Protein Kinase C Is Required for Long-Lasting Synaptic Enhancement by the Neuropeptide DRNFLRFamide in Crayfish

Rainer W. Friedrich1, G. F. Molnar1, Michael Schiebe2, and A. Joffre Mercier1

1 Department of Biological Sciences, Brock University, St. Catharines, Ontario L2S 3A1, Canada; and 2 Abteilung für angewandte Physiologie, Universität Ulm, D-89069 Ulm, Germany

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Friedrich, Rainer W., G. F. Molnar, Michael Schiebe, and A. Joffre Mercier. Protein kinase C is required for long-lasting synaptic enhancement by the neuropeptide DRNFLRFamide in crayfish. J. Neurophysiol. 79: 1127-1131, 1998. The FMRFamide-related neuropeptide AspArgAsnPheLeuArgPhe-NH2 (DRNFLRFamide, DF2) induces a long-lasting enhancement of synaptic transmission at neuromuscular junctions on the crayfish deep abdominal extensor muscles. Here we investigated the function of protein kinase C (PKC) in this effect because PKC has been implied in the control of long-term synaptic modulation in other systems. The general kinase antagonist staurosporine reduced both the initial increase in excitatory postsynaptic potential (EPSP) amplitude and the duration of synaptic enhancement. Unlike staurosporine, the selective PKC inhibitors, chelerythrine and bisindolylmaleimide, augmented the initial EPSP increase. However, like staurosporine, they also reduced the duration of synaptic enhancement. The PKC activator, phorbol-12-myristate 13-acetate, induced a long-lasting synaptic enhancement that was blocked by chelerythrine. These results show that synaptic enhancement by DF2 is mediated by different intracellular signaling systems that act in temporal sequence. The initial increase in EPSP amplitudes is negatively regulated by PKC and involves another, staurosporine-sensitive, kinase; whereas, the maintenance of synaptic enhancement requires PKC.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Long-lasting changes in synaptic transmission underlie important physiological and developmental processes such as learning, memory formation, and the establishment of synaptic connections. In the temporal domain, long-term synaptic modulation can be separated into sequential phases that are mediated by distinct intracellular signaling systems, as found in model systems such as serotonin-induced facilitation in crustaceans (Dixon and Atwood 1989a,b; Goy and Kravitz 1989) and Aplysia (Braha et al. 1993; Byrne and Kandel 1996; Sugita et al. 1992) and long-term potentiation (LTP; Bliss and Collingridge 1993) and depression (Linden and Connor 1995) in vertebrates.

In crayfish, a long-lasting enhancement of synaptic transmission is induced by the neuropeptide AspArgAsnPheLeuArgPhe-NH2 (DRNFLRFamide, DF2) at the deep abdominal extensor neuromuscular junction (Mercier et al. 1993). This synaptic enhancement is the result of an increase in the amount of transmitter released (Skerrett et al. 1995). DF2, which belongs to the family of FMRFamide-related peptides (Price and Greenberg 1992), also has cardioexcitatory effects and probably functions as a neurohormone, because it is released into the circulation (Mercier et al. 1993; Skerrett et al. 1995). The physiological roles of DF2 might include the adaptation of neuromuscular function to low temperature (Friedrich et al. 1994) as well as other, not yet identified, functions.

In a previous study it was shown that inhibition of calcium/calmodulin-dependent protein kinase II (CaMK II) delays the increase in excitatory postsynaptic potential (EPSP) amplitude induced by DF2 at the neuromuscular junction but does not affect long-term synaptic enhancement (Noronha and Mercier 1995). The maintenance of synaptic enhancement by DF2 therefore appears to be mediated by other signaling systems. Here we have examined whether or not these signaling systems involve protein kinase C (PKC), because PKC has been implicated in the control of long-term synaptic modulation in some nonpeptidergic forms of synaptic modulation (e.g. Bliss and Collingridge 1993; Byrne and Kandel 1996; Dixon and Atwood 1989a,b). Pharmacological inhibition of PKC abolished the maintenance but not the initial phase of synaptic enhancement by DF2, showing that PKC is required for long-lasting synaptic enhancement by DF2. Preliminary results were published in abstract form (Friedrich et al. 1993).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Experiments were performed on the deep abdominal extensor muscle L1 in segment 4 of the crayfish, Procambarus clarkii (2-4 cm carapace length). EPSPs in muscle cells were evoked by electrical stimulation (0.1 or 0.2 Hz) of axon 3 in segment 3 and recorded intracellularly as described by Mercier and Atwood (1989). EPSP amplitude was measured every 30 s and the values averaged within 1.5- or 5-min time bins. All experiments were performed in a perfused recording chamber at 8 ± 0.3°C (SE) except those involving phorbol-12-myristate 13-acetate (PMA), which were perfomed in a static bath at 15 ± 0.5°C for safety reasons. To exclude possible seasonal variations (Lnenicka and Zhao 1991), sets of experiments using each chemical agent were performed within 1 mo. Each set of experiments was compared statistically to control experiments with DF2 alone that were performed in the corresponding time of the year (within 1 mo before or after the experiments with that chemical agent). Chemical agents were applied 10-40 min before addition of DF2 and removed at the end of DF2 application.

The following stock solutions were prepared and diluted into saline directly before use: DF2 (synthesized by T.S. Chen of the Biotechnology Service Centre, Toronto, Canada), 100 µM in water or saline; PMA (Sigma), 1 mM in dimethyl sulfoxide (DMSO); staurosporine (Sigma), 1 mM in DMSO; chelerythrine chloride (LC Services Woburn, MA), 10 mM in water; and bisindolylmaleimide 1 (Calbiochem-Nova Biochem, La Jolla, CA), 5 mM in water. Final DMSO concentrations were kept constant throughout any given experiment and did not exceed 0.1%, which had no effect in control experiments. Control recordings in the absence of DF2 were carried out with each drug for an extended period of time to detect possible effects of the drug alone. None of the drugs used had an effect on the resting membrane potential. Statistical comparisons were performed by using a Mann-Whitney U test unless otherwise noted. Values are means ± SE.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

A 15- to 20-min application of 200 nM DF2 elicited a long-lasting increase in EPSP amplitudes (Fig. 1A) that amounted, on average, to 146 ± 12% (n = 44). In three experiments, recordings were continued for 80 min after onset of DF2 application. EPSPs were still elevated at that time, showing that synaptic enhancement outlasts DF2 application by >1 h.


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FIG. 1. Time course of synaptic enhancement by AspArgAsnPheLeuArgPhe-amide (DRNFLRFamide, DF2) and effect of protein kinase inhibitors. A: time course of increase in excitatory postsynaptic potential (EPSP) amplitude induced by 200 nM DF2 applied during time indicated by shaded area (n = 13). Inset: examples of EPSPs recorded before (lower trace) and at end (upper trace) of DF2 application. Scale bars, 4 mV, 15 ms. B-D: time course of synaptic enhancement in presence of general kinase inhibitor, staurosporine (100 nM or 1 µM; n = 10) and protein kinase C (PKC) inhibitors chelerythrine (10 µM; n = 7) and bisindolylmaleimide (BIM; 250 nM; n = 6). Inhibitors were applied 10-15 min (staurosporine, BIM) or 30-40 min (chelerythrine) before to addition of DF2 and removed at end of DF2 application. For better comparision of time courses, graphs are normalized to maximum EPSP increase. Effects of inhibitors on maximum EPSP increase are shown separately in Fig. 2. · · ·, level of EPSP increase in control experiments 36 min after onset of DF2.

Protein kinase inhibitors were used to identify components of the intracellular signaling systems that mediate synaptic enhancement by DF2. To analyze the effects of the inhibitors on the temporal development of synaptic enhancement independently from those on the maximum EPSP increase, the time courses of the EPSP increase induced by DF2 were normalized to the maximum EPSP increase for each inhibitor and are compared in Fig. 1. Effects of inhibitors on the maximum EPSP were analyzed separately and are expressed in Fig. 2 as the percentage of the control EPSP increase induced by DF2 in the absence of any drugs.


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FIG. 2. Effect of inhibitors on maximum EPSP increase induced by DF2. Maximum increase in EPSP amplitude during an 18-20 min application of 200 nM DF2 (average 146 ± 12%; n = 44) is indicated by 100% and compared with EPSP increase in presence of staurosporine (stau: left, 100 nM, n = 8; right, 1 µM, n = 9), chelerythrine (chel; 10 µM, n = 8), and BIM (250 nM, n = 6). Levels of significance: P <=  0.05; *** P < 0.0001.

To test for the involvement of protein kinases in synaptic enhancement by DF2 we used the broad spectrum kinase antagonist staurosporine (Ruegg and Burgess 1989). In the absence of DF2, staurosporine alone (100 nM or 1 µM) slightly decreased EPSP amplitudes in 3 of 18 preparations, although by not more than 5%. The EPSP increase induced by DF2 in the presence of 100 nM and 1 µM staurosporine was significantly reduced to 54 ± 4% (n = 8) and 43 ± 5% (n = 9), respectively, of the control increase obtained in the absence of staurosporine (P < 0.0001; Fig. 2). Furthermore, the peak EPSP increase occurred earlier than in the control and EPSP amplitudes subsequently declined significantly more rapidly (Fig. 1B; analysis of variance(ANOVA) followed by post hoc Scheffe's F test; P < 0.001), indicating that staurosporine inhibited the maintenance of synaptic enhancement.

The PKC antagonists, chelerythrine and bisindolylmaleimide (BIM), were used to determine the role(s) of PKC in synaptic enhancement by DF2. These agents inhibit PKC by different mechanisms and were shown not to affect other kinases at the concentrations used here (Herbert et al. 1990; Toullec et al. 1991). Chelerythrine alone (10 µM) decreased EPSP amplitudes in 10 of 14 preparations by an average of 13 ± 5% and EPSPs reached a stable level after 10-15 min. BIM alone (250 nM) had no effect. In contrast to staurosporine, both chelerythrine and BIM increased the maximum effect of DF2 to 138 ± 19% (n = 8, P = 0.05) and 154 ± 25% (n = 6, P < 0.05), respectively, of the control EPSP increase (Fig. 2). Like staurosporine, chelerythrine and BIM shifted the peak EPSP increase to earlier times and significantly reduced the duration of synaptic enhancement by DF2 (Fig. 1, C and D; ANOVA followed by post hoc Scheffe's F test; P < 0.001). These results indicate that PKC is necessary for the maintenance of synaptic enhancement by DF2 but has a negative effect on the initial increase in EPSP amplitudes.

The PKC-activator, PMA, was used to determine the effect of PKC on synaptic transmission in the absence of DF2. PMA (500 nM) induced a progressive increase in EPSP amplitudes, which after 60 min amounted to 313 ± 100% (n = 7; Fig. 3) and could not be reversed by washing for up to one hour. 10 µM chelerythrine almost completely blocked the EPSP increase induced by PMA (32 ± 21% increase after 60 min; n = 6, P < 0.0001; Fig. 3). When preparations were treated first with PMA for 1-3 h, subsequent addition of 10 µM chelerythrine for 30-40 min decreased EPSPs by 34 ± 10% of the amplitudes recorded at the end of exposure to PMA alone (n = 3; data not shown). These data indicate that activation of PKC causes a long-lasting synaptic enhancement, which is consistent with the effect of PKC on other synapses (Dixon and Atwood 1989a; Malinow et al. 1988; Nishizuka 1992; Shapira et al. 1987; Sugita et al. 1992) and with a role for PKC in the maintenance of synaptic enhancement by DF2.


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FIG. 3. Effect of exogenous activation of PKC on EPSP amplitude. Phorbol-12-myristate 13-acetate (PMA, 500 nM) was applied during period indicated by shaded area in absence (open circle ; n = 7) or presence (bullet ; n = 6) of 10 µM chelerythrine. Chelerythrine alone was applied 30 min before addition of PMA.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

We have used the broad-spectrum kinase inhibitor, staurosporine, and the specific PKC antagonists, chelerythrine and BIM, to examine the function of PKC in the long-lasting synaptic enhancement induced by DF2 at the crayfish neuromuscular junction. The specificity of chelerythrine and BIM for PKC is well established (Herbert et al. 1990; Toullec et al. 1991) and further supported by the following observations: 1) the effects of both inhibitors on synaptic enhancement by DF2 were very similar and 2) they do not affect the EPSP increase elicited by the protein kinase A (PKA) activator, Sp-cAMPS, or by the adenylyl cyclase activator, forskolin (M. M. Boldt, R. W. Friedrich, and A. J, Mercier, unpublished results). In the absence of DF2, chelerythrine and staurosporine caused slight reductions in EPSP amplitude. These may be nonspecific effects on synaptic transmission, but the underlying mechanisms are not clear.

In the presence of each of the inhibitors used, the peak increase in EPSP amplitude induced by DF2 occurred earlier and the synaptic enhancement was less sustained. The magnitude of the EPSP increase was reduced by staurosporine but augmented by chelerythrine and BIM. These results show that PKC has a negative effect on the initial EPSP increase but is required for the maintenance of synaptic enhancement by DF2. On the basis of these results, synaptic enhancement by DF2 can be dissociated into at least two phases: 1) an early phase that comprises the initial increase in EPSP amplitudes and is inhibited by staurosporine but augmented by PKC antagonists and 2) a late phase that comprises the maintenance of synaptic enhancement and is abolished by both staurosporine and PKC inhibitors. These phases are not obvious without pharmacological dissection because no clearcut transition is apparent in the time course of synaptic enhancement in the absence of inhibitors.

The increase in EPSP amplitude induced by DF2 in the presence of either staurosporine or the PKC inhibitors peaked within 10 min and thereafter declined while DF2 was still present, indicating that the early phase is transient (total duration <= 40 min). Without inhibitors, EPSP amplitudes increased asymptotically during the period of DF2 application and thereafter remained elevated for at least 1 h. This suggests that the late phase, which requires PKC, overlaps with the early phase but progresses more slowly. This is consistent with the slow increase in EPSP amplitude induced by PMA and with the time course of synaptic enhancement in the presence of a CaMK II antagonist, which appears to inhibit the early phase (Noronha and Mercier 1995; see below).

The early phase of synaptic enhancement by DF2 was antagonized by staurosporine but not by PKC inhibitors, indicating that it involves at least one kinase other than PKC. This kinase may be CaMK II, because inhibition of CaMK II delays the EPSP increase induced by DF2 by 10-15 min and the peak by about 40 min without affecting prolonged synaptic enhancement (Noronha and Mercier 1995). Thus, inhibitors of CaMK II and PKC have complementary effects on synaptic enhancement by DF2 and appear to selectively inhibit the early and late phases, respectively. However, it cannot be concluded that CaMK II and PKC account for the entire effect of DF2 because other protein kinases (such as PKA) could also be involved.

PKC inhibitors augmented the initial EPSP increase induced by DF2, suggesting that PKC acts as a negative regulator of the early phase. Because the early phase requires CaMK II (Noronha and Mercier 1995), it is likely that it involves an increase in intracellular [Ca2+]. In various cell types, agonist-induced PKC activity develops more slowly than changes in intracellular [Ca2+] and participates in feedback mechanisms that lead to the the subsequent down-regulation of intracellular [Ca2+] (Nishizuka 1992). Thus it may be speculated that Ca2+-dependent activation of CaMK II during the early phase is gradually antagonized by a concomitant, but slower, activation of PKC, which may eventually terminate CaMK II activity. This hypothesis would parsimoniously explain the data obtained to date, although direct experimental support is not available. The function of such a negative regulatory effect of PKC may be to down-regulate the early phase while the late phase is progressing, thereby regulating the transition between the two phases.

Synaptic enhancement by DF2 is induced by a neuropeptide that probably functions as a neurohormone (Mercier et al. 1993). Like other, nonpeptidergic forms of long-lasting synaptic modulation, synaptic enhancement by DF2 is mediated by multiple intracellular signaling systems that act in temporal sequence. A comparison of the data available indicates that the signaling systems actually involved in different model systems show considerable diversity; however, PKC often appears to be required for the long-term nature of synaptic modulation. For example, in serotonin-induced facilitation in crayfish, PKC might be responsible for linking the signaling systems that mediate the first phase and the second (maintenance) phase (Dixon and Atwood 1989a,b). In serotonin-induced facilitation in Aplysia, inihibitors of PKC selectively block those components that are associated with the maintenance of synaptic modulation with little or no effect on the early components (Braha et al. 1993; Byrne and Kandel 1996; Sugita et al. 1992). In LTP, pharmacological inhibition of PKC converts the long-term (>40 min) effect into a transient response (Ben-Ari et al. 1992; Bliss and Collingridge 1993; Lovinger et al. 1987; Malinow et al. 1988; Reymann et al. 1988), similar to the effect on synaptic enhancement by DF2. These observations suggest that PKC may play a decisive role in the control of the transition from short-term to long-term synaptic modulation (Schwartz 1993) in diverse systems. Despite the apparent functional similarity of the role of PKC however, it is not clear to what degree PKC signaling is equivalent on the molecular level. Further work is required to examine the regulation of PKC, the signaling downstream of PKC, and the events that ultimately lead to modulation of synaptic function.

    ACKNOWLEDGEMENTS

  We thank Dr. Jens R. Coorssen for advice on pharmacology and comments on the manuscript.

  This work was supported by grants from the National Sciences and Engineering Research Council of Canada (to A. J. Mereier) and the Deutsche Forschungsgemeinschaft (to M. Schiebe).

    FOOTNOTES

   Present address of R. W. Friedrich: Max-Planck-Institut für Entwicklungsbiologie, Abteilung Physikalische Biologie, Spemannstr. 35, D-72076 Tubingen, Germany.

  Address for reprint requests: A. J. Mercier, Brock University, Dept. of Biological Sciences, St. Catharines, Ontario L2S 3A1, Canada.

  Received 18 August 1997; accepted in final form 7 November 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society