Serotonin persistently activates the extracellular signal-related kinase in sensory neurons of Aplysia independently of cAMP or protein kinase C

Neuroscience 116 (2003) 13–17

LETTER TO NEUROSCIENCE SEROTONIN PERSISTENTLY ACTIVATES THE EXTRACELLULAR SIGNAL-RELATED KINASE IN SENSORY NEURONS OF APLYSIA INDEPENDENTLY OF cAMP OR PROTEIN KINASE C J. R. DYER,a F. MANSEAU,b V. F. CASTELLUCCIb AND W. S. SOSSINa*

In Aplysia, activation of gene expression is required for long-term facilitation of sensory to motorneuron synapses, but gene expression is not required for short-term facilitation of these synapses. A number of transcription factors have been implicated in the specific requirement for longterm facilitation (Alberini et al., 1994; Bartsch et al., 1995; Bartsch et al., 1998; Bartsch et al., 2000). The extracellular signal-related kinase (ERK) is an important signal that is thought to be important for the activation of the transcription factors that lead to the new gene expression required for long-term facilitation (Martin et al., 1997; Yamamoto et al., 1999). ERKs have also been implicated in long-term synaptic changes in many different systems other than Aplysia (Sweatt, 2001). ERK activity is important for the activation of the transcription factor cyclic AMP response elementbinding protein (CREB) through activation of a CREB kinase in vertebrate neurons (Xing et al., 1996). Also, ERK can phosphorylate other transcription factors, and modulates synaptic strength through the phosphorylation of ion channels (Sweatt, 2001). There are a number of mechanisms by which ERKs can be activated downstream of neurotransmitter-gated G protein coupled receptors. In Aplysia, the neurotransmitter serotonin (5-HT) causes synaptic facilitation mainly through activation of the kinase protein kinase A (PKA) by cAMP and activation of the kinase protein kinase C (PKC) by diacylglycerol (DAG) (Byrne and Kandel, 1996). cAMP can activate ERK through either PKA phosphorylation of b-raf (Vossler et al., 1997) or directly through cAMP-gated guanine nucleotide exchange factors (GEFs) (de Rooij et al., 1998; Kawasaki et al., 1998). In both vertebrate and Aplysia neurons, cAMP and PKA activation are also important in the translocation of ERK to the nucleus (Martin et al., 1997). PKC activation has been shown to be important for ERK activation through phosphorylation of the upstream Raf kinase (Cai et al., 1997; Marais et al., 1998) and DAG can directly activate ERK through the DAGactivated GEF, RasGrp (Lorenzo et al., 2001). There are also other mechanisms by which ERK can be activated downstream of G protein-coupled receptor (Dikic and Blaukat, 1999; Pierce et al., 2001). We examined how 5-HT activates ERK in Aplysia sensory neurons. We find that sustained activation of ERK by 5-HT is independent of cAMP or PKC suggesting that

a Department of Neurology and Neurosurgery, McGill University, Montreal Neurological Institute, Room 776, 3801 rue University, Montreal, Que., Canada H3A 2B4 b Laboratoire de Neurobiologie et comportement, Centre de Recherche en Sciences Neurologiques, De´partement de physiologie, Universite´ de Montre´al, Montreal, Que., Canada

Abstract—Activation of the extracellular signal-related kinase is important for long-term increases in synaptic strength in the Aplysia nervous system. However, there is little known about the mechanism for the activation of the kinase in this system. We examined the activation of Aplysia extracellular signal-related kinase using a phosphopeptide antibody specific to the sites required for activation of the kinase. We found that phorbol esters led to a prolonged activation of extracellular signal-related kinase in sensory cells of the Aplysia nervous system. Surprisingly, inhibitors of protein kinase C did not block this activation. Serotonin, the physiological transmitter involved in long-term synaptic facilitation, also led to prolonged activation of extracellular signal-related kinase, but inhibitors of protein kinase A or protein kinase C did not block this activation. We examined whether the protein synthesis-dependent increase in excitability stimulated by phorbol esters was dependent on phorbol ester activation of extracellular signal-related kinase, but increases in excitability were still seen in the presence of inhibitors of extracellular signal-related kinase activation. Our results suggest that prolonged phosphorylation of extracellular signal-related kinase in the Aplysia system is not mediated by either of the classic second messenger activated kinases in this system, protein kinase A or protein kinase C and that extracellular signal-related kinase is not important for phorbol ester induced long-term effects on excitability. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: signal transduction, long-term facilitation, excitability, MAP kinases, learning and memory, diacylglycerol.

*Corresponding author: Tel: ⫹1-514-398-1486; fax: ⫹1-514-3988106. E-mail address: [email protected] (W. Sossin). Abbreviations: CREB, cyclic AMP response element binding protein; DAG, diacylglycerol; ERK, extracellular signal-related kinase; 5-HT, 5-hydroxytryptamine or serotonin; GEF, guanine nucleotide exchange factor; PDBu, 4␤-phorbol ester 12,12 dibutyrate; PKA, protein kinase A; PKC, protein kinase C.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 5 6 6 - 3

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additional signal transduction pathways are important for the stimulation of long-term facilitation in Aplysia.

EXPERIMENTAL PROCEDURES Isolation of neuronal components Aplysia californica (50 –250 g) were obtained from Marine Specimens Unlimited at Pacific Palisades, California, and maintained in an aquarium for at least 3 days before experimentation and sensory cell clusters were dissected from the isolated pleural ganglia as done previously (Dyer and Sossin, 2000).

Treatments of sensory cell clusters with pharmacological reagents The dissected sensory cell clusters were incubated in resting media for 3 h at 15°C before addition of pharmacological agents. At the end of the experiments, tissue was homogenized in lysis buffer (5% 2-mercaptoethanol, 20 mM Tris at pH 8.8, 50 mM NaF, 5 mM NaPyrophosphate, 1 ␮M microcystin, 5 mM benzamidine, 100 ␮M leupeptin, and 20 ␮g/ml aprotinin). In all experiments, control and experimental ganglion consisted of paired components from the same animal.

Immunoblotting Western blots with ERK and phospho-ERK antibodies were performed as described (Dyer and Sossin, 2000). Immunoblots were scanned and analysis performed using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nihimage/). We calibrated our data with the uncalibrated OD feature of NIH Image which transforms the data using the formula [y⫽log10(255/(255⫺x)] where x is the pixel value (0 –254). Control experiments demonstrated that after this calibration, values were linear with respect to amount of protein over a wide range of values.

Statistics Each experiment was evaluated using a paired t-test between control cluster and 5-HT- or 4␤-phorbol ester 12,12 dibutyrate (PDBu)-treated cluster. In the presence of inhibitors the paired t test was between inhibitor-treated cluster and inhibitor plus 5-HTor phorbol ester-treated cluster. To determine where there was a difference between the presence or absence of an inhibitor an unpaired t test was performed on the percentage changes in the presence or absence of an inhibitor. Changes of ⬍0.05 were considered significant.

Excitability measurements Excitability was measured as described (Manseau et al., 1998). Briefly, 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. Action potential threshold was determined rapidly, and starting from that value, a total of five pulse stimuli increasing by 0.05 nAMPs were applied at 1-min intervals. At the end of the recording, the intracellular electrodes were removed, and either PDBu and vehicle or PDBu and 10 ␮M U0126 were added for 5 min. Washing was done manually with 10 times the bath volume and a second series of identical depolarizing steps and recordings was started 24 h later. 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.

Immunocytochemistry Sensory cells were cultured as described (Manseau et al., 2001), treated with 1 ␮M PDBu or DMSO for 1 h, and then fixed and immunostained as described (Martin et al., 1997). The anti-MAP kinase antibody was a kind gift from Kelsey Martin. To allow double labeling we used an anti-chicken PKC antibody that was raised to the same epitope as the rabbit PKC. This antibody appears indistinguishable from the rabbit antibody on immunoblots and immunocytochemistry (data not shown). Confocal pictures were taken at a level where the nucleus was clearly distinguishable. The mean pixel density was calculated for membrane (defined as the first two microns from the outside), cytoplasm, and nucleus for each cell and ratios calculated (nucleus/cytoplasm or membrane/cytoplasm) for each cell. Quantitation was done blind to the experimental condition.

RESULTS To monitor ERK activation, we monitored phosphorylation of ERK, which is necessary and sufficient for ERK activation (Payne et al., 1991). We used a regular and a phosphopeptide antibody to mammalian ERK that both crossreact with Aplysia ERK (Dyer and Sossin, 2000). Activation was calculated as the percentage increase in ERK phosphorylation standardized to levels of total ERK. In sensory cell clusters, a 60-minute treatment with 1 ␮M PDBu was very effective in causing a sustained increase in phosphorylation of ERK (Fig. 1A). Surprisingly, this effect appears independent of PKC activation because it was not blocked by a PKC inhibitor chelerythrine (Fig. 1B) that has been shown to block PKC mediated effects in the Aplysia nervous system (Manseau et al., 1998; Dyer and Sossin, 2000; Sutton and Carew, 2000). Nor was the increase in ERK phosphorylation caused by the ability of PDBu to increase cAMP levels (Sugita et al., 1997) because RpcAMP also did not block the ability of PDBu to increase ERK phosphorylation (Fig. 1B). Even using a smaller concentration of PDBu (100 nM), the effect of PDBu was not blocked by RpcAMP or chelerythrine (Fig. 1C, quantitated in Fig. 1D). Higher concentrations of chelerythrine (50 ␮M) also did not block the effects of 100 nM PDBu (data not shown). Thus, PDBu increases ERK phosphorylation through a pathway that is independent of either cAMP or PKC. A continuous 90-min application of 5-HT led to a similar persistent increase in ERK phosphorylation (Fig. 2A, C). 5-HT still persistently increased ERK phosphorylation in the presence of RpcAMP, chelerythrine, or both inhibitors (Fig. 2A, B) such that there was no significant difference between the increase in ERK phosphorylation in the presence or absence of these inhibitors (Fig. 2C). Therefore, neither 5-HT not phorbol esters activate sustained ERK phosphorylation through the standard second messengers known to be involved in synaptic facilitation in Aplysia. Phorbol esters only stimulate transient changes in synaptic facilitation in Aplysia (Braha et al., 1990; Wu et al., 1995), but can stimulate prolonged increases in excitability that depend on protein synthesis (Manseau et al., 1998). To examine whether these effects are mediated through ERK we examined whether an inhibitor of the upstream activator of ERK, U0126, could block the ability of phorbol

J. R. Dyer et al. / Neuroscience 116 (2003) 13–17

Fig. 1. Activation of ERK phosphorylation by PDBu is independent of PKC or PKA activation. (A) Immunoblots of homogenates of sensory cell clusters treated for 60 min with either 1 ␮M inactive 4-␣-PDBu (⫺) or the active isoform 1 ␮M PDBu (⫹). The membrane was probed with the antibody to phosphorylated ERK (ERK-P), stripped, and re-probed with the antibody to regular ERK. (B) Immunoblots of homogenates of sensory cell clusters treated with 1 ␮M inactive 4-␣-PDBu (⫺) or the active isoform 1 ␮M PDBu (⫹). The clusters were preincubated with either vehicle, 10 ␮M chelerythrine or 500 ␮M RpcAMP for 30 min prior to PDBu treatment and during the incubation with PDBu. (C) The same as (B) but with 0.1 ␮M PDBu instead of 1 ␮M PDBu. (D) The percent change in ERK phosphorylation was calculated by first calculating a phosphate ratio (intensity of ERK-P blot/intensity of ERK blot) then calculating the percent change in this ratio from the control paired cluster. Results are the mean⫾S.E.M. for PDBu alone (A, n⫽9) for PDBu in the presence of RpcAMP (B, n⫽6) and for PDBu in the presence of chelerythrine (C, n⫽6). Results with 0.1 and 1 ␮M PDBu and with 10 ␮M and 50 ␮M chelerythrine were pooled, as there were no significant differences between these treatments. (*P⬍0.05, paired t test between control and treated cluster. NS, P⬎0.1 unpaired t test between percent change in the absence or presence of inhibitors).

esters to increase long-term excitability. However, even in the presence of U0126, there was a strong increase in excitability 24 h after PDBu treatment (19.8⫾11.8 increased spikes after PDBu, n⫽11) not significantly different to that seen with PDBu alone (21.6⫾9.2, n⫽14). Thus, ERK activation is not required for the PDBu increased change in excitability. It is surprising that PDBu activates ERK, but does not stimulate long-term facilitation (Wu et al., 1995). One possibility is that PDBu increases phosphorylation of ERK, but not nuclear translocation of ERK. We measured the ability of PDBu to translocate ERK to the nucleus in cultured sensory cells using an antibody to Aplysia MAP kinase. PDBu did not translocate ERK to the nucleus and indeed, caused a translocation to the membrane of the cells, similar to translocation observed for PKC (Fig. 3A; Fig. 3B). Thus different ways of activating ERK may lead to differential localization of ERK and thus, downstream effects of ERK activation.

DISCUSSION Phorbol esters activate ERK in many systems and in many cases this is through PKC activation. However, other phorbol ester binding proteins may also lead to ERK kinase activation. In particular, phorbol esters directly stimulate

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Fig. 2. Activation of ERK phosphorylation by 5-HT is independent of PKC or PKA activation. (A) Immunoblots of homogenates of sensory cell clusters either treated for 90 min with resting medium (⫺) or 20 ␮M 5-HT (⫹). The clusters were pre-incubated with vehicle, 10 ␮M chelerythrine, or 500 ␮M RpcAMP for 30 min prior to 5-HT treatment and during the incubation with 5-HT. The membrane was probed with the antibody to phosphorylated ERK (ERK-P), stripped, and re-probed with the antibody to regular ERK. (B) Same as (A) but preincubation was with both Chelerythrine and RpcAMP. (C) The percent change in ERK phosphorylation was calculated as in Fig. 1. Results are the mean⫾S.E.M. for 5-HT alone (A, n⫽14) for 5-HT in the presence of RpcAMP (B, n⫽10) for 5-HT in the presence of chelerythrine (C, n⫽11) and for 5-HT in the presence of chelerythrine and RpcAMP (D, n⫽11). *P⬍0.05, paired t test between control and treated cluster. NS, P⬎0.1 unpaired t-test between percent change in the absence or presence of inhibitors.

RasGrp, which activates ERK by acting as a Ras GEF (Lorenzo et al., 2001). However, in the related mollusk Hermissenda PKC inhibitors did block phorbol ester-mediated increases in ERK activation and ERK activation by serotonin was partly dependent on PKC (Crow et al., 2001). The neurons being assayed, as well as the time of measurement (5 min vs. 60 or 90 min) differ between this study and ours. It may be that the initial activation of ERK can be mediated by PKC activation, but prolonged activation requires other pathways. We did not see a role for cAMP in ERK activation, but there is clearly an important role for cAMP in ERK translocation to the nucleus (Martin et al., 1997). Forskolin has been reported to increase ERK activity in Aplysia, but these experiments used an in-gel kinase assay and were based on a small number of replications (Michael et al., 1998). We did not see a significant increase in ERK phosphorylation by forskolin (data not shown) and furthermore, RpcAMP did not inhibit the ability of 5-HT to stimulate sustained ERK phosphorylation (Fig. 2). Using pleural ganglia isolated from animals, it is impossible to avoid an injury response (Alberini et al., 1994). Thus, an important caveat for our results is that they take place in an “injury-primed” model. This may explain the relatively high levels of ERK phosphorylation in control samples. Another Map kinase, RISK-1, which is distinct from Aplysia ERK is activated by injury (Sung et al., 2001). It is unlikely that we are measuring activation of this kinase, since we are using a different antibody than the one used

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dent in hippocampal neurons (Patterson et al., 2001). Indeed, PDBu did not translocate ERK to the nucleus in cultured sensory neurons (Fig. 3). The lack of effect of an inhibitor of the ERK kinase on PDBu mediated changes in excitability suggests other pathways are probably important for this long-term change. Indeed, PKC inhibitors did block the effects of PDBu on excitability (Manseau et al., 1998). Interestingly, 5-HT does not appear to lead to long-term changes in excitability (Liao et al., 1999a), but other pathways activated by injury, some of which activate PKC, do lead to long-term excitability changes (Liao et al., 1999b). It will be interesting to discover which transcription factors activated by PKC are important for these changes. Acknowledgements—This work was funded in part by a FCAR fellowship to F.M.; MRC of Canada MT-14,142 grant to V.F.C.; CIHR grant MT 12,046 to W.S.S.; W.S.S. is a CIHR investigator and a Killiam Scholar.

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Fig. 3. PDBu does not translocate ERK to the nucleus. (A) Confocal images of cultured sensory neurons incubated for 1 h with DMSO (control) or 1 ␮M PDBU (PDBu). Cells were stained either with chicken anti-PKC (PKC) or a rabbit anti-Aplysia MAP kinase (Martin et al., 1997). (B) For each experiment five cells were imaged, and the membrane/cytoplasm or nuclear/cytoplasm (see Methods) ratio for both MAP kinase staining and PKC staining for each cell was averaged. A percentage change in the average ratios between control and PDBu treated cells was then calculated for each experiment. Results are given with the mean and S.E.M. from four experiments.

to recognize RISK-1 and we are doing experiments in a time frame before injury activated RISK-1 would be translocated to the cell body. Furthermore, it has been reported that 5-HT does not activate RISK-1 (Sung et al., 2001). In Aplysia sensory neurons, phorbol esters activate ERK (Fig. 1), but do not lead to long-term facilitation of sensory to motor-neuron synapses (Braha et al., 1990; Wu et al., 1995). Thus, one can presume that ERK kinase activation is insufficient to induce the transcription factors that lead to long-term facilitation. Mechanistically, this could be explained if application of PDBu activated ERK in the cytoplasm, but in the absence of cAMP signaling, ERK would not be translocated to the nucleus and would not activate transcription factors. In support of this, ERK translocation and activation have been shown to be indepen-

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(Accepted 1 August 2002)