Roles of PKA and PKC in facilitation of evoked and spontaneous transmitter release at depressed and nondepressed synapses in aplysia sensory neurons

Neuron,

Vol. 9, 479-489,

September,

1992, Copyright

0 1992 by Cell Press

Roles of PKA and PKC in Facilitation of Evoked and Spontaneous Transmitter Release at Depressed and Nondepresd Synapses in Aplysia Sensory Neurons Mirella Chirardi,* Orit Braha,+ Binyamin Hochner,+ Pier Ciorgio Montarolo,* Eric R. Kandel,+ and Nicholas Dale+ * Dipartimento di Anatomia e Fisiologia Umana C. so Raffaello 30 10125 Torino Italy +Howard Hughes Medical Institute and Center for Neurobiology and Behavior College of Physicians and Surgeons Columbia University New York, New York 10032

Summary Two second messenger pathways, one that uses the CAMP-dependent protein kinase A (PKA), the other that uses protein kinase C (PKC), have been found to contribute to the short-term presynaptic facilitation of the connections between the sensory neurons in Aplysia and their target cells, the interneurons and motor neurons of the gill-withdrawal reflex. To study their relative contributions as a function of the previous history of the neuron’s activity, we have examined the effects of inhibiting PKA (using Rp-CAMPS) and PKC (using H7) on the shortterm facilitation of spontaneous release as well as of the evoked release induced by serotonin at nondepressed, partially depressed, and highly depressed synapses. Our results suggest that whereas activation of PKA is sufficient to trigger the facilitation of nondepressed synapses, activation of both PKA and PKC is required to facilitate depressed synapses, with the contribution of PKC becoming progressively more important as synaptic transmission becomes more depressed. Introduction Sensitization is an elementary nonassociative behavioral modification in which an animal learns to enhance its defensive reflexes after receiving a noxious stimulus. Thus, in Aplysia, a strong or noxious sensitizingstimulustothetailcausestheanimaltoenhance its gill-withdrawal reflex to tactile stimulation of the siphon. Strong tail stimuli will also enhance a gillwithdrawal reflex that has been previously habituated, giving rise to the related behavioral modification called dishabituation. Since both sensitization and dishabituation involve an enhancement of the gillwithdrawal reflex following a noxious stimulus, the two behavioral modifications share a number of behavioral features (Carew et al., 1971; Cohen et al., 1991, Sot. Neurosci., abstract, and personal communication). Nevertheless, they differ somewhat in their cellular mechanisms (Hochner et al., 1986) and can be distinguished behaviorally in the siphon component

of the reflex (Marcus et al., 1988). Thus, the same stimuli to the tail of Aplysia that lead to the release of serotonin (5-hydroxytryptamine; 5-HT) can bring about dishabituation and sensitization. This modulatory transmitter contributes to both dishabituation and sensitization by acting at a number of sites within the neural circuit of the gill-withdrawal reflex, including the monosynaptic connections between siphon sensory and gill motor neurons, where 5-HT produces facilitation of transmitter release (Glanzman et al., 1989). Within the sensory neurons, however, 5-HTtriggers two distinct cellular mechanisms that contribute differentially to the facilitation of transmitter release depending upon whether the synapse has or has not been previously depressed. In synapses that have not undergone prior depression, the facilitation is associated with, and appears to depend importantlyon, closure of two types of K+ channels (1~s and I&, which cause spike broadening, enhanced entry of Ca2+ during the action potential, and thus an increase in transmitter release (Klein et al., 1980; Klein and Kandel, 1980; Siegelbaum et al., 1982; Baxter and Byrne, 1989, 1990; Eliot et al., unpublished data; Hochner and Kandel, unpublished data). This facilitatory mechanism, referred to as the first process (Hochner et al., 1986), parallels behavioral sensitization. Several lines of evidence based on the injection of CAMP, the catalytic subunit of CAMP-dependent protein kinase(PKA), and specific inhibitors of PKA suggest that PKA may play an important role in the modulation of both classes of K+ channels and hence in controlling the first process in response to tail stimuli or brief pulses of 5-HT (Brunelli et al., 1976; Castellucci et al., 1980, 1982; Siegelbaum et al., 1982; Schuster et al., 1985; Hochner and Kandel, unpublished data; B. A. Goldsmith and T. W. Abrams, personal communication). After depression of the synapses between the sensoryand motor neurons, as occurs with habituation, a second mechanism becomes evident. As the synapse becomes progressively more depressed, spike broadening alone becomes gradually less effective at enhancing transmitter release. However, 5-HT still causes presynaptic facilitation and now does so by a mechanism independent of the modulation of either Ca2+ influx or other ionic currents (Hochner et al., 1986; Gingrich and Byrne, 1985). This reversal of synaptic depression parallels behavioral dishabituation and is referred to as the second process (Hochner et al., 1986). Both the first and the second processes presumably represent not a single mechanism, but an integrated set of mechanisms. Dale and Kandel(1990) delineated one direct physiological correlate of the second process, a 5-HT-induced enhancement of spontaneous transmitter release. Their studies suggested that the second process involves in part a direct modulation of spontaneous transmitter release

that is independent of changes in the resting levels of Ca2+ or modulation of Ca2+ influxes. While CAMP appears to function as an important second messenger for the first process, the intracellular messengers important for the second process are still unclear. Braha et al. (1990) provided the first evidence on the second process when they found that phorbol esters simulate this process and H7 (l-(5 isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) blocks a component of it. These studies suggested that the second process requires the actions of protein kinase C (PKC). This finding is consistent with those of Sacktor and Schwartz (1990) and Sossin and Schwartz (1992), that 5-HT leads to the translocation of one of the four isoforms of PKC existing in the Aplysia sensory neuron. What remains unclear, however, is the degree to which the two second messenger systems contribute to each of these processes. Specifically, to what degree does PKC contribute to the first process and PKA to the second? To determine these relative contributions, the effect of inhibiting the two second messenger systems on the facilitation of synaptic transmission produced at various levels of depression was examined. As a first step in this direction, Goldsmith and Abrams (1991) have recently examined partially depressed synapses and found that inhibitors of adenylyl cyclase block the ability of 5-HT to produce facilitation, suggesting that the CAMP cascade contributes importantly to the second process, perhaps more importantly than PKC. To evaluate more precisely the relative contributions of PKA and PKC to the two processes of facilitation as a function of the neuron’s previous activity and the consequent level of synaptic transmission, we examined isolated sensory and motor neurons in dissociated cell culture and studied the effects of 5-HT on synapses that were not depressed, on synapses that were moderately depressed, by 65%, so that they were only 35% of their initial value, and on synapses that were severely depressed, by 90% or more, so that they were only 10% of their initial value. To evaluate the contribution of the two kinases to facilitation under each of these three conditions, we used the Rp diastereoisomer of thecyclicadenosine3’,5’-monophosphorothioate(RpCAMPS) and H7. Rp-CAMPS is a specific blocker of PKA that binds competitively to the CAMP-binding site of the regulatory subunit of the CAMP-dependent protein kinase and prevents it from binding CAMP. H7 is ageneral kinase inhibitor that has been found to show a preference for blocking PKC in intact Aplysia neurons in a variety of different biochemical and pharmacological studies (Conn et al., 1989; Taussig et al., 1989; Braha et al., 1990; Braha et al., unpublished data). As an independent check on the second process, we studied not onlyevokedrelease, but also spontaneous release, so as to be able to analyze the second messengers that are involved in triggering one known component of the second process.

Our results suggest that the participation of two second messenger pathways is determined by the neuron’s previous activity. Facilitation of the nondepressed synapse seems to require only PKA. However, the facilitation of depressed synaptic connections requires the additional activation of PKC. Moreover, the relative contribution to facilitation of PKC is not fixed but the magnitude of its contribution varies as a function of the history of the neuron’s previous activity, so that PKC becomes progressively more important as synaptic transmission becomes progressively more depressed by homosynaptic activity. Results An Inhibitor of PKA Selectively Blocks Facilitation of Release of Nondepressed Synaptic Connections Application of 5-HT to sensory-motor synapses evokes short-term facilitation regardless of whether or not the synapse is first depressed by homosynaptic stimulation. Previous studies have shown that PKA contributes importantly to the enhancement of transmitter release at nondepressed synapses. To examine quantitatively the contribution of PKA to the facilitation of nondepressed synapses, we used Rp-CAMPS, a cell-permeant competitive antagonist of CAMP that has been shown to inhibit PKA activity in different cellular systems, including cultured sensory neurons of Aplysia (Rothermel et al., 1984; Erneux et al., 1986; Pereira et al., 1987; Backsai, Hochner, Kaang, Kandel and Tsien, unpublished data). To confirm the specificity of Rp-CAMPS, we studied its effect on two actions of 5-HT in cultured sensory neurons mediated by PKA: the increase in excitability and spike broadening. RpCAMPS almost completely blocked both the excitability increase and the spike broadening (Figure I). As we shall show below and elsewhere (Braha et al., unpublished data), this inhibitor does not seem to affect cellular processes that are dependent on PKC. To examine the effects of Rp-CAMPS on facilitation of nondepressed synapses, we first documented the effect of 5-HT alone. A single test stimulus to the sensory cell was given, followed by application of 10 PM 5-HT, and afurthertest stimulus wasgiven 5 min later. The 5-HT caused an increase in the amplitude of the synaptic potential of 54.5% (i: 15.7% SEM, n = 5; Figure 2) measured 5 min later. In control synapses, in which the sensory cell was given two stimuli separated by 5 min without the addition of 5-HT, there was only a slight decrease in synaptic strength (-17.9% f 3.0% SEM, n = 7). This reflects a homosynaptic depression, a depression of transmission that occurs at these synapses even when they are stimulated as infrequently as once every 5 min. If the homosynaptic depression is taken into account, 5-HT leads to a total facilitation of 88%. We next examined the effect of Rp-CAMPS on the facilitation induced by 5-HT in two ways. First, we applied Rp-CAMPS (500 PM) for 30 min prior to applying 5-HT for 1 min. This was then followed by washout of

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(Al) A 500 ms depolarizing pulse was delivered to the sensory neurons in culture to monitor excitability. The membrane potential of the sensory neuron was kept at -45 mV. The anti-accommodation effect of 5-HT is demonstrated by the ability of the 500 ms current pulse to generate 15 spikes during the entire pulse duration, while in control the same pulse generated only 3 spikes at the beginning of the pulse. In the presence of Rp-CAMPS, the addition of 5-HT fails to increase the cell excitability. (A2) Bar graph summarizing the percent increase in the number of spikes after 5-HT application to the control sensory neurons and in the presence of Rp-CAMPS. (Bl) Superimposition of the action potential recorded in sensory neurons before and after EHT application. The action potential was generated by a current pulse of 5 ms duration. The membrane potential was kept at -45 mV. The spike durations were determined by measuring the time from the peak of the spike to repolarization of the spike to 25% of its amplitude. S-HT produces a noticeable spike broadening. The presence of RpcAMPS blocks the 5-HT-induced spike broadening in the sensory neurons. (82) Bar graph summarizing the percent increase in spike duration after 5-HT application in control sensory neurons and in the presence of Rp-CAMPS.

the 5-HT so that the synapse was tested 5 min later in the absence of 5-HT. This test revealed a complete block of the facilitation induced by 5-HT. Rp-CAMPS reduced the excitatory postsynaptic potential (EPSP) to a level below the initial baseline, consistent with the homosynaptic depression that occurs in the absence of 5-HT (-8.9% f 6.3% SEM, n = IO). Second, we applied Rp-CAMPS (500 PM) for 30 min prior to applying 5-HT. But now we did not wash out the 5-HT, so that the EPSP was retested in the presence of 5-HT. Even with 5-HT continually present during the test, Rp-CAMPS still completely blocked the 5-HT-evoked facilitation (-5% + 4.6% SEM, n = 4; data not shown).

Moreover, Rp-CAMPS applied alone did not significantly affect the normal synaptic depression that occurswhen the synaptic potential is retested 5 min after the first stimulus (-22.8% of: 3.72% SEM, n = 5). These results are summarized in Figure 2. By contrast, H7, a kinase blocker that in Aplysia neurons preferentially inhibits the responses mediated by PKC, had no effect on the nondepressed synapses (see also Braha et al., 1990). In the presence of 400 WM H7,5-HT still produced a significant facilitation of 56.4% (+ 10.7% SEM, n = 5; Figure 2). H7 applied alone caused a small decrease in EPSP amplitude in four experiments when retested 5 min after the first

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stimulus (-26.3% f 3.4% SEM). This was comparable to the homosynaptic depression seen in the control. When corrected for homosynaptic depression, the total facilitation in the presence of H7 is 112%. These data on H7 confirm previous work using a completely different protocol (Braha et al., 1990), which indicated that H7 had no effect on facilitation of nondepressed synapses.

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(A) Examples of 5-HT-evoked short-term facilitation of nondepressed synapses in the presence of PKC inhibitor, but not in the presence of PKA inhibitor. The figure shows the EPSP before and 5 min after 5-HT application (the arrow indicates 5HT application) recorded in a control experiment (no 5-HTwas added and the resting potential of the motor neuron was -46 mV), in an experiment with 5-HT without inhibitors (resting potential -47 mV), in experiments with 5-HT in the presence of RpcAMPS (resting potential -50 mV), and in experiments with 5-HT in the presence of H7 (resting potential -48 mV). (B) Summary of the short-term facilitation of nondepressed synapses. The height of each bar is the mean percent change + SEM in the amplitude of EPSP retested 5 min after the first stimulus. IHT (IO NM) was applied immediately after the first stimulus for 1 min. In thecontrol, in the Rp-CAMPS alone, and in the H7alone experiments, no 5-HT was added. A one-way analysis of variance indicates a difference with treatment, F = 30.4, df 5,30, p < 0.001. A comparison of the means (Newman Keuls’ multiple range test) shows that treatments with 5-HT and 5-HT + H7 significantly increase the EPSP amplitude relative to control (p < O.OOl), while treatments with 5-HT + Rp-CAMPS, RpcAMPS alone, and H7 alone are not significantly different from control.

When synaptic connections were moderately depressed to 35% of their initial level with low frequency stimulation of the sensory cell, 5-HT nevertheless produced significant facilitation. In fact, the peak facilitation achieved in relation to the initial level, prior to depression, was comparable to that of nondepressed synapses (43% above control). Because the amplitude of thedepressed synaptic potential was much smaller, the relative facilitation compared with the depressed level was substantially greater (about 300% measured as the peak facilitation reached after 5-HTapplication). To compare the experiments of depressed synapses statistically, we evaluated the facilitation by comparing the percent change in the mean amplitude of the first two EPSPs evoked after the application of 5-HT (stimuli 6-7; see Figure 3) with that of the two EPSPs evoked immediately before application of 5-HT (the responses to stimuli 4-5). Thus, application of 5-HT afterthefifthstimulusevokedafacilitationoftheEPSP of 286% (k 45.6% SEM, n = 7; Figure 3). In the absence of 5-HT, the control showed only further depression (-8.6% & 11.6% SEM, n = 7; Figure3). Incubation with Rp-CAMPS (500 PM for 30 min) greatly reduced the effects of 5-HT. The blockade was particularly effectiveon the initial responses after 5-HT (response 6 and to a lesser degree 7), suggesting that PKA is particularly important in the early phase of facilitation in depressed sequences. In the presence of Rp-CAMPS, 5-HT increased responses 6 and 7 only by 52.3% (+ 25.0% SEM, n = 7) instead of 286%. By contrast, 5-HT produced a significantly greater increase in the synaptic responses to stimuli 7 and 8 of 97% (+ 29% SEM, n = 7, p < 0.02 compared with Rp-CAMPS alone). Thus, the later components of facilitation appeared more resistant to Rp-CAMPS. Conversely, when synaptic connections had been depressed to 35% of their initial level, application of 400 PM H7, which had no effect when the synapses were not depressed, reduced the facilitation evoked by 5-HT from 286% to 88.0% (* 14.1% SEM, n = 7). Since each inhibitor produced a partial effect, we also examined the effects of using both inhibitors. When both inhibitors were present, the facilitation produced by 5-HTwas blocked completely (-2.1% k 13.7%

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SEM, n = 8; Figure 3). Conversely, when the partial effects attributable to PKA and PKC on the basis of these blocker experiments are combined, the magnitude and the time course of dishabituation closely match the effect seen with 5-HT alone. When applied in the absence of 5-HT, neither H7 nor Rp-CAMPS significantly affected the time course and the degree of depression of the EPSP produced by repeated stimulation of the sensory cell (-9.8% +_ 5.0% SEM, n = 4, for the Rp-CAMPS, and -7.4% & 3.8% SEM, n = 5, for H7; Figure 3). Thus, unlike the case of the nondepressed synaptic connection, facilitation of the mod’erately depressed synapses seems to require that PKA and PKC act together in an additive fashion, with PKA contributing slightly more than PKC especially in the early responses following the onset of facilitation.

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Blockade of PKC Has Its Greatest Effect on Facilitation of Release at Synapses That Are Highly Depressed Since H7 is more effective in blocking release at depressed as compared with nondepressed synapses, we next examined synaptic connections that had been severely depressed to about 10% of control levels. This degree of depression is difficult to establish in culture and requires 40 consecutive stimuli to the sensory neuron compared with the 5 used in the previous experiments. With this degree of depression, 10 PM 5-HT evoked a relative facilitation of 629.2% (k 56.7% SEM, n = 4; Figure4). Here, indeed, H7 had itsgreatest inhibitory effect. In the presence of H7,5-HT evoked a facilitation only of 71.7% (f. 28.6% SEM, n = 6). This increase was not significant compared with the control and was very different from the facilitation pro-

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(A) Examples of the facilitation evoked in depressed synapses by 5-HT in the presence and in the absence of PKA and PKC inhibitors. The figure shows two EPSPs before EHT application (IV-V) and the two first EPSPs after 5-HT application (VI-VII). The arrow indicates 5-HT application. The resting potential of the motor neuron was -48 mV, -45 mV, -50 mV, -46 mV, and -47 mV in the control, 5HT, 5-HT + RpcAMPS, 5-HT + H7, and 5-HT + Rp-CAMPS + H7 examples, respectively. (6) Each bar represents the percent change of the mean amplitude of the EPSPs evoked by the VI and VII stimuli compared with the mean amplitude of the IV and V EPSPs (* SEM). 5-HT (10 PM) was applied immediately after the V stimulus. A one-way analysis of variance indicates a difference with treatment, F = 21.3, df 6,37, p < 0.001. A comparison of the means indicates that the 5-HT treatment significantly increases the facilitation relative to control (p< 0.001); in the presence of RpcAMPS, 5-HT induces an EPSP increase significantly smaller than the facilitation obtained with 5HT alone (p < 0.001) and not significantly different from thecontrol. In the presenceof H7, the5-HT-induced facilitation is significantly different compared with the facilitation observed with 5-HT without H7 (p < 0.001) and compared with the experiments in the presence of H7 without 5HT (p < 0.05). (C) Summary of the short-term facilitation of the depressed synapses in the presence and absence of PKA and PKC inhibitors. (SEM ranged between 2.28 and 11.98.)

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duced by 5-HT alone (p < 0.001). By contrast, RpCAMPS allowed the facilitation induced by 5-HT to reach the initial (predepressed) level (Figure 4). Thus, in the presence of Rp-CAMPS, the EPSP was increased 526% (+ 47.3% SEM n = 6), and this increase is not significantly different from that of 5-HT alone. H7 Reverses the increase in Spontaneous Release Produced by Serotonin If PKC is primarily effective on depressed synapses, one might expect that its effect would be directed to the mechanisms that contribute to the second process. Dale and Kandel(l990) found that 5-HT produces a 5-to8-fold increase in thefrequencyof spontaneously occurring miniature EPSPs (mEPSPs). This increase seemed to reflect a component of the second process, since it did not require Ca2+ influx. To determine the relative contribution of PKA and PKC to the modulation of spontaneous release by 5-HT, we first preincubated a culture with Rp-CAMPS for 30 min and then applied 10 t.rM 5-HT. In all five cultures tested, RpCAMPS had no effect. Application of 5-HT still caused a large enhancement of spontaneous release (Figure 5A) comparable to that seen in the absence of the blocker. The mean time interval between consecutive mEPSPs decreased 5-fold to 19.6% of the control (f 5.7% SEM, n = 5, p < 0.02). We therefore conclude that the enhancement of spontaneous release is triggered by a second messenger pathway that is independent of PKA. To determine whether PKC contributed to this enhancement, we next examined the effects of H7 on

enhancement of spontaneous release by 5-HT (Figure 5B). We found that 10 PM 5-HT reduced the mean time interval between mEPSPs to 18.5% of the control (k 3.5% SEM; p < O.OOl), while 200-400 PM H7applied to the same cells during the application of 5-HT producedasignificant blockade(Figure5B)and increased the time interval between mEPSPs back to 54.9% of control (*10.8% SEM, p < 0.02 compared with 5-HT, and p < 0.01 compared with control). Phorbol Dibutyrate Increases the Frequency of Spontaneous Transmitter Release in a Dose-Dependent Manner To explore further the actions of PKC on spontaneous release, we examined the effects of the phorbol ester phorbol dibutyrate (PDBu). In normal sea water, application of PDBu caused an increase in spontaneous release. To determine whether this increase was independent of Ca2+ influx, as is the effect of 5-HT (Dale and Kandel, 1990), we tested the effect of PDBu on the spontaneous release in CaL+-free sea water that included the Ca 2+ buffer BAPTA (1,2-bis(o-aminophenoxy) ethane-N,N,-N’,N’-tetraacetic acid) at 1 mM. Under these conditions, the mean time interval between consecutive mEPSPs was 3.0 s (k 0.7 s SEM, n = 5). During the application of 10 nM PDBu to the same five synapses, the mean time interval between mEPSPs was reduced to 0.64 s (k 0.3 s SEM, p < 0.02, compared with control), equivalent to20% of control. Addition of 5-HT in the presence of PDBu did not cause a further increase in the mEPSP frequency (Figure 6Al). Application of 100 nM a-PDBu, an inactive phorbol ester that does not activate PKC, did not significantly affect the occurrence of the mEPSPs (data not shown); it increased the time interval between consecutive mEPSPs by 30% from control (+ 27.4% SEM, n = 3, p > 0.1). We next examined the dose-response relation of PDBu. Applications of PDBu (I-50 nM) increased the occurrence of mEPSPs in a dose-dependent manner, with the optimal dose of 10 nM causing a 5-fold increase (Figure 6A2). The dose dependence of the increase in mEPSPs in response to PDBu was very similar to that of the evoked EPSP (see Figure 28 in Braha et al., 1990). PDBu Does Not Change the Mean Amplitude of the mEPSPs A histogram of the amplitudes of the mEPSPs before and after application of IO nM PDBu in saline containing zero Ca2+ revealed that the mEPSP amplitudes were distributed in a multimodal manner (Figure 68). The first peak corresponds to the mean amplitude of the unit potential, while the other two peaks are integral multiples of the first amplitude peak and probably represent coincidence of spontaneous release. Application of PDBu did not change the size of the mean amplitude of the unit potential. In an average of seven different synapses, IO nM PDBu caused a small, but statistically not significant, decrease in the

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(Al) The PKA inhibitor has no effect on spontaneous release. Five consecutive 1 s long traces. After a 30 min incubation of the sensory motor fL7) culture with 500 uM Rp-CAMPS, application of 10 KM 5-HT significantly increased spontaneous release. After washout of Rp-CAMPS, 5-HT application caused an enhancement of spontaneous release comparable to that seen in the presence of the PKA inhibitor. (AZ) Bar graph summarizing the mean time interval between mEPSPs (T) from five experiments in which EHT was applied in the presence of Rp-CAMPS. The effect of the drugs in each experiment was normalized to the control T. Error bar represents SEM. (Bl) H7 reverses the increase in spontaneous release produced by 5-HT. Four consecutive 1 s long traces measured from LFS motor neurons, cultured with a sensory neuron before application of 5-HT (Control). Application of IO BM 5-HT increased spontaneous release, and 400 uM H7 in the presence of 5-HT reversibly decreased the spontaneous release. (62) Bar graph summarizing the mean time interval between mEPSPs (T) from eight experiments in which 10 PM 5-HT was applied and then 200-400 BM H7 was applied in the presence of 10 frM 5-HT. The effect of the drugs in each experiment was normalized to the control T. Error bars represent SEM.

mean amplitude of the mEPSP (0.96-fold f 0.04 SEM, p > 0.2).

of control,

H7 Also Reverses the Increase in Spontaneous Release by PDBu Figure6C2 shows an example from a synapse in which PDBu caused an increase in the occurrence of the mEPSPs, and application of 200 PM H7 reversed this increase. A summary of five such experiments is shown in the histogram (Figure 6C2), in which the increase in occurrence is measured as a decrease in the mean time interval between consecutive mEPSPs. In five experiments, IO nM PDBu decreased the time interval between mEPSPs from 3 s (+ 0.7 s SEM) to 0.64 s (+ 0.3 s SEM), equivalent to 18.8% of control (+ 5.4% SEM, p < 0.02). Addition of 200 PM H7 on the same cells increased the time interval back to 1.8 s

(k 0.5 SEM), equivalent to 55.4% of control (* 8.5 SEM, p < 0.05 compared with PDBu and p < 0.02 compared with control). When applied alone (Figure 5B2), 200 PM H7 caused a small increase in the incidence of spontaneous release, reducing the mean time interval between mEPSPs to 92% of control (& 15.1% SEM, n = 6, p > 0.2). The Peptide SCP, Which Fails to Activate the Second Process, Also Does Not Cause an Increase in Spontaneous Release The peptide SCP can evoke facilitation of sensorymotor synapses in Aplysia by elevating CAMP and thus activating PKA (Abrams et al., 1984). However, Schacher et al. (1990) demonstrated that SCP appeared to facilitate only nondepressed synapses and was apparently incapable of triggering the second

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PDBu+ H7

Release

(Al) PDBu increases mEPSP frequency in Aplysia saline containing no added Cal’. Five consecutive 1 s long traces before application of PDBu (Control); a few mEPSPs can be clearly detected. Application of 10 nM PDBu caused an increase in the spontaneous release. Application of IO RM 5-HT in the presence of PDBu did not cause a further increase of the spontaneous release. (AZ) The increase in spontaneous release by PDBu is dose dependent, with an optimal dose of 10 PM. The frequency of the spontaneous release was measured as the mean time interval between consecutive mEPSPs 0. Thus, an increase in the mEPSP frequency will correspond to a decrease in T. Each point is an average of five experiments (in saline containing no added Ca”). Error bars represent SEM. (B) PDBu does not change the mean amplitude of the mEPSP. The amplitudes of mEPSPs observed in saline containing no added Cali were measured from an LFS motor neuron cultured with a sensory neuron. Amplitude histograms are shown after application of 10 nM PDBu (Bl) and in the control condition (82). The continuous line is the best fitted Gaussian distribution. In both cases three Caussians were fitted. The mean amplitudes of the smaller peaks (larger amplitude of mEPSPs) are close to integral multiples of the mean amplitude of the first peak and were interpreted as synchronous spontaneous release. In control, the mEPSP amplitude was measured over a period of 60 s. In the presence of PDBu, the frequency of mEPSPs increased and the mEPSP amplitudes were measured during a 15 s period, which gave mEPSPs comparable in number to those measured in control. (C) H7 reverses the increase in the spontaneous release produced by PDBu. (Cl) Five consecutive traces from one cell before and after application of 10 nM PDBu and after application of 200 PM H7 in the presence of PDBu (all in zero Ca2+ saline). PDBu increased the frequency of mEPSPs, and application of H7 in the presence of PDBu reduced it. (C2) Average of five experiments. The frequency of the spontaneous release was measured as the mean time interval between consecutive mEPSPs (T) and was normalized to the T in control for each experiment. Error bars represent SEM.

Second 487

Messengers

in Presynaptic

Facilitation

process to enhance transmission at depressed synapses. To explore this finding further, we studied the effects of SCP on a physiological correlate of the second process: modulation of spontaneous release. In artificial seawater containing 10 mM Ca2+, application of 10 uM SCP resulted in a modest increase in spontaneous release (the mean time interval between mEPSPs decreased to 56.8% of control, f 6.0% SEM, n = 4). This increase was almost certainly due to the depolarization of the sensory neurons caused by SCP. When SCP was applied in artificial seawater containing no added Ca2+ and 1 mM BAPTA, a solution in which 5-HT still causes a 5- to 8-fold increase in frequency, the mean time interval between mEPSPs increased slightly to 116% of control (k 10.4% SEM, n = 4). We therefore conclude that SCP does not increase the rates of spontaneous release by a mechanism independent of a Ca2+ influx and thus cannot activate the second process. Discussion Specificity of the Antagonists of PKA and PKC Much of our argument as to which second messengers control the two processes of facilitation rests on the specificity of the blockers of PKA and PKC that we have used. There is good evidence from a variety of other systems that Rp-CAMPS is specific to PKA. Our data on Aplysia sensory neurons in culture are consistent with this conclusion (see also Braha et al., unpublished data; Tsien et al., personal communication). Thus, we have found that Rp-CAMPS seems to affect only processes known to be mediated by CAMP by other, independent, criteria. Rp-CAMPS blocks antiaccommodation and spike broadening of sensory neurons by 5-HT in culture, but does not affect processes attributable to PKC, such as the increase in spontaneous release and the modulation of the L-type CaL+ current (Braha et al., unpublished data). On the other hand, H7 is known from other systems to be a much less specific kinase inhibitor than Rp-CAMPS. Nevertheless, in Aplysia, at the doses we have used in sensory neurons, Braha et al. (unpublished data) have found that H7 does not antagonize the modulation of the S currents by 5-HT or the effects of CAMP injection on accommodation and spike broadening. However, H7 significantly reduces the facilitation of the EPSP evoked by phorbol esters (Brahaet al., 1990). Similarly, as we here show, H7 reducesthe increase in spontaneous release evoked both by phorbol esters and by 5-HT. In Aplysia H7 therefore appears not to block the actions of PKA and seems to possess selectivity for PKC, as first suggested by the studies of Conn et al. (1989). Although we cannot exclude the possibility that H7 could be blocking kinases other than PKC, the use of these two compounds has allowed us to dissociate further the role of relative contributions of second messengers on the first and second processes of facilitation.

Second Messengers for Facilitation We have systematically investigated the effects of antagonists of PKA and PKC on spontaneous release and on the facilitation of evoked release at both nondepressed and depressed synapses. We find that the contributions of the two second messenger pathways are differentially recruited as a function of the neuron’s recent history of activity. Inhibitors of PKA fully prevent facilitation of nondepressed synapses and, block a substantial part of the facilitation of moderately depressed synapses, but they block only a small component of the facilitation of severely depressed synapses. Moreover, Rp-CAMPS has no effect on the modulation of spontaneous release by 5-HT. Conversely, agents that elevate CAMP can facilitate both nondepressed synapses and partiallydepressed synapses, but cannot increase spontaneous release. By contrast, H7, which we believe blocks PKC in the sensory neurons, has no effect on facilitation of nondepressed synaptic potentials and only partially blocks (less than 50%) synapses that are moderately depressed. However, H7 blocks a large part of the facilitation of highly depressed synapses and blocks almost completely the enhancement of spontaneous release by 5-HT. Consistent with a role for kinases in facilitation of depressed synapses, combined application of inhibitors for both PKA and PKC blocked completely the facilitation of depressed synapses. Thus, we conclude with Braha et al. (1990) that two separate second messenger pathways are required to evoke the two processes that underlie synaptic facilitation. Activation of PKA is required to activate the first process, a process that is associated with spike broadening due to a reduction in K’ currents (IK5 and I& and enhanced transmitter release at nondepressed synapses. Activation of PKC appears to be necessary to trigger the second process that enhances transmitter release at depressed synapses in a way that does not require spike broadening and CaL+ influx. A key question is whether the two kinase pathways act independently (in parallel) or in series. If the two kinases acted in series, antagonists of the particular kinase that initiates the cascade should block the second process as well as the first. To the contrary, we have found that the first process and the second processcould be blocked independently(with Rp-CAMPS and H7, respectively). Our results therefore suggest that the two second messengers seem to act in parallel to trigger the two mechanisms of facilitation. Finally, our results with inhibitors as well as the biochemical results of Sossin and Schwartz (1992) explain the actions of phorbol esters that previously seemed paradoxical. Phorbol esters are powerful stimulators of transmitter release and enhance release from the sensory neurons as well as from many other neurons in Aplysia that we have examined. In the sensory neurons, phorbol esters enhance transmission of nondepressed as well as depressed synapses, yet all of our

data and those of Braha et al. (1990) using the blockers of PKA and PKC are internally consistent in showing that PKC seems not to have a significant role in enhancing release from nondepressed synapses. This might now be explained by the finding of Sossin and Schwartz (1992), that while phorbol esters translocate all isoforms of PKC, both 5-HT and natural tail stimuli translocate only one of these isoforms. A corollary to this finding is that phorbol esters may not be ideal agents for studying the actions of transmitters that work through individual isoforms of PKC. Ultimately, the several interacting processes that contribute to facilitation will have to be dissected precisely, by using genetic techniques such as dominant-negative mutations or antisense oligonucleotides to block specific PKC isoforms, as well as specific 5-HT receptors, second messenger kinases, and K’ channels. Experimental

Procedures

Cell Culture and Electrophysiology Sensory cells (1 or 2) isolated from the pleural ganglia of adult animals (80-100 g) were cocultured with a single motor neuron, L7, from the abdominal ganglia of juvenile animals (l-4 g), as described by Schacher and Proshansky (1983). After 4-5 days in culture, the strength of the synaptic connection between the sensory and motor cells was tested. The motor cell was recorded intracellularlywith a microelectrode filled with 2.5 M KCI (10 Mbl), and the membrane potential was hyperpolarized 30 mV below its resting potential. The EPSP was evoked in L7 by stimulating the sensory cell with an extracellular electrode (Montarolo et al., 1988). Results were stored on a four channel tape recorder (Racal). Short-Term Facilitation of Nondepressed Synapses Toassessfacilitationof nondepressedsynapses,asingledepolarizing stimulus was applied to the sensory neuron and the initial EPSP amplitude was recorded. Immediately, 5-HT (creatine sulfate salt; Sigma), was applied to the bath to a final concentration of 10 PM for 1 min, followed by washout of the bath solution (1 mllmin). The EPSP amplitude was remeasured 5 min after the treatment. In four experiments, the bath solution was not washed out and the EPSP was retested in the presence of 5-HT. Short-Term Facilitation of Depressed Synapses To measure short-term facilitation of depressed synapses, the sensory cell was stimulated at 20 s intervals. After the fifth stimulus, when the EPSP was depressed by 30%-40% of its initial level, IO PM 5-HT was added to the bath. In the experiments on highly depressed synapses, the sensory cell was stimulated 40 times, achieving a depression to IO%-20% of control level, before adding 5-HT. The sensory cell was given up to 4 additional stimuli to determine the extent of facilitation. PKA and PKC Inhibitors To block the PKA activity, we used the Rp-CAMPS (Biolog). RpCAMPS is a CAMP competitive antagonist and prevents dissociation of the kinase holoenzyme into catalytic and regulatory subunits, resulting in a complete loss of phosphorylating activity (Rothermel and Botelho, 1988). It was dissolved in distilled water at 10 mM, and aliquots were lyophilized and frozen. In pilot experiments wefirstdetermined the concentration of Rp-CAMPS and the timeof incubation with Rp-CAMPS that gave the maximal level of inhibition of the facilitation of nondepressed synapses. In all reported experiments, Rp-CAMPS was applied at a final concentration of 500 1M 30 min before recording the initial EPSP. To block the PKC-mediated effects, we used H7 (Seikagaku America), a kinase blockerthat has been reported to inhibit preferentially PKC in intact Aplysia neurons (Corm et al., 1989; Taus-

sig et al., 1989). H7 was dissolved in water at IO mM, and aliquots were lyophilized and stored at 4OC. It was applied directly in the bath at a final concentration of 200-400 PM immediately before the 5-HT application. Analysis of Data of Evoked Release The effects of various treatments were calculated as the percent change of EPSP amplitude after treatment from the initial EPSP amplitude before treatment. All the data are presented as mean and SEM. A one-way analysis of variance and Newman Keuls’s multiple range test were used in Figures 2,3, and 4 to measure significance of the EPSP changes produced by the various treatments. mEPSP Recording Spontaneous mEPSPs can be clearly observed when recording intracellularly from the follower cell. High resistance intracellular electrodes containing 3 M KCI were beveled to give a resistance of 15-20 Mbl. In most of the experiments we used the LFS as follower cells; their high input resistance (compared with L7) caused the mEPSPs to be larger in amplitude (mean amplitude 180-600 PV for LFS; 60-120 PV for L7; Dale et al., 1988). Some of the LFS cells showed large voltage fluctuations, which made the reading of the mEPSPs harder; thosewere discarded. The voltage of the motor neuron was recorded at both high and low gain. The low gain record was filtered at 3 kHz (-3 dB) and was DC coupled; the high gain trace was filtered at 300 Hz (-3 dB) and was AC coupled. In experiments involving PDBu (L. C. Services), active (f3) and inactive (a) isomers of PDBu were dissolved in 100% dimethyl sulfoxide at 0.05 M and kept at 2O“C. Before an experiment, they were diluted in recording medium and applied directly to the bath, while in experiments that included 5-HT and H7onlya microperfusion system was used. In most experiments solutions of zero CaZ+ were used, which were made with no added Cal+ and addition of 1 mM BAPTA to chelate contaminating Ca2+ in the water. The amplitude and occurrence of mEPSPs were analyzed using methods described by Dale and Kandel (1990). Statistical comparisons of the effects of drugs on mEPSP frequency were performed using the paired sample two-tailed t test. Acknowledgments We thank Drs. Thomas Abrams, John Byrne, Marc Klein, Steven Siegelbaum, and Wayne Sossin for their critical reading of the manuscript. We also thank Fang Wu for help in preparing cell cultures, Harriet Ayers and Andy Krawetz for typing the manuscript, and Sarah Mack and Charles Lam for preparing the figures. This work was supported by the Howard Hughes Medical Institute, Columbia University, and MURST Fellowships. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenf” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

March

25, 1992; revised

June

22, 1992

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