 |
INTRODUCTION |
Many studies on memory formation show that short-term forms of plasticity (on the order of minutes) rely on covalent modifications of preexisting proteins, whereas long-term forms (on the order of days) require the synthesis of new macromolecules (Bailey et al. 1992
; Davis and Squire 1984
; Montarolo et al. 1986
; Nguyen et al. 1994
). Although this traditional view has allowed great progress in the field, the process of memory formation is probably more complex and involves multiple overlapping temporal phases. Recent studies have provided evidence for temporal periods of memory formation that can be distinguished from the classic short- and long-term forms based on pharmacological, genetic, and electrophysiological experiments (Andersen and Hvalby 1991
; Bliss and Collingridge 1993
; Colley et al. 1990
; DeZazzo and Tully 1995
; Matthies 1994
).
In Aplysia, it long has been recognized that the sensorimotor synapses contributing to withdrawal reflexes undergo synaptic facilitation in response to sensitizing stimuli or exposure to serotonin (5-HT), an endogenous neuromodulator. Traditionally, the study of this modulation has been addressed only in the context of a strict dichotomy between short- and long-term processes. Recently however, a new intermediate-term stage of facilitation (ITF) has been described (Ghirardi et al. 1995
). Its time course is distinct from that of short- and long-term changes (Mauelshagen et al. 1996
), and it differs from the former and the latter in being, respectively, protein synthesis dependent and mRNA synthesis independent (Ghirardi et al. 1995
). Protein kinases C (PKC) may play an important role in ITF because PKC activation by 5-HT has a similar time course to ITF (Sossin 1997
; Sossin et al. 1994
). Furthermore pharmacological activation of PKC is sufficient to cause a transient facilitation of sensorimotor synapses (Wu et al. 1995
).
Although PKC(s) have been suggested to play a key role in plasticity of vertebrate (Ben-Ari et al. 1992
; Klan et al. 1993
; Malinow et al. 1989
; Sacktor et al. 1993
) and invertebrate (Farley and Shuman 1991
) nervous system, the multiplicity of isoforms and the absence of specific inhibitors have limited the understanding of their function. By contrast, there are only two PKC isoforms
the Ca2+-activated Apl I, and the Ca2+-independent Apl II
present in the nervous system of Aplysia californica (Kruger et al. 1991
; Sossin et al. 1993
). The simplicity of this distribution makes Aplysia an attractive model for testing the idea that each PKC isoform may have distinct physiological functions; a hypothesis supported by the differential regulation of these isoforms in a number of paradigms (Sossin and Schwartz 1992
; Sossin et al. 1994
, 1996
).
This study examines the contribution of PKC to long-lasting changes in excitability. Although we initially were testing the ability of 4
-phorbol ester 12,13-dibutyrate (PDBU) to induce changes in facilitation at intermediate time points, we noted an increase in the excitability of sensory neurons that were treated with phorbol ester (PE). This was unexpected because earlier results examining short-term plasticity reported no effects of PDBU on excitability. In this report, however, we provide evidence that pharmacological activation of PKC produces long-lasting (from 3 to 24 h) enhancement of excitability in sensory neurons (SNs). Moreover, this modulation can be dissociated in two stages based on the requirement for protein synthesis. Intermediate (3 h)- and long-term (24 h) changes were, respectively, independent and dependent on protein synthesis.
Our findings extend the current view that although they are activated independently (Sacktor and Schwartz 1990
), the two multifunctional kinases
protein kinase A (PKA) and PKC
cooperate in a state- and time-dependent manner to modulate properties of the neurons in the reflex pathway (Byrne and Kandel 1996
).
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METHODS |
Cell culture preparation
For all experiments, A. californica (Marine Specimens, Venice, CA and Aplysia Resource Facility, University of Miami, FL) weighing between 90 and 300 g were anesthetized with an injection of isotonic MgCl2 solution. Animals then were dissected in a solution containing equal volumes (1:1) of isotonic MgCl2 and artificial seawater (ASW). Pleuropedal and abdominal ganglia were removed from the intact nervous system, pinned to the bottom of a silicone elastomer (Sylgard)-coated dish and desheathed with fine forceps. Ganglia were incubated in ASW and 1% protease for ~1.5 h at 36°C; they were rinsed with normal ASW. For experiments on cell excitability, sensory neurons from the ventrocaudal (VC) cluster of pleural ganglia were isolated. They were dissociated mechanically and transferred to separate dishes containing L-15 (modified for Aplysia) (Schacher and Proshansky 1983
), hemolymph (7-10%), and bovine serum albumin (0.01%), keeping the cells from sticking to the bottom of the dish. Neurites were allowed to retract for 1-4 days, and cells were plated on dishes containing 3 ml of L-15 just before recording. Preparation of sensorimotor synapses in culture was essentially as described by Klein (1994)
. Briefly, after 1-3 days in culture (once the cells had retracted their neurites), sensory neurons were paired individually with siphon motoneurons of the LF cluster (LFS) from the abdominal ganglion, and a minimum rest of 24 h was initiated to allow the formation of new synaptic connections (soma to soma contacts). Shortly before recording, each pair of neurons was transferred to a dish containing 4 ml of ASW.
Electrophysiology
All recordings were done at room temperature (21-24°C) using Axoclamp-2A and Axoprobe-1A amplifiers in the current-clamp configuration (bridge mode). Two different types of experiment were done.
CHANGES IN EXCITABILITY.
Cell excitability changes were determined by comparing the total number of action potentials evoked by a series of step depolarizing currents before and after treatment. Cells were impaled with beveled electrodes (10-15 M
, KAc 2 M), and their resting potential was adjusted to
50 mV with constant current injection. Intracellular stimulation was made by superimposing 1-s depolarizing current pulses starting at +0.05 nA; these pulses were increased gradually by 0.05-nA steps throughout the protocol. Action potential threshold was determined rapidly, and starting from that value, a total of five pulse stimuli were applied at 1-min intervals. At the end of the recording, the intracellular electrodes were removed, and PDBU or its inactive isomer 4
-PDBU was applied. Because PE concentrations ranging from 10 to 200 nM have been shown to mimic physiological effects of neuromodulators in cultured Aplysia neurons (Braha et al. 1990
, 1993
; Ghirardi et al. 1992
), we used a final concentration of 100 nM. Washing was done manually with 10 times the bath volume, and a second series of identical depolarizing steps and recordings was started 3 or 24 h later. All current intensities that were used for a single cell before treatment, including the ones that were subthreshold, were repeated in this second recording (see Fig. 1A).This procedure was designed to evaluate changes in excitability over a range of depolarizing stimuli and to reveal possible changes in the firing threshold. For each cell, the total number of spikes evoked before treatment was subtracted from the total number of spikes produced after the treatment. The mean of these differences was used as an index of excitability changes. Each culture dish contained from 3 to 10 sensory cells and contributed to a single score. Sensory cells that had a membrane resistance <100 M
or an unstable membrane potential after impalement were discarded. Data were acquired and analyzed digitally using a modified version of pCLAMP (Axon Instruments), provided by Dr. M. V. Storozhuk.

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| FIG. 1.
Phorbol ester (PE) induces a lasting increase in the excitability of cultured sensory neurons. A: sample recordings from a typical experiment on the excitability change of sensory neurons. Membrane potential was initially set to 50 mV (baseline). Depolarizing current pulses were applied by a step increase of 0.05 nA, once threshold was reached (in this case: 0.2 nA), 4 additional steps were used. In this example, a total of 16 spikes were evoked. Three hours after the cell had been treated with 4 -phorbol ester 12,13-dibutyrate (PDBU; 100 nM, 90 min), the same sequence was repeated and a total of 51 spikes were observed. B: mean number of spikes is plotted against the series of pulses (normalized to the initial firing threshold of each cell). Notice that after treatment, some cells fired at lower current intensities than during the initial recording. Lowering of the threshold occurred more frequently for PDBU-treated cells (50% of all tested cells) than for 4 -PDBU-treated cells (25% of tested cells). C: bar graph summarizing the effect of 4 -PDBU and PDBU (100 nM, 90 min) on excitability of sensory neurons tested 3 h after a 90-min exposure. Height of each bar represents the mean difference(± SE) between the number of spikes recorded after vs. before treatment (for example, 51 16 in A). A significant increase of excitability was observed in PDBU-treated cells relative to 4 -PDBU-treated cells (n = number of culture dishes).
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A modified protocol was used to examine excitability changes in isolated ganglia. In this set of experiments, treated and control cells were recorded only once. Briefly, pleuropedal ganglia were removed from the animal and desheathed in 1:1 isotonic solution. Each hemiganglion then was incubated directly in ASW in the presence of PDBU or 4
-PDBU (1 µM). At the end of a90-min exposure, the ganglia were transferred to separate dishes with additional exchange of solution and allowed to rest at room temperature. The recording session started 3 h later. Intracellular electrodes (glass pipette 10-15 M
, KAc 2M) were used to record from individual sensory neurons of the VC cluster. In each experiment, a fixed series of 500-ms depolarizing current pulses (0.1-4 nA) was injected in ~10 neurons (resting potential adjusted to
50 mV). The total number of action potentials was summed for each cell, and these values were averaged for each ganglion contributing to a single score. Cells that had a membrane resistance <25 M
were discarded. These experiments were performed using a blind procedure. The experimenter did not know which ganglion was exposed to active or inactive isomer of PE until the end of data acquisition.
CHANGES IN SYNAPTIC TRANSMISSION.
Motoneurons were impaled with beveled electrodes (glass pipette 10-15 M
filled with KAc 2M) and hyperpolarized to
80 mV to prevent spike generation. Membrane potential was allowed to stabilize for 5 min while the tip of an extracellular stimulating electrode was positioned close to the sensory neuron cell body (Montarolo et al. 1988
). Briefly, the glass pipette had a 15-mm tip diameter, it was filled with the bath solution, coated with silver paint at the tip, and wrapped partially with aluminum foil to permit the contact for the second pole of the electrode; the first pole being provided by the filament wire in the pipette. Two threshold pulse stimuli (1 ms, 5-10 V; interstimuli interval = 2 min) were delivered while excitatory postsynaptic potentials (EPSPs) were recorded in the motoneuron. Electrodes then were removed and cells were exposed to PDBU or 4
-PDBU for 90 min. The bathing solution was exchanged by means of a peristaltic Gilson pump (40 ml/15 min). Half an hour after the end of phorbol ester exposure, motoneurons were reimpaled and another series of EPSPs was recorded. For statistical analysis, we only used the first EPSPs recorded before and after treatment. Membrane resistance of the motoneuron was measured at the start and at the end of each recording. The criteria for the selection of healthy pairs were a membrane resistance >25 M
until the end of the experiment and a stable resting potential. Pairs also were excluded from the final analysis (~22%) when action potentials or local responses in the motoneuron prevented the accurate measurement of EPSPs in the first part of the experiment (before treatment). Data were stored simultaneously on tape (Vetter, Rebersburg, PA) and on computer with the Axotape program (Axon Instruments).
Drugs and solutions
For experiments with cultured cells, we used standard ASW containing (in mM) 460 NaCl, 10 KCl, 11 CaCl2, 50 MgCl2, and 10 tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 7.6). Commercial ASW (Forty Fathoms, Baltimore, MD; filtered at 0.2 m), was used for the experiments with the intact ganglia. 4
-Phorbol ester 12,13-dibutyrate (PDBU) and its inactive isomer (4
-PDBU), both from Sigma (St. Louis, MO), were dissolved in dimethyl sulfoxide (DMSO) to make stock solutions of 2-10 mM and were kept at
20°C. Once thawed and mixed with the recording medium at the desired concentration, aliquots were used within 2 days. The final concentration of DMSO in the bath did not exceed 0.005% (vol/vol) for cultured neurons and 0.05% for intact ganglia. Chelerythrine from Calbiochem (La Jolla, CA) was dissolved in distilled water at 10 mM and stored at
20°C. Anisomycin from Sigma was dissolved in ethanol and stored at 4°C. Protease type IX was purchased from Sigma. Solvent effects (DMSO and EtOH) were assessed by exposing control preparations to concentrations similar to the final diluted drug solution used for experimental preparations. The final ethanol concentration was 0.05%.
Data handling
All data are expressed as means ± SE. Comparisons of means were done with Student's t-test (paired or unpaired when required).
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RESULTS |
Phorbol ester induces a lasting increase in excitability
To find PKC-induced changes that would be expressed during the intermediate time period between short- and long-term effects, we first measured the evoked spiking of isolated SNs before and after a 90-min treatment with PE. In preliminary experiments, the same treatment was effective at producing an intermediate-term facilitation of EPSPs from cultured sensorimotor connections. The mean percent increase above the pretreatment control value was 151.1 ± 43.0% for PDBU-treated pairs of neurons (n = 15 dishes; paired t-test, P < 0.01). By contrast, the mean percent increase for the control pairs treated with the inactive isomer 4
-PDBU was not significant (+5.6 ± 10.8%, n = 16). Although there were small differences between the experimental protocol of Wu et al. (1995)
and ours (treatment duration = 2 vs. 1.5 h; posttreatment test time = 1 vs. 0.5 h), the percent mean increase in amplitude of the evoked EPSPs was similar in both sets of experiments.
To estimate the excitability index of each neuron, we used a series of depolarizing pulses given intracellularly in a stepwise fashion (+0.05-nA steps). In contrast to other studies (Dale et al. 1987
; Klein et al. 1986
), the particular series of current pulses used for a single SN depended on its activation threshold (see METHODS). By normalizing the "number of spikes/current" responses of each cell to its own threshold value, one can make a point to point comparison of the pre- versus posttreatment responses. The curves thus generated show that after PDBU treatment, cells tend to fire more spikes at every current intensity (Fig. 1B). When the change in excitability is expressed as the difference between the total number of spikes evoked in a cell after and before treatment [(post)
(pre)], statistical analysis indicates that PDBU significantly increased the excitability of SNs at 3 h after treatment (Fig. 1C). By contrast, there was no significant change of the cell membrane resistance after treatment with either active or inactive isomers of PDBU (4
-PDBU: 253 ± 26 to 254 ± M
; PDBU: 255 ± 26 to 222 ±20 M
). We also have tried to examine if the change in excitability could be explained in part by a reduced accommodation. To estimate the change of accommodation, we have measured the interval between the occurrence of the last evoked action potential and the end of the pulse. With this measure, a larger value indicates a greater accommodation. In PDBU-treated cells, there was less accommodation at all current intensities, the mean interval value was reduced by 202 ± 28 ms after the treatment. By contrast, in control cells, the accommodation was increased at the second recording session, the interval value was increased by118 ± 7 ms.
Changes in excitability are observed in the intact nervous system
To examine the generality of our observations, we tested the effect of PDBU on excitability in the intact nervous system. In these experiments, we used PDBU at 1 µM, instead of the 100 nM used on sensory neuron cultures because high concentrations (500 nM to 3 µM) are required to produce physiological effects in intact ganglia (Goldsmith and Abrams 1992
; Sugita et al. 1992
, 1994
); this is presumably due to a poor penetration of PDBU in intact tissue and perhaps also to additional regulatory constraints present in the intact ganglion (Sossin and Schwartz 1994
). In this experiment, there was no pretreatment recording of the sensory cells. We made instead, posttreatment comparisons between sensory neurons of the two hemiganglia, each treated with either active or inactive (
) isomer of PDBU. In the intact ganglia, as was the case for the isolated cells, we found that 3 h after treatment with PDBU (90 min), the mean number of spikes evoked by a series of depolarizing currents in sensory neurons was increased significantly relative to control 4
-PDBU cells (Fig. 2).

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| FIG. 2.
Lasting increase in the excitability of sensory neurons is induced by PDBU in intact pleural ganglia. Mean number of spikes plotted as a function of the current injection level. Inset: bar graph summarizing the total number of action potentials evoked at all current levels in PDBU and 4 -PDBU-treated (control) cells. Three hours after exposure, PDBU-treated cells fired more spikes than the control cells in response to the same series of depolarizing current pulses.
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Long-term plasticity can be induced by brief exposure to PE
In Aplysia there is general agreement that while short-term neuromodulation or memory only lasts a few minutes, long-term effects should last
24 h. Hence, labeling a process as a short-, long-, or even intermediate-term effect depends primarily on the time period at which it is expressed. However, other discriminating aspects usually are found. For instance, long-lasting forms of neural plasticity generally differ from short-term forms by the longer duration of their critical induction phase. In a typical experiment, brief exposure to 5-HT will lead to, at best, a 30-min facilitation of sensorimotor junctions (Mauelshagen et al. 1996
). Because in our experiments, a 90-min exposure to PDBU produced excitability enhancement at 3 h, we tried using a brief exposure to PE as an independent control, thinking that it should not have any effect on cultured sensory neurons. Surprisingly, this also led to an increase in excitability 3 h later that was just as large as that seen with 90-min PDBU (Fig. 3A). One may argue that in the case of a brief exposure to PE, the compound may remain concentrated in the lipid membrane of cells even after the end of the washout period. We think that it is unlikely, especially in the case of PDBU, because it was shown in an earlier study that the rate of release of PDBU from cultured cells is very rapid; the time constant is ~1 min (Szallasi et al. 1994
).

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| FIG. 3.
Brief exposure to PDBU is sufficient to induce long-lasting increase in excitability. Cultured neurons were exposed for 5 min to active or inactive isomer of PE. At 3 h (A) and 24 h (B) after treatment, a significant increase of excitability was observed in PDBU-treated cells relative to 4 -PDBU-treated cells.
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Because the increase in excitability is expressed at 3 h after treatment, it is now tempting to label it as an intermediate-term effect. It in fact could parallel the expression of PE-induced intermediate-term facilitation of sensorimotor connections (Wu et al. 1995
and our own results). However, one key property of the ITF described by Wu et al. is its transient character; it is not expressed 24 h after treatment. By contrast, we found that the change in excitability induced by PE lasted 24 h (Fig. 3B), demonstrating that PKC activation does have long-term effects.
Involvement of PKC in excitability modulation
We next wanted to examine the specificity of the PE effect on the excitability change of sensory neurons and the possible involvement of PKC. This was done by using chelerythrine, a cell-permeant inhibitor targeted to the substrate binding site of PKC that is highly effective in the Aplysia nervous system (Sossin 1997
; Yanow et al. 1998). When cells were pretreated with chelerythrine [(10 µM) bath applied 30 min before and left during the 5-min exposure to PE], SNs did not show an increase of their excitability in response to PDBU (Fig. 4). Although chelerythrine by itself had a depressing effect on excitability (the number of spikes before and after treatment is significantly different; P < 0.05), the change in the excitability of the control group (4
-PDBU) was not different from that of the experimental one (PDBU). These results and the observation that inactive PE was ineffective at inducing excitability changes suggest that the effects of PDBU are likely to be mediated by PKC.

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| FIG. 4.
Protein kinase C (PKC) inhibitor chelerythrine blocks the effect of PDBU exposure. Isolated cells were treated with 4 -PDBU (control) or PDBU (5 min) in the presence of chelerythrine (10 µM; applied 30 min before treatment). The posttreatment recording was done 3 h later. Control and the experimental groups were not different from each other.
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Protein synthesis inhibition reveals two distinct stages of excitability enhancement by PE
Previous studies have established the requirement for new protein and mRNA synthesis in the induction of long-term neuronal plasticity and memory in Aplysia (Bailey et al. 1992
; Castellucci et al. 1989
; Dale et al. 1987
; Montarolo et al. 1986
). Although 5-HT can induce both short- and long-term changes in the excitability of SNs, only the latter are blocked by protein synthesis inhibition (Dale et al. 1987
; Goldsmith and Abrams 1992
; Klein et al. 1986
). Because PKC may mediate some part of the 5-HT effect on excitability, we next examined whether the effect of PE showed similar requirements. Sensory cells were pretreated with the general protein synthesis inhibitor anisomycin (10 µM, present 1 h before and during PE treatment) and tested at 3 or 24 h later. This procedure, which has been shown to disrupt the induction of long-term plasticity by 5-HT, did not alter the PDBU-induced increase in excitability at the 3 h time point (Fig. 5A). However, it effectively blocked the long-term effect at 24 h (Fig. 5B). To summarize, these data suggest that PDBU can stimulate lasting changes in excitability through mechanisms that are independent of protein synthesis for
3 h but that become dependent on it over a 24 h period.

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| FIG. 5.
Dependence on protein synthesis reveals 2 distinct stages in the PE effect on excitability. A: pretreatment of cultured neurons with anisomycin (10 µM; present 1 h before and during phorbol ester incubation) did not prevent the PDBU-induced increase in excitability at 3 h. B: same treatment blocked the induction of the long-term change in excitability.
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DISCUSSION |
Alterations in the membrane properties and excitability of sensory neurons are associated with sensitization of withdrawal reflexes (Clatworthy and Walters 1993
, 1994; Dale et al. 1987
; Klein et al. 1986
; Walters 1987
). By increasing the number of spikes produced in response to a stimulation, these changes may contribute to the enhanced behavioral response.
Pharmacological activation of PKC leads to long-lasting changes in excitability
In this report, we present new evidence that the excitability of sensory neurons can be modulated by PKC. We show that PE exposure increases the excitability of sensory neurons from cultures or intact ganglia and that even a brief(5-min) pulse induces long-lasting changes that are blocked by the application of a specific PKC inhibitor: chelerythrine. These findings were unexpected because in previous studies, changes in excitability generally have been associated with PKA function (Dale et al. 1987
; Goldsmith and Abrams 1992
; Klein et al. 1986
; Scholz and Byrne 1988
).
Although Braha et al. (1990
, 1993)
reported that pharmacological activation of PKC had no short-term effect on the excitability of sensory neurons in culture, Sugita and colleagues (1992) had noticed previously that PDBU causes a small increase in firing that develops slowly during a15-min period in the intact ganglion. Even though this effect was significant, it was judged too small to contribute to the 5-HT-induced modulation, which could be mimicked almostentirely by a adenosine 3
,5
-cyclic monophosphate(cAMP) analogue. Our results, however, indicate that this small effect continues to increase after the initial inducing stimulus and that it actually can reach a magnitude that is comparable with the 5-HT-induced long-term change in excitability (see Dale et al. 1987
).
Possible involvement of PKC in learning-induced changes in excitability
When activated through a receptor-coupled G protein, PKC and PKA are thought to induce the response of a target sensory neuron to the neuromodulators released during sensitizing stimuli. Although short-term changes often are mediated by the direct phosphorylation of effector proteins (ionic channels, receptors, etc.), long-lasting changes may involve indirect pathways. There is now considerable evidence that PKA contributes to long-term changes by activating the synthesis of new proteins which reconfigure specific properties of the neuron. In the current model, this step is triggered by PKA-dependent phosphorylation of transcription factors (Alberini et al. 1994
; Bartsch et al. 1995
). In contrast, activation of PKC by PDBU does not lead to long-term changes in facilitation (Wu et al. 1995
) presumably because PKC does not induce such phosphorylations. However, 5-HT-stimulated long-term facilitation and long-term changes in excitability recently have been dissociated because inhibitors of transforming growth factor-beta (TGF-beta) signaling block the former changes (facilitation) but not the latter (excitability) (Zhang et al. 1997
). Thus it is possible that activation of PKC may phosphorylate a subset of the transcription factors that are involved in regulating the long-term changes in excitability, but not the long-term changes in facilitation.
There is no doubt that PKA contributes more than PKC to short-term changes in excitability. However, what should be reconsidered in the light of our data, is the possibility that the contribution of PKC to long-lasting changes is important.
Effect of a brief PKC activation
Short exposure to 5-HT or brief tactile stimuli to the animal skin only leads to short-term forms of neuronal plasticity or learning in Aplysia. In contrast, a short (5-min) exposure to PDBU was sufficient to induce intermediate- and long-term changes in the excitability of sensory neurons. If PKC is activated through a second-messenger pathway coupled to 5-HT receptors, why does a brief exposure to 5-HT not lead to long term changes in excitability? Our data do not allow us to give a direct answer to this question, but it may be related to the fact that there are differences at the biochemical level between the pharmacological effect of phorbol esters, which can activate both isoforms of PKC, and neuromodulators such as 5-HT, which activate only one isoform of PKC during a short exposure (Sossin and Schwartz 1992
). Thus the long-term effect on excitability may be induced by the stimulation of the Ca2+-independent PKC Apl II, which is not stimulated by short-term application of 5-HT. This would suggest two physiological models for stimuli that would induce long-term changes in excitability (Fig. 6): Apl II is activated after prolonged treatments with 5-HT in a process that itself requires protein synthesis (Sossin 1997
; Sossin and Schwartz 1994
) and Apl II is activated by signals that work through receptor tyrosine kinases (Sossin et al. 1996
). Hence, PKC-induced changes in excitability may be important for modulation by neuromodulators other than 5-HT. Activity of L29 interneuron for example, has been reported to have modulatory effects at the sensorimotor junction (Hawkins et al. 1981
), but the neuromodulator and the second messenger systems involved are still unidentified. Nerve injury also causes a long-lasting increase in excitability possibly because of a neuromodulator released by hemocytes (Clatworthy et al. 1994
).

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| FIG. 6.
Schematic summary of 2 proposed models of PKC action. A: serotonin (5-HT)-induced change in translation causes the late transient activation of the Ca2+-independent isoform of PKC (Apl II), which in turn produces the long-lasting change in excitability. By using PDBU, we are directly activating this step. B: a growth factor or an unknown transmitter interacts with a tyrosine kinase receptor upstream of Apl II and induces long-lasting changes in excitability.
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Intermediate-term changes depend on covalent interactions
Because the increase in excitability expressed at 3 h after treatment is independent of protein synthesis, it must depend on covalent modifications of preexisting proteins. The time course of PKC activity during and after phorbol ester exposure has not been extensively tested, but it is possible that constitutive kinase activity is initiated. This activity could be needed for the maintenance of intermediate-term changes in excitability. However, there are alternative scenarios. In peptidergic neurons of Aplysia, Conn et al. (1989)
have shown that a transient PKC activation can trigger lasting (20-40 min) excitability changes that persist in the absence of PKC activity. Their proposal that PKC could induce the expression of a covert calcium channel by stimulating its translocation from intracellular vesicles to the plasma membrane (see Knox et al. 1992
) is supported by the recent findings from Ehlers et al. (1995)
. These authors found that PKC phosphorylation of specific serine residues within the NR1 subunits of N-methyl-D-aspartate receptors causes their relocalization from the plasma membrane to the cytoplasm.
In a few species, there is evidence that some forms of lasting plasticity do not require de novo protein synthesis. These have been proposed to underlie intermediate-term forms of memory (DeZazzo and Tully 1995
). Accordingly, we think that the PDBU induced change in excitability observed at 3 h after treatment should be seen as an intermediate-term effect. It is distinct from short-term changes in excitability that are mediated primarily by PKA, and it is distinct from long-term changes that depend on protein synthesis.
Mechanism for the modulation of excitability by PKC
A likely explanation for the results presented here would be that PKC alters the excitability of sensory neurons by modulating certain ionic channels. Short-term changes in excitability for instance, result primarily from a cAMP-mediated closure of
S
potassium channels mediating the IK,S current (Klein et al. 1982, 1986). Because this effect is accompanied by an increase in the input resistance of sensory neurons, the fact that in our experiments the membrane resistance of cells was unchanged between the pre- and posttreatment conditions could be an indication that different channels are being modulated by PKC.
Although Scholz and Byrne (1987)
provided evidence that modulation of IK,S contributes to the long-term reduction in outward currents observed in sensory neurons 24 h after sensitization, they did not exclude the possibility that part of it was mediated through other K+ currents. Alteration of Ia, for example, would increase excitability, without changing membrane resistance. In addition to the changes in potassium conductance, modulation of other types of ionic channels could be involved in long-lasting excitability changes. Although modulation of Na+ currents has not been examined extensively, some transcripts of Aplysia sodium channels contain consensus PKC phosphorylation sites (Dyer et al. 1997
). As suggested above, the persistence of the excitability change could result from a more or less irreversible insertion of channels into the membrane (as in the bag cells). New channel proteins also could be synthesized to ensure long-term changes in sensory neurons. Voltage-clamp studies will have to be done to determine what current is being modulated at intermediate- and long-term time points after pharmacological activation of PKC.
Summary
To summarize, we have demonstrated that activation of PKC leads to intermediate and long-term changes in the excitability of sensory neurons in Aplysia. These results strengthen the evidence that PKC is important for intermediate changes in sensorimotor connections and are the first indication that PKC also has long-term, protein synthesis dependent effects at this connection. In the future, it will be important to determine the mechanism(s) underlying these actions of PKC.