INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Modulation of synaptic strength has been associated with learning in both vertebrates and invertebrates (Abel and Kandel 1998; Bailey et al. 2000a; Byrne and Kandel 1996; Huang et al. 1996; Lechner and Byrne 1998; Milner et al. 1998). In Aplysia, changes in the strength of synaptic connections between sensory neurons and motor neurons that participate in the gill and siphon withdraw reflexes can be produced by behavioral training and are mimicked by application of the modulatory neurotransmitters, serotonin (5HT) and small cardioactive peptide (SCP). Depending on the concentration, duration, and pattern of application, 5HT can produce short-, intermediate-, and long-term facilitation (Ghirardi et al. 1995; Mauelshagen et al. 1996, 1998; Montarolo et al. 1986; Muller and Carew 1998; Sherff and Carew 1999; Sutton and Carew 2000). Brief applications and low concentrations of 5HT produce short-term effects while applications of longer durations or higher concentrations produce long-term effects. Although intermediate- and long-term changes in synaptic plasticity have been extensively studied at central synapses, they have not been well studied in the periphery. Neuromuscular synapses between central neurons and the buccal muscles involved in feeding also exhibit persistent modulation by 5HT that resembles the intermediate-term effects of 5HT on sensory neurons (Fox and Lloyd 1997, 1998). Here we investigated the mechanisms that underlie the persistent modulation of these neuromuscular synapses. In these studies, we used the results obtained from experiments on intermediate- and long-term facilitation of sensory neuron synapses in the central ganglia of Aplysia as a guide.

The short-term modulation at neuromuscular synapses of buccal muscles has been examined extensively in Aplysia (Weiss et al. 1992, 1993; Whim et al. 1993). Many of the motor neurons that produce movements of buccal muscles during feeding have been identified, and all of them express modulatory peptide cotransmitters (Church and Lloyd 1991, 1994; Cohen et al. 1978; Gardner 1971). We have chosen to study a muscle (termed the intrinsic anterior muscle 3; I3a) that participates in the closing of the jaws and is innervated by two identified excitatory motor neurons B3 and B38. Both neurons use glutamate as their fast transmitter but express different modulatory peptide cotransmitters; B3 expresses FMRFamide and B38 expresses SCP (Church and Lloyd 1991; Church et al. 1993; Fox and Lloyd 1999). In addition, I3a is also innervated by a pair of modulatory serotonergic neurons termed the metacerebral cells (MCCs) that have extensive central and peripheral synaptic outputs (Eisenstadt et al. 1973; Weinreich et al. 1973; Weiss and Kupfermann 1976; Weiss et al. 1978). Both neuronal release and exogenous application of 5HT and SCP act peripherally to modulate excitatory junction potentials (EJPs) and contractions of I3a (Church et al. 1993; Fox and Lloyd 1997, 1998, 2001; Keating and Lloyd 1999; Lotshaw and Lloyd 1990). The short-term effects of 5HT and SCP on I3a are similar. Both modulators increase the amplitude and relaxation rate of contractions evoked by either B3 or B38, selectively facilitate B38-evoked EJPs and decrease the latency between the onset of a motor neuron burst and the onset of the resulting contraction much more for B38 than for B3. However, the time courses of their effects are very different. The effects of SCP readily reverse on washout while those of 5HT persist for many hours (Fox and Lloyd 1997, 1998). Thus neuromuscular synapses in I3a are modulated by the same transmitters that facilitate synapses of the sensory neurons that participate in the withdrawal reflexes and the facilitation produced by 5HT can be persistent in both systems. We investigated the mechanisms that might underlie the difference in persistence in the I3a neuromuscular system. First we considered the possibility that the duration of the facilitation produced by SCP was attenuated by desensitization of the SCP receptor or that the duration of 5HT's effects was prolonged by its uptake and re-release. Desensitization of SCP receptors does not appear to underlie the difference in persistence, but we could not eliminate re-release of 5HT as a possible mechanism. Next we examined processes downstream of 5HT receptor activation that might underlie persistence. In sensory neuron synapses, new protein synthesis, the cAMP pathway, and the protein kinase C (PKC) pathway are all thought to contribute to intermediate- and long-term facilitation. Our results suggested that protein synthesis was not required for persistent facilitation of EJPs in I3a. We next concentrated on the cAMP pathway as 5HT and SCP cause large increases in cAMP levels in I3a and many of the short-term effects of these modulators appear to be mediated by this pathway (Fox and Lloyd 2000; Lotshaw and Lloyd 1990). The cAMP pathway, however, did not appear to mediate persistent effects of 5HT. Instead, the persistent effects of 5HT were mirrored by the application of phorbol, suggesting that protein kinase C or an Aplysia homologue of unc13 may mediate these effects (Betz et al. 1998; Sossin et al. 1994; Sugita et al. 1992). Although SCP does not activate the second-messenger system(s) involved in persistence in I3a, it did cause a persistent increase in cAMP levels that could be important behaviorally. The similarities between our results in I3a and the results from Aplysia sensory neurons suggest that some of the mechanisms that underlie intermediate- and long-term facilitation at central sensory synapses may also reside in peripheral synapses and contribute to the change in gain of the motor response reflex.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

Aplysia californica (60-300 g) were obtained from Marinus (Long Beach, CA), maintained in circulating artificial sea water (ASW) at 16°C, and fed dried seaweed every 3 days.

Neuron stimulation

Detailed experimental methods have been described previously (Fox and Lloyd 1997). Briefly, animals were immobilized with an injection of isotonic MgCl₂ and the dissection carried out in high-Ca²⁺ (33 mM; 3× normal), high-Mg²⁺ (165 mM; 3× normal) ASW (termed high-Ca-Mg ASW). The buccal mass and buccal ganglia were removed, and the mass was bisected along the midline. Buccal nerve 2, which contains the peripheral axons of B3 and B38 was left intact (nerve designations from Gardner 1971; muscle nomenclature from Howells 1942; also see Lloyd 1988). The ganglia were desheathed and superfused with low-Ca²⁺ (0.5 mM; 0.05× normal), high-Mg²⁺ (110 mM; 2× normal) ASW (termed low-Ca ASW). B3 and B38 were identified by their position, size, and muscle innervation patterns (Church et al. 1993). Neurons were normally impaled with two microelectrodes (2-4 M; filled with 3 M K acetate), one to inject current and one to monitor membrane potential. Individual spikes in motor neurons were driven by brief (10-20 ms) depolarizing current pulses. Many experiments were carried out by alternatively stimulating bursts in B3 and B38 at 50 s intervals (100 s intervals for each neuron). These long interburst intervals were used to minimize release of endogenous peptide cotransmitters and posttetanic potentiation (Church et al. 1993; Lotshaw and Lloyd 1990; Whim and Lloyd 1990). All experiments were performed at room temperature (~22°C).

Measurement of I3a contractions

The bath containing the I3a muscle was superfused with ASW and was separated from the ganglia by a partition through which ran the intact nerve 2. Transmitters and/or pharmacological agents were dissolved in ASW and applied via the superfusion. Typical application periods were 20 min to ensure adequate penetration into the muscle. Note that the partition prevented the ganglia from being exposed to these substances. Contraction amplitudes were monitored with an isotonic transducer (Harvard Apparatus) and submaximal contractions were evoked by stimulating B3 or B38. The frequency of action potentials within a burst was usually 16 Hz, and burst durations were adjusted so that contractions evoked by B3 and B38 were similar in amplitude. There was no relationship between the burst length and the magnitude of the responses to 5HT, SCP, or other pharmacological agents.

Measurement of I3a EJPs

EJPs were recorded with a perfusion electrode (Church et al. 1993; Fox and Lloyd 1997). The perfusion electrode consisted of a small chamber (100 µl) with an aperture (~1.5 mm) that was positioned to press firmly down on a portion of the muscle (see Figure 1 in Church et al. 1993). The inside of this electrode was perfused with ASW while the rest of the preparation was superfused with low-Ca ASW to suppress synaptic transmission and muscle contractions. This procedure confined the contractions to the small area of the muscle covered by the recording chamber and thus markedly reduced movement artifacts in the recordings. The earliest evoked muscle contractions occur after the sixth EJP so the early EJPs in a burst are recorded in the absence of any movement. EJPs were recorded by extracellular electrodes placed inside and just outside the wall of the perfusion apparatus. Signals were amplified using a Grass P15D AC amplifier. Transmitters and/or pharmacological agents were applied in ASW to the inner chamber of the perfusion electrode so the ganglia were not exposed to these substances. Typical application periods were 20 min to ensure adequate penetration into the muscle. This procedure permits us to simultaneously record from a population of muscle fibers thereby reducing sampling bias. The frequency of action potentials within a burst was usually 16 Hz, and burst durations were adjusted so that compound EJPs evoked by B3 and B38 were similar in amplitude. 5HT antagonists were applied for 10 min after 30 min washout from 1 µM 5HT to test their effects on persistence. Antagonists were also simultaneously applied with 0.1 µM 5HT to test if they inhibited the short-term effects of 5HT.

Measurement of cAMP

Muscle segments were dissected in high-Ca-Mg ASW, weighed (~3 mg each), and washed in several changes of ASW for 1 h. Segments were incubated in ASW, transmitter, or pharmacological agents for 20 min (the same period used in most physiological experiments) and then either extracted immediately or after washing in ASW for a series of different periods. Because rapid and complete washout of transmitter was important in these experiments, we took several precautions to ensure that this occurred. All washes and incubations were carried out in 4 ml ASW on a rotary shaker. This volume was 1,000-fold larger than the muscle volume. At the end of incubation and wash periods, muscle segments were picked up using clean forceps, rinsed with ASW, and transferred to new dishes containing ASW. For the longer washes (>1 h), muscle segments were transferred every 15 min. After incubation, the muscle segments were homogenized in 2% 2 N HCl in ethanol at 30°C and centrifuged at 10,000 g. The supernatants were used for duplicate cAMP determinations using a commercial radioimmunoassay (Biomedical Technologies, Norwood, MA). Data were normalized to the cAMP levels of muscle segments from the same animals incubated in ASW (control saline) for 20 min.

Measurement of 5HT uptake

Muscle segments were prepared as described in the previous section. Segments were incubated for 90 min in ASW containing 1 µM unlabeled 5HT and 30 nM ³H-5HT (99 Ci/mmol, Amersham, diluted 300-fold from stock) in the absence or presence of putative 5HT uptake inhibitors clomipramine (also called chlorimipramine), fluoxetine, imipramine, 6-nitroquipazine. Uptake was linear over this period. Of these, fluoxetine was the most effective and was studied further. At the end of the incubation periods, muscle segments were washed twice for 15 min each in low-Ca ASW (to inhibit release) then extracted in 500 µl of 10 mM trifluoroacetic acid (containing 10 nmol unlabeled 5HT) at 100°C for 10 min. Extracts were run on HPLC to determine whether the labeled 5HT, taken up by the muscle, was metabolized or remained as 5HT. Extracts were analyzed on a Microsorb MV column using a gradient from 10 to 30% CH₃CN. The solutions contained 10 mM heptafluorobutyric acid to increase retention of 5HT. The location of the 5HT peak was monitored by absorbance at 280 nm, and the radioactivity in the peak was quantified by liquid-scintillation counting.

Measurement of labeled substances recovered from muscle

I3a muscle was separated from the desheathed buccal ganglia by a partition through which ran the intact buccal nerve 2. The muscle alone was loaded with ³H-5HT by incubating it with ASW containing 1 µM unlabeled 5HT and 30 nM ³H-5HT (99Ci/mmol, Amersham) for 2 h using a recirculating peristaltic pump at 1 ml/min. All aspects of these experiments were done in the absence of 5HT-uptake inhibitors. The muscle bath was then turned over completely four times with ASW in 30 min, the partition was removed, and a perfusion electrode placed on the muscle. Motor neurons B3 and B38 were impaled with electrodes and stimulated using the same paradigm as the physiological experiments described in the preceding text. EJPs were also recorded from the muscle. Perfusate samples (10 ml ASW over 10 min) were collected for 60 min. One milliliter of each crude sample was counted, and the 5HT and other labeled substances in the remainder of the sample were extracted on a solid phase cartridge (Sep-Pak, C18, Millipore), eluted with 50% CH₃CN, the elute dried, and analyzed by HPLC as described in the previous section to determine what fraction of the recovered radiolabel was actually ³H-5HT.

Measurement of protein synthesis

Muscle segments were prepared and washed as described above. Muscle segments were divided into two groups; an experimental in which all solutions contained anisomycin (10 µM) and a control. Solutions for the labeling and washing of the samples were prepared in ASW/hemolymph (1:1). Samples were washed with ASW/hemolymph for 1 h and then labeled with 10 mCi/ml ³⁵S-methionine for 1 h (Amersham). The samples were then chased with unlabeled methionine (1 mM) for 4 h, homogenized in 0.2 ml of phosphate-buffered saline (100 mM sodium phosphate pH 7.5, 400 mM sodium chloride), and centrifuged at 10,000 g for 5 min at 4°C. Proteins in the supernatant (50 µl) were precipitated with 1 ml of cold 10% trichloroacetic acid (TCA), incubated on ice for 1 h, and centrifuged. The pellet was washed with cold TCA, centrifuged, resuspended, and counted using a scintillation counter.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Both 5HT and SCP selectively facilitated B38-evoked EJPs and potentiated contractions evoked by both B3 and B38 (Fig. 1). The effects of SCP reversed on washout while those of 5HT persisted for several hours.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Facilitation of excitatory junction potentials (EJPs) and potentiation of contractions by 1 µM serotonin (5HT) or small cardioactive peptide (SCP). Compound EJPs and contractions in the I3a muscle fibers were evoked by alternately stimulating bursts of action potentials (16 Hz) in B3 and B38 at 50-s intervals (100-s intervals for each neuron). Initial burst durations were adjusted for each neuron to evoke EJPs and contractions of similar amplitude. Motor neuron bursts are not shown, bursts in B3 are indicated (). A: application of SCP or 5HT (|) dramatically facilitated B38-evoked EJPs and had little effect on the B3-evoked EJPs. Note that the facilitation of EJPs produced by SCP began to reverse soon after it was washed out, whereas the facilitation of EJPs produced by 5HT was persistent. For the SCP application, B3 bursts were 0.7 s and B38 bursts were 0.5 s. For the 5HT application, B3 bursts were 1.0 s and B38 bursts were 0.3 s. B: application of SCP or 5HT (|) dramatically potentiated contractions evoked by both B3 and B38. Note that the potentiation of contractions produced by SCP began to reverse soon after it was washed out while the potentiation of contractions produced by 5HT was persistent for both motor neurons. For the SCP application, B3 bursts were 1.75 s and B38 bursts were 3.0 s. For the 5HT application, B3 bursts were 1.3 s and B38 bursts were 1.1 s.

SCP receptors did not desensitize

We first investigated the possibility that the apparent reversal of the SCP's effects on washout might actually be due to desensitization of the SCP receptor. If desensitization of SCP receptors is very slow, then the affects of desensitization might not be apparent during the SCP application and could resemble the reversal of the SCP's effects on washout. Indeed, this phenomena has been observed previously at central neuronal synapses and peripheral heart tissue in Aplysia where 5HT and SCP have similar effects, but the effects of SCP slowly desensitize while those of 5HT do not (Abrams et al. 1984; Lloyd et al. 1985). B38-evoked EJPs were used to assay for receptor desensitization because they are dramatically facilitated by either 5HT or SCP. A perfusion electrode was used to record the EJPs from a small portion of the I3a muscle that was perfused with ASW while the remainder of the muscle was superfused with low-Ca ASW to suppress synaptic transmission and muscle contractions (Church et al. 1993). This procedure permitted stable long-term recordings, rapid solution turnover, and simultaneous recordings from a population of fibers thereby reducing sampling bias. EJPs from individual experiments were normalized to their maximum facilitated amplitude (set at 1) and the results from several experiments were pooled. Initially, we tried increasing the duration of the SCP (1 µM) application from 20 to 60 min. This increased EJP amplitude to a maximum of 807 ± 238% (mean ± SE) over control, and the facilitation observed, at the end of a 60 min SCP application, was still 84 ± 9% of the maximum in these experiments (n = 4). Thus very little desensitization occurred over the 60 min application. Often higher concentrations of agonist accelerate receptor desensitization. Accordingly, as a second test for desensitization, we applied a higher concentration of SCP (10 µM) for 20 min. This strongly facilitated EJPs 1,011 ± 580% above control, and facilitation at the end of the 20 min SCP application was 97 ± 2% of the maximum (n = 3; Fig. 2). As one might expect, the higher concentration of SCP produced a more rapid facilitation of EJPs and somewhat slower reversal during washout. In conclusion, the reversal of the effects produced by SCP was not due to receptor desensitization.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Time course of the effects of 1 or 10 µM SCP on the amplitude of B38-evoked EJPs. In this and the following time course figures, EJPs were normalized to their maximum amplitude (set at 1), and the results from several experiments were pooled. With this method of normalization, the maximum EJP amplitude of the pooled data was often <1 because of variablilty in the timing of the peak facilitation between experiments. This variation was normally <300 s and was probably due to differences in the muscle thickness. Application of 1 or 10 µM SCP (|) facilitated B38-evoked EJPs. Percentage increases in EJP amplitude at the end of the SCP application (time = 0 min) were 587 ± 174% (mean ± SE, n = 7) for 1 µM SCP and 1,011 ± 580% (n = 3) for 10 µM SCP. Note that the effects of both concentrations of SCP did not desensitize significantly during their application. In addition, the facilitation produced by 10 µM SCP reversed more slowly during washout than that produced by 1 µM, but its effects were still not persistent. Bursts in B38 were fired at 100 s intervals. Peak amplitude of the compound EJPs was used in this and the following time course figures. Graphed values are means ± SE.

Persistent facilitation of EJPs probably does not depend on 5HT uptake and re-release

The persistent effects of 5HT could be due to the uptake and slow release of 5HT from elements of I3a muscle. This possibility was tested in two ways. First the release of 5HT from muscles incubated in ³H-5HT was measured directly, and the effects of pharmacological agents, which inhibit 5HT uptake or 5HT receptors, on the persistent facilitation of EJPs were determined. Muscles were incubated in ³H-5HT, and release was monitored by collecting perfusate during the washout and analyzing it by HPLC. We found that the muscle perfusate did release radiolabeled substances; however, only a small proportion of the released label was 5HT (4 ± 1%; mean ± SE, n = 3). By contrast, extracts of the muscle indicate that I3a did take up labeled 5HT and maintained most of it unchanged (>50%).

Because there was some release of labeled 5HT, we examined the effect of inhibiting 5HT uptake on persistence. First, the effectiveness of several 5HT uptake inhibitors (clomipramine, fluoxetine, imipramine, and quipazine-6-nitro) were tested on I3a muscle segments. Of the substances tested, fluoxetine (100 µM) was the most effective at inhibiting ³H-5HT uptake (reduced by 95 ± 2% from control; n = 4). However, at this concentration fluoxetine selectively and persistently facilitated B38-evoked EJPs. We found that a 40 min application of 100 µM fluoxetine reduced B3-evoked EJPs by 4 ± 3% (n = 3), whereas it facilitated B38-evoked EJPs by 495 ± 84% (n = 5) when measured just before washout. The facilitation produced by fluoxetine was persistent (Fig. 3A). Indeed, in three of the five experiments, facilitation of B38-evoked EJPs produced by fluoxetine was larger 1 h after it was washed out than during its application. Fluoxetine appears to act, at least partially, through receptors linked to adenylyl cyclase because it elevated the level of cAMP in the I3a muscle segments by 4.4 ± 0.8 fold (n = 4; 20 min application). Fluoxetine is a competitive inhibitor of 5HT uptake as well as some 5HT receptors (Ni and Miledi 1997; Sanchez and Hyttel 1999; Wong et al. 1995). Therefore the most likely explanation for the effects we observed is that fluoxetine is acting as a weak 5HT agonist, but we cannot rule out the possibility that it is acting at other receptors. Another alternative mechanism is that spontaneous release of endogenous 5HT becomes more effective at facilitating EJPs when reuptake is blocked with fluoxetine. Thus fluoxetine was the most effective substance at inhibiting 5HT uptake, but experiments using fluoxetine were difficult to interpret because it activated the cAMP pathway and probably was a weak 5HT receptor agonist in the I3a preparation.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Effects of the 5HT uptake inhibitor fluoxetine and 5HT antagonists on the facilitation of B38-evoked EJPs. A: application of the 5HT uptake inhibitor fluoxetine (100 µM; |) persistently facilitated B38-evoked EJPs. Percentage increase in EJP amplitude at the end of the fluoxetine application (time = 0 min) was 495 ± 84% (n = 5). Bursts in B38 were fired at 100 s intervals. B: 5HT antagonists did not inhibit the persistent facilitation of B38-evoked EJPs produced by 1 µM 5HT. The antagonists were applied 30 min after 5HT was washed out. A 10 min application of the antagonists (10 µM) tended to slow the reversal of the persistent facilitation. Indeed, the rate of decay of EJP amplitude was slower in the presence of the antagonists than it was in control saline (ASW) alone (n = 3 for all antagonists; P  0.05 for Met, NAN, and Spip; P  0.08 for Cyp, Ket, and Rit). Graphed values are means ± SE of the 3rd EJP of the motor neuron bursts. Cyp, cyproheptidine; Ket, ketanserin; Met, methiothepin; NAN, NAN-190; Rit, ritanserin; Spip, spiperone. C: 5HT antagonists did not inhibit the short-term facilitation of B38-evoked EJPs produced by 0.1 µM 5HT. Addition of the antagonists (10 µM) to 5HT did not inhibit the facilitation of EJPs by 5HT. EJPs were normalized to the peak amplitude of the EJPs during the application of 5HT alone (set at 100%). Graphed values are means ± SE. Cyp (n = 4); Ket (n = 2); Met (n = 4); NAN (n = 4); Rit (n = 2); Spip (n = 4).

Next we examined whether 5HT antagonists inhibited the persistent facilitation of EJPs. Persistent increases in the release of 5HT from modulatory neurons is believed to contribute to the expression of the long-term excitability of the sensory neurons in Aplysia (Liao et al. 1999). Several 5HT antagonists, including cyproheptidine, ketanserin, methiothepin, NAN-190, ritanserin, and spiperone, have been shown to inhibit some of the effects of 5HT on Aplysia sensory neurons, extrinsic buccal muscles, synaptosome preparations, and cloned 5HT receptors expressed in mammalian cell lines (Angers et al. 1998; Emptage and Carew 1993; Li et al. 1995; Liao et al. 1999; Mercer et al. 1991; Ocorr and Byrne 1986; Ram et al. 1994). We hypothesized that if the persistent facilitation of EJPs was caused by 5HT released from muscle elements, then it should be inhibited by 5HT antagonists. This hypothesis was tested by persistently facilitating EJPs with a 20 min application of 1 µM 5HT and then applying the antagonists 30 min after 5HT was washed out. At 10 µM, none of the antagonists significantly reduced EJP amplitude. Instead, the reversal of the facilitation produced by 5HT was slower during the application of the antagonists, suggesting that they themselves might facilitate EJPs (Fig. 3B).

These results prompted concerns about the ability of these antagonists to inhibit the short-term effects of 5HT at the neuromuscular synapses of I3a, so we tested whether the antagonists inhibited the facilitation of EJPs produced by a lower 5HT concentration. Application of 0.1 µM 5HT facilitated B3- and B38-evoked EJPs, and this was not significantly inhibited by any of the antagonists at 10 µM (Fig. 3C). Higher concentrations of the antagonists also did not inhibit the effects of 5HT. Indeed, at 100 µM, many of the antagonists facilitated B38-evoked EJPs. For example, methiothepin, a commonly used 5HT antagonist that inhibits the short-term hyperexcitability in sensory neurons and inhibits 5HT receptors expressed in cell lines, facilitates EJPs in the I3a preparation. Because the antagonists did not inhibit the short-term effects of 5HT, it was impossible to determine if the persistent facilitation was due to the release of 5HT from elements in the muscle. However, one result that appears clear is that the pharmacology of the serotonin receptors in I3a appears to be very different from that of the receptors in sensory neurons involved in the withdrawal reflexes.

Persistent facilitation was not dependent on new protein synthesis

In Aplysia sensory neuron synapses that contribute to the withdrawal reflexes, 5HT causes short-, intermediate-, and long-term facilitation (Abrams et al. 1984; Brunelli et al. 1976; Byrne and Kandel 1996; Walters et al. 1983). The long-term facilitation and a component of the intermediate-term facilitation is dependent on new protein synthesis (Alberini et al. 1994; Ghirardi et al. 1995; Montarolo et al. 1986; Muller and Carew 1998). Inhibition of protein synthesis significantly reduced facilitation produced by 5HT measured as early as 0.5 h after 5HT washout (Ghirardi et al. 1995). Because the persistent facilitation of B38-evoked EJPs by 5HT lasts 3 h in I3a, we tested whether new protein synthesis was required for this effect. Anisomycin (10 µM) has been used extensively to inhibit protein synthesis in Aplysia (Castellucci et al. 1986; Ghirardi et al. 1995; Montarolo et al. 1986). We tested it on the muscle segments and found that it reduced incorporation of ³⁵S-methionine into newly synthesized proteins to 12 ± 4% of control (n = 2). To ensure that protein synthesis was effectively inhibited, application of anisomycin was started 30 min before the application of 5HT and was continued until 5HT was washed out. Anisomycin itself had a small inhibitory effect on facilitation. During the initial superfusion, it reduced the B38-evoked EJPs by 29 ± 5% (n = 4). Anisomycin did not, however, affect the persistent facilitation produced by 5HT (Fig. 4). B38-evoked EJPs were still 91 ± 6% (n = 4) of the maximum facilitation after 1 h of wash and 82 ± 8% (n = 4) after 2 h. Indeed, in one experiment using anisomycin, recordings were maintained for 5 h after 5HT washout, and the facilitation persisted with little decrement after the first hour (declined to 75% of maximum after 1 h and 63% of maximum after 5 h). Therefore new protein synthesis does not appear to be required for persistent facilitation, at least, during the first few hours.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Time course of the effects of 1 µM 5HT on the facilitation of B38-evoked EJPs in the absence or presence of 10 µM anisomycin. Application of 5HT (from 20 to 0 min) persistently facilitated B38-evoked EJPs. Inhibiting protein synthesis with anisomycin (applied 30 min before the application of 5HT and continued until 5HT was washed out) had little effect on the time course of the persistent facilitation produced by 5HT. Increases in EJP amplitude were 566 ± 108% (n = 7) for 5HT and 412 ± 93% (n = 4) for the application of anisomycin and 5HT (Aniso/5HT). Bursts in B38 were fired at 100 s intervals. Graphed values are means ± SE.

Persistent facilitation was not dependent on persistent elevation of cAMP levels

5HT and SCP increase cAMP levels in buccal muscles and many of their short-term effects in these muscles are mediated by the cAMP pathway (Brezina et al. 1994a,b; Fox and Lloyd 2000; Lloyd et al. 1984; Lotshaw and Lloyd 1990; Probst et al. 1994). Activation of the cAMP pathway is also important for short- and long-term facilitation of sensory neuron synapses that contribute to the withdrawal reflexes. The magnitude and the duration of this facilitation appears to depend on the amplitude of the increase in the cAMP concentration. For example, application of SCP alone to cultured sensory neuron synapses caused only short-term facilitation while SCP application in the presence of IBMX, a phosphodiesterase inhibitor that should prolong and increase the effect of SCP on the cAMP levels, produced long-term facilitation (Schacher et al. 1990). Therefore we examined if potentiating the effects of SCP with IBMX persistently facilitated EJPs in buccal muscle. We determined the time course of facilitation produced by SCP alone on B38-evoked EJPs and then the time course of facilitation produced by co-application of SCP and 100 µM IBMX on the same preparation (Fig. 5). To ensure that the phosphodiesterases were effectively inhibited, IBMX application began 30 min before the SCP application and was continued for an additional 30 min after SCP washout. IBMX alone facilitated EJPs and also slowed the time course of the reversal of facilitation caused by SCP somewhat, but the time course was still rapid in comparison to persistent facilitation caused by 5HT. The slowed washout in the presence of IBMX resembled the washout of higher concentrations of SCP (10 µM; Fig. 2). The inability of SCP to persistently elevate EJPs was not due to a reduced effectiveness of SCP during its second application. When SCP alone was applied twice to the same preparation, the facilitation caused by the second application was 0.88 ± 0.10 (n = 3) of that caused by the first application. In addition, SCP application does not inhibit persistent facilitation of EJPs caused by subsequent 5HT applications (Fox and Lloyd 1997). Therefore prolonging the elevation of cAMP with IBMX did not persistently facilitate B38-evoked EJPs.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Time course of the effects of 1 µM SCP on the facilitation of B38-evoked EJPs in the absence or presence of 100 µM IBMX. Application of SCP (top black bar) facilitated B38-evoked EJPs. Inhibiting phosphodiesterases with IBMX before, during, and after the SCP application (bottom black bar) prolonged the time course of the washout of the facilitation produced by SCP, but the facilitation still was not persistent. Note that IBMX alone facilitates EJPs. Increases in EJP amplitude were 640 ± 465% for SCP alone and 676 ± 324% (n = 3) for IBMX and SCP (IBMX/SCP). Bursts in B38 were fired at 100 s intervals. Graphed values are means ± SE.

Next we determined if persistent facilitation could be produced by direct activation of the cAMP pathway. We tested the effects of membrane permeable cAMP analogues and the adenylyl cyclase activator forskolin. Both methods have been shown to cause short-term facilitation (Fox and Lloyd 2000). Application of cpt-cAMP (500 µM) alone or the co-application of a lower concentration cpt-cAMP (100 µM) with IBMX (100 µM) selectively facilitated B38-evoked EJPs, but it was not persistent (Fig. 7). Most of the facilitation reversed during the first hour of wash for both cpt-cAMP alone (reduced to 22 ± 4% of the maximum, n = 3) and the simultaneous application cpt-cAMP with IBMX (reduced to 13 ± 3% of the maximum, n = 5). Therefore cpt-cAMP does not cause persistent facilitation of B38-evoked EJPs.

Forskolin (10 µM) also selectively facilitated B38-evoked EJPs (Figs. 6 and 7). This facilitation appeared to be produced by the activation of adenylyl cyclase because forskolin increases the level of cAMP in I3a (Fox and Lloyd 2000) and a forskolin analogue that does not activate adenylyl cyclase but has many of forskolin's nonspecific effects (10 µM 1,9-dideoxy-forskolin) had little effect on EJPs (Fig. 6). However, unlike cpt-cAMP, the effects of forskolin appear to persist for several hours after washout (Figs. 6 and 7). Although the persistent facilitation of EJPs by forskolin appears to contradict the results from the cpt-cAMP experiments, there are interpretations of these data that are consistent with both results. For example, forskolin may induce persistence by producing a large increase of cAMP levels in a restricted muscle compartment, whereas the diffusion of cpt-cAMP may be more widespread and not reach the threshold necessary to induce persistence. However, it is also possible that forskolin's persistent effects are due to its slow washout from the preparation even though the effects of forskolin appear to reverse readily in other Aplysia muscles (Lloyd et al. 1985, 1988). Accordingly we tested a forskolin analogue, 7-deacetyl-6-(N-acetylglycyl)-forskolin (DAAG-forskolin), that is more water soluble than forskolin and was shown to activate the cAMP pathway without producing the nonspecific effects of forskolin in sensory neurons isolated from the pleural ganglia of Aplysia (Baxter and Byrne 1990b). Application of DAAG-forskolin (75 µM) selectively facilitated B38-evoked EJPs to a similar degree as forskolin, and its effects readily reversed during washout (Fig. 6). We do not know if the difference in the persistence between the two analogues is due to the difference in their hydrophobicity and rate of washout or in their activation of different adenylyl cyclase isozymes. At least 10 different adenylyl cyclase isozymes have been identified, and they exhibit different sensitivity to forskolin and its analogues (Hanoune and Defer 2001; Onda et al. 2002).

View larger version (30K): [in this window] [in a new window]   Fig. 6. Facilitation of EJPs by forskolin analogues. Compound EJPs were evoked by alternately stimulating bursts of action potentials (16 Hz) in B3 () and B38 at 50 s intervals (100 s intervals for each neuron). Initial burst durations were adjusted to evoke EJPs of similar amplitude for both neurons. Application of forskolin (10 µM) and 7-deacetyl-6-(N-acetylglycyl)-forskolin (DAAG-forskolin, 75 µM; |) dramatically facilitated B38-evoked EJPs and had a smaller effect on the B3-evoked EJPs. Note that the facilitation produced by forskolin persisted during washout, whereas that produced by DAAG-forskolin reversed. Application of dideoxy-forskolin (10 µM; |), a forskolin derivative that does not activate adenylyl cyclase, had little effect on EJPs evoked by either neuron, suggesting that forskolin and DAAG-forskolin act by activating the cAMP pathway. B3 bursts were 0.25 s for dideoxy-forskolin, 0.3 s for forskolin, and 0.45 s for DAAG-forskolin. B38 bursts were 0.15 s for all the forskolin analogues. Recordings were from the same preparation and forskolin analogues were applied in order from top to bottom.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Time course of the effects of forskolin (10 µM) or cpt-cAMP and IBMX (each at 100 µM) on B38-evoked EJPs. Application of forskolin or cpt-cAMP/IBMX (from 20 to 0 min) facilitated B38-evoked EJPs. The facilitation produced by forskolin was persistent, whereas that produced by cpt-cAMP/IBMX was not. Increases in EJP amplitude were 556 ± 141% (n = 7) for forskolin and 410 ± 121% (n = 5) for the cpt-cAMP/IBMX. Bursts in B38 were fired at 100 s intervals. Graphed values are means ± SE.

The persistent facilitation produced by 5HT might be due to a persistent increase in cAMP levels. To test for this, we measured cAMP concentrations in I3a muscle segments during the washout of 5HT and SCP. Muscle segments were incubated with either 1 µM 5HT or SCP for 20 min, a standard protocol that reliably causes persistent facilitation of EJPs by 5HT. Both agents increased the level of cAMP over 200-fold above control. To ensure effective washout of the modulators, muscle segments were agitated vigorously in dishes containing large volumes of ASW and transferred to a new dish every 15 min. The level of cAMP produced by both agents declined rapidly during the first hour of washout but stabilized at a level well above control (Fig. 8). Surprisingly, SCP caused a more persistent elevation of cAMP levels than did 5HT. Indeed, for SCP the cAMP levels tended to be slightly elevated 24 h after its washout (by 64 ± 26%, n = 3). Lower concentrations of SCP (0.1 µM) also produced persistent increases in cAMP levels even though the increases in cAMP were much smaller. 5HT was not tested at this lower concentration because it only slightly increases cAMP levels (Fox and Lloyd 2000). Therefore 5HT and SCP caused an increase in cAMP levels that persisted long after they were washed out. It is also interesting to note that these data suggest that the threshold level of cAMP necessary to facilitate EJPs may be relatively high or increase after the application of SCP because the cAMP levels are still significantly elevated (~10-fold) long after the facilitation of EJPs has completely reversed.

View larger version (14K): [in this window] [in a new window]   Fig. 8. Time course of the effects of 5HT or SCP on cAMP levels in I3a muscle. A: application of 1 µM 5HT or SCP for 20 min increased cAMP levels >200-fold. The increase in cAMP produced by both modulators declined rapidly during the 1st hour of washout but then plateaued well above control values (ASW). Note that SCP caused a somewhat more persistent elevation of cAMP levels than 5HT. B: the effects of SCP on cAMP levels were concentration dependent. SCP (0.1 µM) also produced persistent increases in cAMP levels even though the increases in cAMP were much smaller than for 1 µM SCP. Data were normalized to the cAMP levels of muscle segments from the same animals incubated in ASW for 20 min. Graphed values are means ± SE (n = 8 for 5HT; n = 4 for 0.1 and 1 µM SCP; n = 3 for ASW).

Potentiation of contractions by cpt-cAMP was larger after the I3a muscle was exposed to SCP

We were interested in determining whether the persistent elevation of cAMP by SCP might have physiological or behavioral consequences. The long-term elevation of cAMP by SCP was quite large (~10-fold at 3 h after washout), indicating that it occurred predominantly in muscle fibers because neuronal axons make up a very small percentage of tissue volume. We looked at the potentiation of contractions because this effect is known to have a prominent postsynaptic component in muscle fibers (Fox and Lloyd 1997). Potentiation of contractions produced by activation of the cAMP pathway with the membrane permeable cAMP analogue, cpt-cAMP (500 µM), alone or at a lower concentration (100 µM) in combination with IBMX (100 µM), were compared before and after I3a was exposed to SCP. When applied alone, cpt-cAMP potentiated B3- and B38-evoked contractions more effectively 1 h after the muscle was exposed to SCP than before. The ratios of the effects of cpt-cAMP after SCP to those before SCP were 3.3 ± 0.6 (n = 3) for B3 and 2.7 ± 0.4 (n = 4) for B38 (Fig. 9A). The simultaneous application of cpt-cAMP and IBMX also potentiated B3- and B38-evoked contractions more effectively 1 h after SCP than before the muscle was exposed to SCP. These ratios were 1.7 ± 0.2 for B3 and 2.1 ± 0.4 for B38 (n = 7 for each; Fig. 9B). Exposure to SCP appears to be important for this effect because contractions are not significantly potentiated after two consecutive applications of cpt-cAMP and IBMX (each at 100 µM). The ratio of the second application to the first application was 1.0 ± 0.1 for B3 and 1.5 ± 0.4 for B38 (n = 4). Therefore activators of the cAMP pathway produced a larger potentiation of contractions after the I3a muscle was exposed to SCP. This effect is likely to be due, at least in part, to the persistent elevation in the cAMP levels and could be important behaviorally.

View larger version (34K): [in this window] [in a new window]   Fig. 9. Potentiation of contractions by substances that activate the cAMP/PKA pathway were larger after I3a was exposed to SCP. Contractions of I3a were evoked by alternately stimulating bursts of action potentials (16 Hz) in B3 () and B38 at 50 s intervals (100 s intervals for each neuron). Initial burst durations were adjusted to evoke contractions of similar amplitude for both neurons. Generally, longer bursts were required to produce contractions of similar amplitude later during an experiment, but there was no relationship between burst length and the magnitude of the responses to 5HT, SCP, and agents that activate the cAMP pathway. A: application of 500 µM cpt-cAMP (|) potentiated contractions evoked by both B3 and B38. SCP (1 µM) was applied to the preparation for 20 min. After it was washed out for 60 min, cpt-cAMP was more effective at potentiating contractions. B3 bursts were 1.5 s and B38 bursts were 1.6 s before the SCP application and B3 bursts were 2.7 s and B38 bursts were 2.1 s after. B: application of cpt-cAMP/IBMX (each at 100 µM; cpt-cAMP/IBMX; |) potentiated contractions evoked by B38, but had little effect on those evoked by B3. SCP (1 µM) was applied to the preparation for 20 min. After it was washed out for 60 min, cpt-cAMP/IBMX was more effective at potentiating contractions for both neurons. B3 bursts were 1.45 s and B38 bursts were 1.45 s before the SCP application and B3 bursts were 2.1 s and B38 bursts were 1.45 s after.

Phorbol persistently facilitated EJPs evoked by both B3 and B38

The PKC pathway mediates a component of intermediate-term facilitation caused by 5HT at Aplysia central sensory neuron synapses involved in the withdrawal reflexes (Manseau et al. 1998; Sossin et al. 1994; Sugita et al. 1992; Sutton and Carew 2000). Therefore we examined if activation of the PKC pathway persistently facilitated EJPs at neuromuscular synapses. Two concentrations of the PKC activator, 4-phorbol 12,13-diacetate (0.3 and 3 µM -PDA), were used and found to persistently facilitate EJPs evoked by both B3 and B38 (Fig. 10). Phorbol facilitated EJPs evoked by both neurons to a similar degree, whereas 5HT, SCP, and agents that activate the cAMP pathway selectively facilitated B38-evoked EJPs. The lower concentration of -PDA (0.3 µM) facilitated B3-evoked EJPs by 289 ± 36% and those of B38 by 280 ± 14% (n = 3). The higher concentration of phorbol (3 µM) facilitated B3-evoked EJPs by 589 ± 28% and those of B38 by 671 ± 209% (n = 3). A component of the facilitation caused by both phorbol concentrations was persistent and lasted for 2 h after washout (Fig. 10); however, this might be due to poor washout of this lipophylic molecule. Similar results were observed with a second phorbol isomer 12-deoxyphorbol 13-isobuterate (DPIB). Application of 0.3 µM DPIB facilitated B3-evoked EJPs by 344 ± 18% and those of B38 by 391 ± 201% (n = 3). A component of the facilitation caused by DPIB persisted for 1 h after it was washed out. These results suggest that phorbol might activate PKC or a homologue of unc13 that may contribute to the persistent facilitation caused by 5HT at I3a neuromuscular synapses.

View larger version (34K): [in this window] [in a new window]   Fig. 10. Facilitation of EJPs by phorbol. Compound EJPs in the I3a muscle were evoked by alternately stimulating bursts of action potentials (16 Hz) in B3 () and B38 at 50 s intervals (100 s intervals for each neuron). Initial burst durations were adjusted to evoke EJPs of similar amplitude by both neurons. The effects of the phorbol isomer that does not activate PKC (4-phorbol 12,13-diacetate; -PDA) and the active isomer (4-phorbol 12,13-diacetate; -PDA) were tested on the same preparation. -PDA was applied and washed out, for 30 min, before the -PDA application. A: application of -PDA (|) dramatically facilitated EJPs evoked by both B3 and B38, whereas -PDA had little effect. Note that the facilitation of EJPs produced by -PDA persisted after washout. B3 bursts were 0.7 s and B38 bursts were 0.5 s for both the - and -PDA applications. B: time course of the effects of 3 µM -PDA on B3- and B38-evoked EJPs. Application of -PDA (|) facilitated EJPs evoked by both neurons to a similar degree. Increases in EJP amplitude were 589 ± 28% for B3 and 671 ± 209% (n = 3) for B38. Note that the facilitation produced by phorbol was persistent for both neurons. Even though the facilitation of EJPs declined somewhat during the 1st hour of washout (B3 evoked EJPs were 52 ± 6% of maximum at 1 h and those of B38 were 39 ± 4% of maximum; n = 3), it reached a plateau that lasted for at least an additional 1 h of washout (B3 evoked EJPs were 40 ± 6% of maximum at 2 h and those of B38 were 44 ± 11% of maximum; n = 3). Bursts in B38 were fired at 100 s intervals. Graphed values are means ± SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We investigated several possible mechanisms that underlie the persistent effects of 5HT. One of these was the possibility that the apparent reversal of SCP's effects was due to desensitization of the SCP receptor. Superfusion for longer periods or with higher concentrations of SCP indicate that the SCP receptors in I3a do not desensitize. We also found that protein synthesis was not required for persistent facilitation of EJPs in the I3a muscle. We found that 5HT was taken up by elements of the muscle, and although our direct measurements indicated that re-release of 5HT was relatively minor, we could not eliminate the possibility that re-released 5HT contributed to the persistent facilitation. Many of the short-term effects of 5HT and SCP are mediated by activation of the cAMP pathway (Fox and Lloyd 2000). However, we could not establish a clear role for this pathway in persistence. We attempted to produce persistent facilitation by potentiating the effects of SCP with IBMX or by directly activating the cAMP pathway, downstream of the 5HT and SCP receptors, with forskolin analogues or membrane-permeable cAMP analogues. Application of SCP with IBMX prolonged facilitation somewhat, but the effects were still not persistent. The agents used to stimulate the cAMP pathway produced similar short-term effects; however, their effects on persistence were different. The effects of cpt-cAMP and DAAG-forskolin washed out while forskolin persistently facilitated EJPs. Taken together, the results suggest that the activation of the cAMP pathway does not mediate persistent facilitation of EJPs. There are two additional observations that support the hypothesis that the persistence may not be mediated by the cAMP pathway. First, at 1 µM, 5HT and SCP caused similar large increases in cAMP levels, but only 5HT caused persistence. Second, 0.1 µM 5HT had persistent effects even though it did not cause a significant increase in cAMP (Fox and Lloyd 2000). Conversely, 0.1 µM SCP caused large increases in cAMP, but its effects reversed readily. Although persistent facilitation of EJPs by forskolin appears to contradict the results from the other experiments, there are interpretations of these data that are consistent with both results. For example it is possible that SCP and 5HT elevated cAMP levels in different muscle compartments. Forskolin and 0.1 µM 5HT may induce persistence by increasing cAMP levels in a restricted element that is the locus for the persistent facilitation of EJPs, whereas SCP, DAAG-forskolin, and cpt-cAMP may activate the cAMP pathway in another element that does not contribute to persistence. This would require that forskolin and DAAG-forskolin act at different sites. This is possible as 10 different isozymes of adenylyl cyclase have been identified in mammals that exhibit different sensitivity to some derivatives of forskolin (Hanoune and Defer 2001; Onda et al. 2002). However, it is possible that forskolin's persistent effects are due to its slow washout from the preparation even though the effects of forskolin appear to reverse readily in other Aplysia muscles (Lloyd et al. 1985, 1988). Finally, the possibility remains that the cAMP pathway may not mediate the short-term or persistent modulation of EJPs and contractions. However, when the results are viewed collectively, we believe that the evidence supporting a role for the cAMP pathway in mediating the short-term actions of 5HT or SCP is very strong (Fox and Lloyd 2000).

Although the cAMP pathway does not appear to mediate persistent effects on EJPs and contractions, 5HT, as well as SCP, actually does cause persistent increases in cAMP levels. Thus there are two mechanisms by which modulation in I3a primes the system to increase the effectiveness of subsequent activity in motor neurons or modulatory neurons. First 5HT release causes a persistent facilitation of EJPs and potentiation of contractions that lasts many hours. Although these effects are more pronounced at high 5HT concentrations (~1 µM), they were also observed with lower concentrations (~0.1 µM) or after stimulation of the serotonergic MCCs in patterns and frequencies observed during feeding-like motor programs (Fox and Lloyd 1998; Kupfermann and Weiss 1982). Second, although SCP does not cause persistent facilitation and potentiation, it does cause a persistent increase in cAMP levels that we have shown increases the responsiveness of the neuromuscular preparation to agents that activate the cAMP pathway. This persistent increase in cAMP levels is prominent at higher concentrations of SCP (~1 µM) but was also observed at lower concentrations (~0.1 µM) similar to the concentrations that are released when B38 fires in patterns and frequencies observed during feeding-like motor programs (Church and Lloyd 1994; Fox and Lloyd 1998). Thus there are two convergent mechanisms that increase the responsiveness of the I3a neuromuscular system for several hours after a period of sustained activity that is similar to that observed during feeding-like motor programs. One mechanism is selectively elicited by 5HT and likely does not involve the cAMP pathway. The other mechanism is elicited by both 5HT and SCP and is mediated by a persistent increase in cAMP levels.

We chose to examine if the persistent effects were mediated by phorbol because the persistent effects of 5HT on EJPs do not appear to be mediated by the cAMP pathway and phorbol causes short-term enhancement of EJPs without detectably increasing cAMP levels in I3a (Fox and Lloyd 2000). Indeed, a phorbol isomer (-PDA) that activates PKC and presumably an Aplysia homologue of unc13 persistently facilitated EJPs evoked by B38. However, the effects of phorbol were different from those of 5HT in that phorbol also persistently facilitated B3-evoked EJPs, whereas 5HT selectively facilitated B38-evoked EJPs. These results suggest that PKC or unc13 may be important for the modulation of synaptic transmission in the I3a neuromuscular preparation. In addition, the facilitation of B3-evoked EJPs by phorbol, but not 5HT, raises the possibility that transmitters other than 5HT can activate PKC or unc13 and modulate B3-evoked EJPs. A candidate for such a transmitter is dopamine which is present in I3a and selectively facilitates B3-evoked EJPs (Fox et al. 1999).

Persistent facilitation of EJPs in I3a appears, at least superficially, to be similar to the heterosynaptic facilitation of sensory neuron synapses that contribute to the gill and siphon withdrawal reflexes in Aplysia. In these systems, application of 5HT and SCP activate adenylyl cyclase, increase the level of cAMP, and facilitate synaptic transmission. In sensory neurons, the concentration of 5HT and the duration or pattern of its application determines the amplitude and duration of the facilitation (Ghirardi et al. 1995; Mauelshagen et al. 1996, 1998; Montarolo et al. 1986; Sherff and Carew 1999). Persistent or intermediate-term facilitation can be induced without new protein synthesis if the 5HT application is paired with activity of the presynaptic neuron (Bailey et al. 2000b; Sutton and Carew 2000). In both systems, 5HT may activate the PKC pathway in addition to activating the cAMP pathway (Byrne and Kandel 1996). However, there appear to be some major differences between the two systems. SCP application in the presence of IBMX produced long-term facilitation of sensory neuron synapses (Schacher et al. 1990), but did not persistently facilitate the neuromuscular synapses of I3a. PKA and PKC inhibitors block some of the effects of 5HT on sensory neurons. However, these inhibitors were not useful in the I3a preparation because both broad-spectrum inhibitors (H7 and staurosporine) and more specific PKC inhibitors (bisindolylmaleimide and chelerythrine) by themselves caused facilitation of EJPs (Fox and Lloyd 2000). Recent evidence suggests that in sensory neurons the cAMP and PKC pathways function in parallel with considerable cross-talk between them. Activation of PKC by phorbol in sensory neurons raises cAMP concentration and also attenuates subsequent responses to 5HT (Sugita et al. 1997). However, this does not appear to occur in I3a because phorbol does not detectably elevate cAMP levels (Fox and Lloyd 2000). Finally, facilitation of the sensory neurons is predominantly presynaptic based on quantal analysis of transmitter release, the increase in cAMP levels, and changes in the spike broadening (Baxter and Byrne 1990a; Bernier et al. 1982; Castellucci and Kandel 1976; Dale et al. 1988; Eliot et al. 1994; Sugita et al. 1997), although it is becoming increasingly clear that the mechanisms underlying the facilitation may also have postsynaptic components (Bao et al. 1997, 1998; Murphy and Glanzman 1997; Schacher et al. 1997; Sherff and Carew 2000). Many of the effects of 5HT or SCP on I3a muscle fibers appear to be mediated postsynaptically. They increase the relaxation rate of contractions, potentiate contractions evoked by the exogenous glutamate to isolated bundles of muscle fibers, and modulate some step(s) in excitation-contraction coupling because they dramatically potentiate B3-evoked contractions even when the B3-evoked EJPs were essentially unchanged (Church et al. 1993; Fox and Lloyd 1997; Lotshaw and Lloyd 1990). It is also possible that 5HT and SCP act presynaptically in I3a because they selectively facilitate B38-evoked EJPs.

In crustacean skeletal muscles, 5HT has been shown to persistently facilitate EJPs (Dixon and Atwood 1985; Dudel 1965; Glusman and Kravitz 1982; Kravitz et al. 1980). The persistent facilitation in crayfish has been divided into two phases, early and late. Although reversal of the late phase is long-lasting, it is less persistent than we observed in the I3a muscle of Aplysia. The same second-messenger systems that underlie the short-term and persistent facilitation in Aplysia, the cAMP and PKC pathways, are also important for early and late facilitation in crayfish. The early phase of facilitation caused by 5HT is mimicked by activators of the PKC pathway while the late phase of facilitation requires the activation of both the cAMP and PKC pathways (Dixon and Atwood 1989a-c).

In conclusion, we have shown that 5HT persistently facilitates EJPs. The mechanisms that underlie the facilitation are complex and probably involve more that one second-messenger system. Activation of the cAMP pathway mimics the short-term effects of 5HT, but it does not appear to cause persistence. Instead, activation of PKC or an Aplysia homologue of unc13 by phorbol produces persistent effects. SCP apparently does not activate the second-messenger system(s) involved in persistence. However, SCP does persistently elevate cAMP levels in the muscle. The increase in the cAMP concentration appears to be a mechanism for the storage of information about previous modulation and potentiates subsequent responses of the muscle to activators of the cAMP pathway. These results, in combination with results from experiments on the sensory neurons that contribute to the withdrawal reflexes in Aplysia, suggest that the mechanisms for intermediate- and long-term facilitation may reside in both central and peripheral components of sensory to motor response reflexes.