were conducted in a high-divalent cation ("hi-di") solution
containing 2.2 times the normal concentration of
Mg2+ and 1.25 times the normal concentration of
Ca2+ (Trudeau and Castellucci
1992
). In experiments where cells or ganglia were maintained
for
24 h, the preparation was placed in culture medium containing L15
(Sigma) dissolved in an iso-osmotic saline solution [(in mM) 400 NaCl;
11 CaCl2; 10 KCl; 27 MgCl2; 27 MgSO4; and 2 NaHCO3)
plus 50% hemolyph by volume. Penicillin G (Sigma, 15 U/ml) and
streptomycin (Sigma, 25 µg/ml) were added. Hemolymph was collected
through a needle inserted into the foot after cooling the animal to
0-2°C. The hemolyph was centrifuged to remove cells and debris, and
passed through a 0.2-µm acetate syringe filter (Nalgene) for sterile filtration.
Electrophysiological tests
Standard procedures were used (Gunstream et al.
1995; Liao et al. 1999
). Briefly, sensory
neurons sampled from similar locations in each ventrocaudal
(VC) cluster (Walters et al. 1983a
) were accepted if they had spike amplitude >70 mV, resting potential greater
than
40 mV, and input resistance >25 M
. Recordings were made at
19-21°C while the preparation was bathed in buffered ASW, hi-di
solution, or a 1:1 mixture of ASW and culture medium (without hemolymph, pH 7.6). Soma spike threshold was measured with a standard series of 20-ms depolarizing pulses. Repetitive firing was quantified by counting the number of spikes evoked by a 1- or 2-s intracellular depolarizing pulse using 2.5 times the threshold current determined with the 20-ms pulse. In some experiments repetitive firing was evoked
by a series of 2-s depolarizing pulses at 1, 2, 3, 4, and 5 nA. To
examine synaptic connections in pleural-pedal ganglia, tail sensory
neurons (SNs) and motor neurons (MNs) were recognized by their
size and position (Walters et al. 1983a
), and excitatory postsynaptic potentials (EPSPs) were elicited by 2-ms depolarizing pulses injected into the SNs.
Dissociated cell culture
Two methods were used. The first was based on those of
Schacher and Proshansky (1983) and Eliot et al.
(1994)
. Briefly, pleural and abdominal ganglia were incubated
at 32-34°C for 2.5 h in 1% protease dissolved in L15 solution.
Ganglia then were desheathed, and individual neurons were impaled and
removed with a segment of axon attached using a glass microelectrode.
Sensory neurons were transferred to a plastic petri dish coated with
poly-lysine and bathed with equal amounts of L15 solution and filtered
hemolyph. The cultures were maintained in an incubator at 16-18°C
for 2-6 days before testing. In the second method, pleural sensory
clusters were excised from pleural ganglia in a solution containing
equal amounts of ASW and isotonic MgCl2. No
protease was used. Cells were dissociated by gently vibrating two
micropipettes within each cluster in L15 solution. The surviving cells
were transferred to a culture dish containing L15 plus 50% filtered
hemolyph and maintained at 16-18°C. Culture medium was changed every
other day.
Nerve stimulation
All pedal nerves were cut as far as possible from the ganglion
(see Gunstream et al. 1995). Except where noted, nerve
stimulation was conducted without anesthetic to permit
activity-dependent plasticity. Only nerves p8 and p9 were stimulated,
using a brief series of 10 (0.5-s) trains of 2-ms pulses (25 Hz)
repeated at either 5-s (Walters and Byrne 1985
) or 1-min
intervals. Nerve shock intensity was five times the threshold for
evoking synaptic activity in large unidentified neurons adjacent to the
anteromedial corner of the VC cluster in the pleural ganglion. These
convenient "monitor" neurons were selected because their thresholds
for synaptic input from pedal nerve stimulation are similar to those of
identified tail motor neurons in the pedal ganglion (which was not
desheathed in these experiments). The five-times threshold nerve
stimulation was two to three times that necessary to activate pleural
sensory neuron axons and caused maximal synaptic input to tail motor
neurons. In some experiments a single 2-ms nerve test stimulus also was delivered after testing each sensory neuron to test for an axon of that
sensory neuron in the nerve. During these tests, the preparation was
bathed in hi-di solution to minimize activation of modulatory neurons
by the nerve test stimuli. In the nerve-crush procedure, each major
pedal nerve except p1 was crushed five times at 15-min intervals,
beginning at the cut end, with each crush successively closer to the ganglia.
Statistical analysis
Unless otherwise indicated, experiments employed paired comparisons (using paired t-tests) in which each animal provided a pair of mean excitability measures, one from each sensory cluster, with 6-20 cells sampled per cluster. In some experiments, comparisons with unpaired t-tests were made in single animals of numerous sampled cells from one cluster compared with those in the contralateral cluster. In these cases, the number of individual animals showing significant between-cluster differences is reported. In one study, a 1-way ANOVA followed by Dunnett's tests was used to compare different groups tested at different times after treatment. In some studies, comparisons were made of average excitability measures from a single cluster per animal, and between-animal comparisons were made to measures from other animals tested during the same period of time using unpaired t-tests. Data that were not normally distributed were analyzed with Wilcoxon and Mann-Whitney U tests, as indicated. All tests were two tailed with the level of significance set at 0.05.
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RESULTS |
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5-HT-induced hyperexcitability of sensory neurons decays rapidly and may involve an indirect pathway
Brief application of 5-HT often has been observed to produce
immediate hyperexcitabilty of Aplysia sensory neurons, both
in ganglion preparations (e.g., Baxter and Byrne 1990;
Klein et al. 1986
; Stark et al. 1996
;
Walters et al. 1983b
) and in dissociated cell culture
(Dale et al. 1987
; Ghirardi et al. 1992
).
We asked how long hyperexcitability lasts after bathing excised ganglia for 2 h with 10 µM 5-HT. Similar exposures to 5-HT are known to induce long-term synaptic facilitation in these cells (Emptage and Carew 1993
; Zhang et al. 1997
) and
biochemical alterations associated with long-term facilitation (e.g.,
Chain et al. 1999
; Greenberg et al. 1987
;
Liu et al. 1997
; Muller and Carew 1998
). Our primary index of excitability was repetitive firing evoked by a 2-s
pulse of depolarizing current 2.5 times the current required to reach
action potential threshold with a 20-ms pulse. Tests were given before
5-HT application, in the presence of 5-HT (near the end of the 2-h
treatment), and at the times after washout of the 5-HT indicated in
Fig. 1 (>3 h). In each animal (n = 4), one
pleural-pedal ganglia complex received the 5-HT dissolved in ASW, and
the pretests and posttests were performed in ASW. The contralateral
pleural-pedal ganglia complex received the 5-HT in hi-di solution, and
all pretests and posttests were performed in hi-di solution (see
METHODS). By greatly reducing the activation of
interneurons (Trudeau and Castellucci 1992
), the hi-di
solution reveals effects of 5-HT on tested sensory neurons that are
likely to be direct.
In the presence of 5-HT, all sensory neurons showed an increase in excitability compared with that displayed in the pretest, regardless of whether hi-di solution also was present (Fig. 1). The somewhat greater hyperexcitability in 5-HT + hi-di relative to 5-HT + ASW is a consequence of the higher action potential thresholds in hi-di solution. Because of our normalization procedure, this resulted in delivery of larger test currents to cells in the hi-di solution. When excitability was measured by injecting the same current (2 nA), into sensory neuron somata, no significant difference was found (5-HT + ASW: 23.6 ± 2.9 spikes; 5-HT + hi-di: 24.4 ± 1.60 spikes; n = 7 cells in each group).
To see how long the hyperexcitability lasted after 5-HT treatment, a one-way ANOVA with subsequent Dunnett's tests was used to compare each test to the pretest in the ASW or hi-di groups (Fig. 1). Different populations of sensory neurons were sampled in each test. In ASW, significant hyperexcitability was observed only in the presence of 5-HT, and 10 min after washout (F7,96 = 10.28, P < 0.0001 overall, and P < 0.01 in 5-HT and P < 0.05 in the first posttest; n = 5 to 20 cells in each test). In hi-di solution, significant hyperexcitability was observed only in the presence of the 5-HT (F7,68 = 13.61, P < 0.0001 overall, and P < 0.01 in 5-HT; n = 5 to 14 cells in each test). These data indicate that hyperexcitability induced by prolonged 5-HT treatment persists <30 min after washout of 5-HT. Moreover, the lack of hyperexcitability during any posttest in hi-di solution indicates that hyperexcitability produced by a direct action of 5-HT on the sensory neurons is even shorter lasting-persisting <10 min after washout. The somewhat longer-lasting hyperexcitability observed in ASW may be the result of indirect effects of 5-HT.
5-HT induces LTF but little or no LTH of sensory neurons in excised ganglia
Emptage and Carew (1993) showed that repeated
application of 2 µM 5-HT induced 24-h LTF but not short-term
facilitation of these cells' synapses. We therefore considered the
possibility that LTH might be produced by prolonged 5-HT exposure even
though short-term hyperexcitability decayed rapidly (see Fig. 1). We first confirmed that both prolonged (2 h) and repeated (5 × 5 min
at 20-min intervals) treatment with 5-HT (5 µM) would produce LTF of
Aplysia sensory neuron synapses in excised ganglia, as previously reported (Clark and Kandel 1993
;
Emptage and Carew 1993
; Zhang et al.
1997
). We measured synaptic connections to tail motor neurons
in the same cells before (day 1) and 24 h after (day 2) 5-HT
treatment (Fig. 2A). Both
prolonged and repeated 5-HT treatment produced significant LTF (Fig.
2B; F2,28= 5.38, P < 0.02 overall, and P < 0.05 for each 5-HT-treated group
compared with the untreated control group). No significant change in
EPSPs from control cells occurred between days 1 and 2. We also
examined soma excitability before and 24 h after the prolonged or
repeated 5-HT treatments (identical to those in which EPSPs were
measured). No significant difference in repetitive firing was found
between either group of sensory neurons treated with 5-HT and controls treated with ASW alone (Fig. 2C), although all groups showed
greater excitability on day 2 than day 1 (cf. Gunstream et al.
1995
). No significant differences between 5-HT-treated sensory
neurons and controls were found in spike threshold, spike amplitude, or resting potential (not shown).
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Given previous indications that 5-HT or other sensitization-related
modulators would induce LTH in pleural sensory neurons (e.g.,
Dale et al. 1987; Scholz and Byrne 1987
),
the failure to observe LTH after prolonged or repeated application of
5-HT was unexpected. Therefore we conducted a series of studies in
which several factors that might influence the induction of LTH by 5-HT were varied. We omitted the pretests and compared the excitability of
cells in untreated ganglia to that of cells in contralateral ganglia
(from the same animals) that had been treated with 5-HT. Again, no
significant difference was found in the excitability of sensory neurons
in control ganglia and those in contralateral ganglia given prolonged
or repeated 5-HT treatment when the treatment and tests were performed
in ASW (Fig. 3A). We wondered
whether the lack of apparent LTH might have resulted from concurrent
release of inhibitory neuromodulators during testing and/or application of 5-HT. However, when activity of modulatory interneurons was reduced
by bathing with hi-di solution during testing (Fig. 3B) or
continuously during 5-HT treatment, overnight culture, and testing
(Fig. 3C), we still failed to find significant LTH. Another way to reduce the potential influences of extrinsic neuromodulators released from interneurons is to treat and test clusters of sensory neuron somata after excision from the ganglia (see Fig. 1,
inset). This isolates the sensory neuron somata from all
other neuronal somata and most of the neuropil. When we gave prolonged
or repeated 5-HT treatment to excised sensory neuron clusters bathed in
ASW, we found no significant LTH 24 h later (Fig. 3D).
Because the soma excision procedure allows axonal injury signals to
reach the soma before testing, these results also suggest that 5-HT treatment does not enhance the ability of slow axonal injury signals to
induce LTH (see Gunstream et al. 1995
).
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No significant LTH was induced by concentrations of 5-HT that typically
have been used in these preparations (Fig. 2) (see Emptage and
Carew 1993; Zhang et al. 1997
). However, we were
curious to see if LTH might emerge if we used very high concentrations of 5-HT, such as have occasionally been used in studies of
Aplysia synapses (e.g., Fitzgerald and Carew
1991
). For example, we wondered whether a high concentration of
exogenous 5-HT might be able to compensate for the lack of other
LTH-inducing signals that might normally act cooperatively with 5-HT
during noxious stimulation. Figure 3E pools the results from
eight experiments in which 100 µM 5-HT was applied to either isolated
ganglia (n = 5) or excised sensory clusters
(n = 3). No overall significant difference was observed. We then took advantage of the large number of cells sampled
in each treated and contralateral control cluster to test for
statistically significant effects of 5-HT treatment within individual
animals (see Gunstream et al. 1995
). One isolated
ganglion preparation displayed identical excitabilities in the treated and control sensory neurons. Two isolated ganglion preparations and one
excised cluster preparation displayed lower excitability in
the 5-HT-treated sensory neurons, and in the latter case the decreased
excitability was significant (6.1 ± 1.0 vs. 10.0 ± 1.1 spikes, n = 18 cells in each group, P < 0.02, treatment with 5 × 5 min applications of 5-HT). Two
isolated ganglion preparations and one excised cluster preparation
displayed significantly greater excitability in sensory neurons treated
with 2-h applications of 5-HT than in contralateral control sensory
neurons (respectively: 11.5 ± 1.2 vs. 7.2 ± 0.7 spikes,
n = 12 and 10 cells, P < 0.01; 14.2 ± 2.2 vs. 4.6 ± 1.4 spikes, n = 6 and
7 cells, P < 0.01; 7.0 ± 1.1 vs. 4.4 ± 0.5 spikes, n = 18 cells in each group, P < 0.05). These animals were all from the same source (Alacrity Marine
Biological Services) and examined during the winter of 1994-1995, so
the dramatic variability cannot be explained by differences in supplier
or season. We did not pursue these studies further because the
physiological significance of such high 5-HT concentrations seems
doubtful. 100 µM 5-HT is approximately 100 times the threshold for
producing immediate hyperexcitability of the sensory neurons or
facilitation of their synaptic connections in excised ganglia
(Emptage et al. 1996
), and much higher than that for
many other effects. For example, it is 100,000 times the threshold for
modulating cardiac and buccal muscle in Aplysia (see
Levenson et al. 1999
for review).
5-HT fails to induce LTH of sensory neurons in dissociated cell culture
The only previous study of 5-HT-induced LTH in Aplysia
sensory neurons treated isolated sensory neurons growing in dissociated cell culture with four 5-min pulses of 1 µM 5-HT (Dale et al. 1987). We first attempted to extend this finding by seeing if a
single 2-h pulse of 5-HT also would induce LTH. Repetitive firing was
measured with a series of 2-s depolarizing pulses (1, 2, 3, 4, and 5 nA) injected into the cell soma before and 24 h after a single 2-h
pulse of 5 µM 5-HT. The pretest and 5-HT application were given 4-7
days after dissociation. Figure 4 shows
both the mean responses to the 1-nA pulse and the means of the maximal responses observed during the entire series of pulses (1-5 nA). Maximal firing was often evoked by currents lower than the 5-nA maximum
(see also Fig. 5), presumably because the
largest pulses depolarized the sensory neuron to levels where
Na+ channels became inactivated. Excitability in
both the 5-HT-treated and control cells was higher 24 h after the
pretest as predicted by previous demonstrations that excitability
progressively increases for ~1 wk after dissociation (Liao et
al. 1999
). However, no significant differences were found
between the excitability of the 5-HT-treated sensory neurons and the
ASW controls at any current level in either the pretest or posttest,
regardless of which day the 5-HT treatment was given.
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Similar results were obtained using a procedure in which dissociated sensory neurons were tested 24 h after 5-HT treatment in the absence of a pretest (not shown). Sensory neurons (n = 50) treated with either 5 or 10 µM 5-HT (for 1.5-3 h) 2-4 days after dissociation responded with 12.7 ± 3.7 (SE) spikes in the 1-nA test, whereas corresponding control cells (n = 55) responded with 11.6 ± 2.6 spikes (P = 0.6, not significant). No differences were observed with any of the other test currents. Sensory neurons from only one of the six animals used in this set of experiments showed any trend for 5-HT-induced LTH (26.3 vs. 15.6 spikes at 1 nA, n = 6 and 7 cells, respectively, P = 0.14). However, cells from the other animals, including two treated identically (5 µM 5-HT given 4 days after dissociation), showed no hint of a 5-HT effect.
Our failure to induce LTH in dissociated sensory neurons with a
prolonged pulse of 5-HT led us to reexamine the long-term effects on
sensory neuron excitability of repeated pulses of 5-HT. We used the
same culture methods and stimulation protocols described by Dale
et al. (1987); in particular, four 5-min pulses of 1 µM 5-HT
delivered at 20-min intervals. Excitability was tested with 1-, 2-, 3-, 4-, and 5-nA current pulses before and 24 h after 5-HT treatment.
Only the results of the 1- to 3-nA tests are presented because higher
currents often caused clear decreases in spike amplitude and frequency,
presumably because of Na channel inactivation. Figure 5 summarizes the
results from 54 different sensory neurons dissociated from the pleural
ganglia of five different animals. Like Dale et al.
(1987)
, we found that excitability was greater 24 h after
5-HT treatment than during the pretest on day 1. However, control cells
that were treated identically but without exposure to 5-HT showed a
similar increase in excitability. No significant differences were found
between these groups when comparisons were made between the difference
in number of spikes evoked in the pretest on day 1 and the posttest on
day 2 (Fig. 5). No differences in the pattern of results was seen among
animals that were taken at different times of the year (January 1998 and May and June 1999). Likewise, no obvious differences were seen
between cells from animals supplied by the NIH-Aplysia
Resource facility (n = 17 cells) and those from
animals supplied by Alacrity Marine Biological Services
(n = 37 cells). Taken together, these results indicate that 5-HT, at concentrations of 1-10 µM, does not
effectively induce LTH in dissociated sensory neurons.
5-HT fails to enhance LTH induced by tetanic nerve stimulation
Although we did not find conditions under which application of
5-HT reliably induces LTH, it seemed likely that 5-HT would interact
cooperatively with stimuli that do induce LTH. Repeated in vitro
stimulation involving high-frequency ("tetanic") stimulation of
Aplysia pedal nerves is reported to cause a modest
depression of net outward current 24 h later (Noel et al.
1991), indicating that tetanic nerve stimulation can cause LTH.
Studies in this laboratory (see Fig. 6) showed that relatively weak LTH
can be induced by applying a brief series of 10 (0.5-s) trains of 2-ms pulses (25 Hz) to pedal nerves p8 and p9 at 5-s intervals. The LTH
generated by this procedure is expressed most prominently in sensory
neurons the axons of which are activated by the tetanic nerve
stimulation (Brou and Walters, unpublished observations). Therefore we
asked whether application of exogenous 5-HT would enhance LTH in
sensory neurons activated by tetanic nerve stimulation.
The excitability of sensory neurons was tested shortly after tetanic stimulation of nerves p8 and p9 and then 24 h later. Tetanic stimulation was delivered in either the presence or absence of 10 µM 5-HT, applied 5 min before the tetanus and left in the chamber for 2 h. To identify sensory neurons with axons in the tetanized nerves, after the soma of each cell was tested, a single, 2-ms, supramaximal shock was delivered to nerves p8 and p9 to see if an axon spike was evoked. This shock was five times the threshold for synaptic input to selected neurons (of unknown function) near the VC cluster (see METHODS). All tests were conducted in hi-di solution to reduce activation of modulatory neurons by the nerve test stimuli. To facilitate comparisons among the groups, variation in background excitability was minimized by expressing repetitive firing in each sensory cluster as the difference between the median test response and the median pretest response. Because some of the data were skewed, the summary results are presented as medians and nonparametric statistics were used. Pre- and posttreatment comparisons within the same cells were performed with Wilcoxon tests with the number of cells indicated on the figure. Comparisons between clusters were performed with Wilcoxon tests if opposite clusters within the same animals received the two treatments, or were performed with Mann-Whitney U tests if comparisons were made between animals. As described in the preceding text (see Figs. 1-3), treatment with 5-HT in ASW for 2 h caused a large increase in repetitive firing during the 5-HT exposure (P < 0.01 in each of 3 clusters, n = 8-14 cells tested in each cluster), but failed to induce LTH in any of the clusters (data not shown). Exposure to ASW alone for an equal period of time caused no immediate or long-term change in excitability (Fig. 6A). High-frequency, tetanic stimulation of nerves p8 and p9 while the preparation was bathed in ASW caused no change in excitability in tests given 10-30 min later (Fig. 6A). Twenty-four hours after tetanic stimulation, the tetanized sensory neurons displayed significantly more repetitive firing than they had during the pretest (Fig. 6A, P < 0.05). Sensory neurons exposed to 10 µM 5-HT during and after tetanic stimulation also displayed LTH (Fig. 6A, P < 0.05). Selected comparisons showed that there was no difference between the groups tetanized in the presence and absence of 5-HT (Wilcoxon test, n = 5 pairs of clusters), whereas both tetanized groups were more excitable than the control group (Mann-Whitney U tests for between-animal comparisons, P < 0.05 in each case). Although 5-HT did not enhance tetanus-induced LTH, the 5-HT did cause a large increase in excitability while it was present (Fig. 6A, P < 0.0001).
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The lack of enhancement of tetanus-induced LTH by 5-HT might be attributed to the activation during pairing of inhibitory systems that oppose the effects of 5-HT on LTH induction. We began to test this possibility by tetanizing the nerves and applying 5-HT while the preparation was bathed in hi-di solution to minimize activation of modulatory interneurons. Although hi-di solution may interfere with some potential mechanisms of synergism, it would not be expected to block direct actions of 5-HT on the sensory neuron. The hi-di solution failed to prevent robust hyperexcitability from occurring during exposure to exogenous 5-HT (Fig. 6B, P < 0.01). Hi-di did, however, prevent LTH induction by tetanic stimulation alone. Similarly, when delivered in hi-di, 5-HT paired with tetanic stimulation failed to produce any LTH. Taken together, these data suggest that 5-HT does not enhance the ability of tetanic stimulation to induce LTH and, conversely, that sensory neuron activation does not enhance a covert capacity of 5-HT to induce LTH. These results also suggest that tetanus-induced LTH of sensory neurons requires activation of interneurons by the tetanic stimulation.
Potent 5-HT antagonist fails to block induction of LTH
In a systematic comparison of 5-HT antagonists that have been used
to characterize 5-HT receptors in mammals, Cohen et al. (1997) found that 20 µM methiothepin completely blocks
adenylyl cyclase activation by 5 µM 5-HT in membranes prepared from
pleural sensory clusters. Methiothepin also is reported to inhibit two cloned 5-HT receptors in Aplysia that are coupled to
phospholipase C (Li et al. 1995
) and a cloned 5-HT
receptor that inhibits adenylyl cyclase (Angers et al.
1998
). We asked whether inhibiting these 5-HT receptors with
methiothepin would interfere with the induction of LTH by repeated
nerve stimulation in excised ganglia preparations. In these
experiments, we omitted the pretests and tested all sensory neurons in
ASW without shocking the nerves during the 24-h tests to look for
sensory neuron axons. In early experiments, we used an unpaired design
(between-animal comparisons) to demonstrate first that tetanic nerve
shock induces LTH of sensory neurons. Two stimulus protocols were used
(10 0.5-s, 25 Hz trains of suprathreshold 0.2-ms pulses delivered to
pedal nerves p8 and p9 at 5-s intervals, and 10 of the same trains at
1-min intervals). No difference was found in the LTH produced by each
protocol, so the results were pooled. As shown with somewhat different
testing procedures (see Fig. 5A) (Noel et al.
1991
), repeated nerve shock in ASW caused significant 24-h LTH
of sensory neurons compared with the excitability of sensory neurons
from unshocked control preparations that were tested during the same
period (Fig. 7A,
left, P < 0.05). Using a paired design, we
later examined the effect of 50-75 µM methiothepin on LTH induced by
tetanic nerve shock. This concentration range significantly reduces
immediate hyperexcitability produced by 5-HT treatment (see next
section). Nerve shock in the presence of either 50 or 75 µM
methiothepin failed to reduce the LTH produced by nerve shock (Fig.
7A, right). Indeed, LTH was greater in the methiothepin-treated sensory neurons than in the contralateral cells
exposed to the effects of shock alone in four of five preparations.
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We also used an unpaired design to examine excitability 24 h after delivering five crushes to each of the major pedal nerves (except p1). In the absence of anesthetic, each nerve crush, like strong nerve shock, causes brief, high-frequency activation of the sensory neurons and widespread activation of other neurons in the ganglia (unpublished observations). The repeated nerve crush in ASW caused significant 24-h LTH of sensory neurons compared with sensory neurons tested in preparations with uncrushed pedal nerves (Fig. 7B, left, P < 0.005). Subsequently, we used a paired design to see if the presence of 50 µM methiothepin during nerve crush would influence LTH induction. In all four preparations, sensory neurons exposed to the effects of nerve crush in the presence of methiothepin were more excitable 24 h later compared with contralateral sensory neurons exposed to the effects of nerve crush without methiothepin, and the overall effect was significant (Fig. 7B, right, P < 0.01). Therefore LTH induced by either shocking or crushing pedal nerves under unanesthetized conditions is not blocked by the presence of a potent 5-HT antagonist during nerve stimulation. Indeed the enhancement by methiothepin of LTH induced by both forms of nerve stimulation suggests that endogenous 5-HT might have inhibitory influences on LTH induction in these preparations.
Methiothepin inhibits expression of LTH
The development of LTH in Aplysia sensory neurons
sometimes can be blocked by inhibiting spike activity for prolonged
periods between nerve injury or noxious stimulation and subsequent
testing (Gasull et al. 1997). This suggested that
neuromodulators such as 5-HT might be released continually long after
the initial trauma, a possibility supported by the recent finding that
5-HT is elevated in hemolymph 24 h after strong shock to the body
surface (Levenson et al. 1999
). Because of
methiothepin's potency in blocking 5-HT receptors, we thought this
agent might provide evidence about potential contributions of
continuing 5-HT release to the expression of LTH during tests 24 h
after noxious stimulation.
We first confirmed that methiothepin reduces sensory neuron
hyperexcitability produced by bath application of 5-HT (see also Cohen et al. 1997). In every preparation tested
(n = 6 animals), significant reduction of
hyperexcitability was observed in clusters exposed to 5-HT plus
methiothepin compared with those exposed to 5-HT alone. Figure
8A, (left) shows
results (P < 0.05) from a representative experiment in
which sensory neurons in one cluster were tested in 5 µM 5-HT and 50 µM methiothepin, whereas sensory neurons in the contralateral cluster
were tested in 5-HT alone. Decreasing the 5-HT concentration and
increasing the methiothepin concentration increased the apparent
inhibition by methiothepin (Fig. 8A, right). We then
examined the effect of applying methiothepin during testing 24 h
after noxious stimulation. Figure 8B shows repetitive firing
evoked 24 h after all major pedal nerves (except nerve p1) had
been crushed five times at 15 min intervals in ASW. Cells tested in 20 µM methiothepin responded with significantly fewer spikes than
contralateral cells tested without methiothepin (P < 0.05).
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We found that 20 µM methiothepin applied to unstimulated ganglia had
no obvious effect on basal excitability (Fig. 8C). However, higher concentrations of methiothepin (50-100 µM) significantly reduced basal excitability in unstimulated ganglia (n = 5, data not shown). This reduction of basal excitability by higher
concentrations of methiothepin might have been caused by decreasing the
effects of background release of 5-HT in the ganglia. Alternatively,
methiothepin might directly depress sensory neuron excitability
independent of its blockade of 5-HT receptors. Indeed, excitability of
dissociated sensory neurons grown in cell culture (in the absence of
5-HT) is reliably depressed by methiothepin at concentrations of 5 µM (Liao and Walters, unpublished observations). Although sensory neurons in ganglia (which showed no effect from 20 µM methiothepin) are much less sensitive to the depressive effects of methiothepin than
are dissociated sensory neurons (Liao and Walters, unpublished observations), the ability of methiothepin to depress excitability of
dissociated sensory neurons in the absence of 5-HT suggests that some
of methiothepin's immediate effects on sensory neuron excitability in
ganglia are not a consequence of the drug's blockade of 5-HT
receptors. This is particularly true when higher concentrations of
methiothepin are used, which always seemed to decrease excitability. For example, in three preparations tested 4-24 h after repeatedly shocking nerve p9, 50 µM methiothepin significantly decreased repetitive firing. In addition, 50-100 µM methiothepin significantly reduced repetitive firing 24 h after intense bilateral pinching of
the intact animal (n = 4 preparations). These
inhibitory effects might involve direct actions on sensory neuron
excitability as well as blockade of 5-HT receptors.
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DISCUSSION |
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5-HT produces immediate but not long-term hyperexcitability of sensory neurons
Pleural sensory neurons in Aplysia function, at least
in part, as nociceptors (Clatworthy and Walters 1993;
Walters and Cohen 1997
; Walters et al.
1983a
). It is thus interesting that 5-HT application
immediately increases the excitability of peripheral terminals both of
these sensory neurons (Billy and Walters 1989b
) and of
nociceptors in mammals (Taiwo and Levine 1992
). As has now been observed in many studies of sensory neurons in
Aplysia ganglia (e.g., Baxter and Byrne 1990
;
Dale et al. 1987
; Klein et al. 1986
;
Stark et al. 1996
; Walters et al. 1983b
)
and dissociated cell culture (Dale et al. 1987
;
Ghirardi et al. 1992
), we found that 5-HT also produces
an immediate increase in excitability of pleural sensory neuron somata.
Robust hyperexcitability was expressed in the presence of 5-HT, even
after nearly 2-h exposure (Fig. 1), so this effect shows little
apparent desensitization. After washout of the 5-HT, the
hyperexcitability decayed within ~30 min. Similarly, Stark and
colleagues (1996)
found that hyperexcitability decayed within
~2 min after a 6-min pulse of 5-HT. In our study, the decay of
hyperexcitability was faster (<10 min) when the 5-HT was applied in a
hi-di cation solution that has been shown to largely block activity of
interneurons (Trudeau and Castellucci 1992
; X. Liao, M. Ungless, and E. T. Walters, unpublished observations). This
suggests that part of the hyperexcitability of sensory neurons observed
in normal ASW after 5-HT washout may be mediated indirectly by
neuromodulators released from interneurons activated by 5-HT. The rapid
decay of hyperexcitability after 5-HT treatment is reminiscent of the
transient early synaptic facilitation produced by repeated pulses of
5-HT in excised ganglia (Mauelshagen et al. 1996
).
Unlike 5-HT-induced synaptic facilitation in excised ganglia, which
reappears within a day after decay of the short-term form (Emptage and Carew 1993; Mauelshagen et al.
1996
), significant LTH did not emerge after 5-HT treatment,
even though the 5-HT produced significant LTF of sensory neuron
synapses measured at the same time (Fig. 2). Repeated 5-min pulses of
5-HT and a single 2-h pulse of 5-HT proved equally ineffective at
inducing LTH in excised ganglia. Only with a very high concentration
(100 µM) of 5-HT did we see LTH induction in some preparations, but
this concentration also caused long-term reduction of excitability in
other preparations, so that the overall effect was not statistically significant (Fig. 3E). These complex results at high 5-HT
concentrations are consistent with other findings of both facilitatory
and inhibitory effects of 5-HT applied to ganglia (Fitzgerald
and Carew 1991
). They suggest that, even at lower
concentrations where the effects may not be as apparent, exogenous 5-HT
has diverse actions on different types of modulatory neurons that
influence sensory neuron excitability.
The complications of 5-HT's potential effects on modulatory neurons
can be avoided by applying 5-HT exclusively to sensory neurons, which
is achieved in dissociated cell culture. We found no significant
induction of LTH when 5-HT was applied in a single 2-h pulse to
dissociated sensory neurons after several days in culture. We also
failed to find LTH when 5-HT was applied in four repeated pulses using
the methods reported by Dale et al. (1987). The
difference in our results and those of Dale and colleagues might
reflect unknown differences in experimental conditions, such as
differences in hemolymph (a critical component of the culture medium)
taken from different animals. Another possibility is that because Dale
and colleagues did not know the excitability of dissociated sensory
neurons progressively increases with time in culture (Liao et
al. 1999
), their apparent 5-HT effect may have resulted from
slight differences between groups of sensory neurons in their time of
testing or from chance differences in their slowly developing responses
to dissociation and culture. Although 5-HT might be able to induce LTH
under conditions that we have not examined, we conclude that 5-HT
treatment is usually not sufficient to induce significant LTH in
Aplysia sensory neurons in either excised ganglia or
dissociated cell culture preparations.
Our failure to induce LTH with 5-HT treatment is also somewhat
surprising in view of the report of Muller and Carew
(1998) that prolonged or repeated treatment of ganglia with 10 µM 5-HT causes a significant increase in PKA activity 20 h later
(see also Greenberg et al. 1987
). A persistent increase
in PKA activity would be expected to enhance sensory neuron
excitability by depressing soma K+ conductances
(e.g., Baxter and Byrne 1990
; Goldsmith and
Abrams 1992
; Klein et al. 1986
;
Siegelbaum et al. 1982
). One possible explanation is
that small procedural differences affect the duration of long-term PKA
activation by 5-HT; for example, Hegde and colleagues (1997)
did not find evidence for 24-h PKA activation
after similar 5-HT treatment. Another possibility is that 5-HT
simultaneously can induce opposing modulatory processes that block the
hyperexcitability that would otherwise be expressed during increased
PKA activity. Finally, direct and indirect effects of 5-HT on PKA
activity might be occluded partially by a persistent increase in PKA
activity that follows nerve injury (Liao et al. 1999
),
produced in these cases by dissection.
Are 5-HT and 5-HT-stimulated cAMP synthesis important for induction of LTH by noxious stimuli?
Although 5-HT alone was generally unable to induce LTH, several
observations suggested that 5-HT would nonetheless have some involvement in the induction of LTH. Noxious stimulation releases 5-HT
into the hemolymph (Levenson et al. 1999) and produces
LTH of pleural sensory neurons (Cleary et al. 1998
;
Scholz and Byrne 1987
; Walters 1987b
).
Moreover, serotonergic varicosities are positioned on the sensory
neuron somata (Zhang et al. 1991
). Thus we predicted
that 5-HT would act synergistically with other signals to promote the
induction of soma LTH during noxious stimulation (Walters and
Ambron 1995
). Intense nerve stimulation was found to produce
significant LTH when sensory neurons were activated by either tetanic
nerve shock or repeated nerve crush in the absence of anesthetic (Figs.
6 and 7). Serotonin should increase the number of sensory neuron spikes
evoked by nerve stimulation (Clatworthy and Walters
1993
; Klein et al. 1986
), and increase
transmitter release from sensory neurons onto modulatory interneurons
(reviewed by Byrne and Kandel 1996
). Conversely, sensory
neuron spike activity evoked by nerve stimulation should enhance cAMP
synthesis and long-term alterations dependent on cAMP synthesis (e.g.,
Abrams and Kandel 1988
; Ocorr et al.
1985
; Scholz and Byrne 1988
). Therefore we were
surprised to find that pairing 5-HT with tetanic nerve stimulation did
not enhance the ability of the tetanic stimulation to induce LTH or to
enhance a covert capacity of 5-HT to induce LTH. This suggested that
5-HT makes little contribution to the induction of LTH by noxious
stimulation. Alternatively, application of exogenous 5-HT may produce
parallel inhibitory effects that obscure synergistic interactions of
5-HT and noxious stimulation that would normally occur during the
induction of LTH.
As an independent test of contributions of 5-HT to the induction of
nociceptive LTH, we used a potent 5-HT antagonist, methiothepin, which
blocks several 5-HT receptor types in Aplysia (Angers
et al. 1998; Cohen et al. 1997
; Li et al.
1995
). Methiothepin was applied during noxious stimulation,
produced either by shocking or crushing exposed nerves in an excised
ganglia preparation. In neither case did methiothepin interfere with
induction of LTH (Fig. 7). Indeed, methiothepin caused a significant
increase in sensory neuron excitability 24 h after
repeated nerve crush, and a similar tendency was observed after tetanic
nerve shock. The same pattern has been seen after methiothepin was
applied to the CNS during extensive pinching of the body in a
semi-intact preparation (X. Liao and E. T. Walters, unpublished
observations). Because methiothepin potently blocks adenylyl cyclase
activation in Aplysia CNS by 5-HT (Cohen et al.
1997
) and also blocks other 5-HT receptors in this animal
(Angers et al. 1998
; Li et al. 1995
), the
failure of methiothepin to interfere with LTH induction adds to our
evidence that 5-HT is not important for induction of LTH in
Aplysia sensory neurons. In fact, it suggests that 5-HT
inhibits LTH induction (despite causing short-term hyperexcitability).
These conclusions, however, depend on methiothepin being specific for
5-HT receptors. It recently has been found that methiothepin
antagonizes dopamine-induced cAMP synthesis in addition to 5-HT-induced
cAMP synthesis (J. E. Cohen and T. W. Abrams, personal
communication). Thus the enhancement of LTH induction in the presence
of methiothepin might be due to a blockade of inhibitory receptors
(e.g., to dopamine or possibly FMRFamide) (Belardetti et al.
1987
; Billy and Walters 1989b
; Mackey et
al. 1987
; Montarolo et al. 1988
) or to a direct
depression of the excitability of inhibitory interneurons co-activated
by nerve stimulation.
Because relatively low concentrations of methiothepin completely block
the activation of adenylyl cyclase in sensory neuron membranes by 5-HT
(Cohen et al. 1997) and by dopamine (J. E. Cohen and T. W. Abrams, personal communication), it seems likely that methiothepin reduced cAMP synthesis caused by intense nerve stimulation (Fig. 6). The failure of this probable reduction in cAMP synthesis to
inhibit LTH induction provides important support for the conclusion that the cAMP/PKA pathway is not required for induction of LTH by
either noxious pinching of the body surface (Lewin and Walters 1999
) or axotomy (Liao et al. 1999
). Given that
the 5-HT-cAMP-PKA pathway is critical for the induction of LTF by
noxious stimulation (for review, see Byrne et al. 1993
),
these data suggest that separate signal transduction pathways may be
responsible for inducing LTH of the sensory neuron soma and for
inducing LTF of the sensory neuron synapses. Recent evidence suggests
that a nitric oxide-cGMP-dependent protein kinase pathway is critical
for induction of LTH by noxious stimulation (Lewin and Walters
1999
).
Despite the pharmacological complications involved in interpreting methiothepin's effects, the simplest interpretation of the failure of methiothepin to block LTH induction is that 5-HT and 5-HT-induced cAMP synthesis are not required for LTH induction. Combined with our observations that 5-HT treatment is not sufficient to induce LTH and that 5-HT fails to enhance LTH induction by nerve stimulation, we conclude that 5-HT release does not induce LTH in Aplysia sensory neurons during noxious stimulation.
Does nociceptive sensitization involve persistent release of 5-HT?
Intense or repeated noxious stimulation of Aplysia
produces two, related types of long-term "sensitization" that
traditionally have been the province of separate disciplines. First,
long-lasting behavioral sensitization-enhancement of defensive
behaviors such as gill, siphon, and tail withdrawal (Cleary et
al. 1998; Frost et al. 1985
; Pinsker et
al. 1973
; Walters 1987a
,b
) largely has been
examined by investigators of learning and memory. Second, long-lasting
sensitization (enhanced sensitivity) of sensory neurons largely has
been studied by pain physiologists in mammalian models but is displayed
clearly in Aplysia sensory neurons (which function as
nociceptors) after noxious stimulation (Billy and Walters
1989b
, Miller and Walters 1998
; Walters
1987b
). In Aplysia, both types of sensitization have
been assumed to represent persistent, intrinsic changes in nociceptive
sensory neurons triggered by relatively transient exposure to 5-HT and
other signals (reviewed by Walters 1994
; Walters
and Ambron 1995
). In the dissociated sensory neuron cultures in
which much of this research has been conducted, it would not be
possible for persistently released extrinsic signals to play a role in
maintaining long-term sensory alterations. However, in more intact
preparations, noxious stimulation might cause a long-lasting increase
in activity of modulatory neurons that could help to maintain long-term
sensitization of both sensory neurons and defensive behavior. We found
that the presence of methiothepin during excitability testing 24 h
after noxious stimulation significantly reduced the expression of LTH
(Fig. 8). Because 5-HT is probably degraded rapidly (e.g.,
Levenson et al. 1999
), this finding suggests that part
of the 24-h memory of noxious stimulation is mediated by continuing
release of 5-HT.
The interpretation of methiothepin's actions are complicated, however,
because the drug has nonspecific effects, including blockade of
dopamine receptors (J. E. Cohen and T. W. Abrams, personal
communication) and direct depression of sensory neuron excitability
(Liao and Walters, unpublished observations). With this reservation in
mind, the possibility that methiothepin antagonizes persisting 5-HT
release is of considerable interest in view of the finding that 5-HT
levels in Aplysia hemolymph are elevated 24 h after
noxious shock to the body surface (Levenson et al. 1999), and the observation that expression of LTH induced by
nerve crush depends partly on persisting PKA activity (Liao et
al. 1999
). Moreover, long-lasting blockade of spike activity in
excised ganglia after nerve crush (using hi-di solution or
tetrodotoxin) can reduce subsequently tested LTH (Gasull et al.
1997
). Together, these three types of observation suggest that
1-day memory of noxious stimulation involves a continuing defensive
arousal mediated by persistent activity of 5-HT-containing
interneurons, and by consequent, ongoing activation of PKA in sensory
neurons. This mechanism should complement the long-lasting activation
of PKA caused by degradation of regulatory subunits, which has been
observed after prolonged 5-HT application (Chain et al.
1999
; Greenberg et al. 1987
). Although persistent activity of modulatory neurons may be one nociceptive memory
mechanism, the fact that long-term changes in sensory neuron excitability after peripheral trauma are expressed under conditions in
which interneuronal activity and neuromodulator release are blocked
(Miller and Walters 1998
; E. T. Walters, C. G. Brou, and M. Ungless, unpublished observations) shows that some
components or phases of nociceptive memory are also stored as lasting
changes intrinsic to the sensory neurons.
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ACKNOWLEDGMENTS |
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We thank E. Kandel and M. Ungless for useful suggestions.
This work was supported by National Institutes of Health Grants NS-35882 and NS-35979 to E. T. Walters. Some of the animals were supplied by the National Center for Research Resources, National Resource for Aplysia, at the University of Miami under NIH Grant RR-10294.
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FOOTNOTES |
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Address reprint requests to E. T. Walters.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 June 1999; accepted in final form 25 August 1999.
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REFERENCES |
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