After finding a connection between a sensory and a motor neuron, testing occurred once every 5 min and consisted of eliciting a single action potential in the sensory neuron with a 30-ms, suprathreshold depolarizing pulse and recording the monosynaptic excitatory postsynaptic potential (EPSP) produced in the motor neuron (Zhang et al. 1994
). During tests, motor neurons were hyperpolarized by ~30 mV to prevent the EPSP from triggering an action potential. Measurements of input resistance of the motor neuron (while the cell was hyperpolarized) were made before each test by intracellularly injecting 1-s, 1- or 2-nA hyperpolarizing constant current pulses. After three baseline trials, ganglia were exposed to KN-62 (10 or 1 µM) or control vehicle (N-{1-[N-methyl-p-(5isoquinolinesulfonyl ) benzyl ]
2 - ( 4 - phenylpiperazine ) ethyl } - 5 isoquinolinesulfonamide) (KN-04); an inactive form of KN-62, 10 or 1 µM) 15 min before the application of 15 µM 5-HT (Fig. 1). The amplitudes of EPSPs were normalized to the baseline EPSP, which was defined as the average of three EPSPs (0, 5, and 10 min) recorded immediately before the application of KN-62 or the control vehicles.

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| FIG. 1.
Experimental protocol. Effects of {1-[N, O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine} (KN-62) were examined by using 9 test stimuli (trials) delivered at an interstimulus interval (ISI) of 5 min. Each test consisted of eliciting a single action potential in a tail sensory neuron with a suprathreshold depolarizing current pulse and recording monosynaptic exitatory postsynaptic potential (EPSP) elicited in follower tail motor neuron. Within 1 min after third stimulus (time = 10 min), experimental or control solution was applied to bath; 15 min after treatment with KN-62 or control, 15 µM serotonin (5-HT) subsequently was applied to bath. Value of each trial was normalized to mean of 3 baseline trials preceding treatment.
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The experiments using CaM inhibitors [calmidazolium, 25 µM and trifluoperazine (TFP), 50 µM] were similar to those using KN-62 except an interstimulus interval (ISI) of 1 min was used. This ISI, which leads to more pronounced synaptic depression, was selected to maximize the possibility of observing a selective effect of CaM on depressed synapses. The amplitude of EPSPs were normalized to the baseline EPSP, which was defined as the average of three baseline trials (0, 1, and 2 min).
Measurements of spike duration and excitability
Clusters of sensory neuron somata were isolated surgically from pleural ganglia and were pinned to the floor of a recording chamber containing ASW (Sugita et al. 1992
, 1994
), and the preparation was maintained at 15 ± 1°C. Two-electrode current-clamp techniques (Sugita et al. 1992
, 1994
) were used. The membrane potential of the sensory neuron was monitored and was adjusted via current injection to a potential of
45 mV ~30 s before each stimulus. Individual action potentials were elicited at an ISI of 5 min. The duration of action potentials elicited by a 3-ms, 5-nA current pulse was measured as the time between the peak of the spike and the point of the repolarizing phase at which the membrane potential was 10% of the peak amplitude of the spike (Sugita et al. 1992
). In a separate series of experiments, excitability was measured as the number of action potentials elicited during 1-s,2-nA constant-current pulses (ISI = 5 min). Measurements of spike duration and excitability were performed in different cells to avoid possible interactions between the two stimulus paradigms.
Chemicals
KN-62 was purchased from Sigma and Seikagaku America.KN-04, an inactive form of KN-62, was purchased from Seikagaku America. KN-62, KN-04, and calmidazolium (Sigma) were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the bath did not exceed 0.5% (vol/vol), a concentration that has no effects on the membrane currents in sensory neurons (Baxter and Byrne 1990b
). TFP (Sigma) and 5-HT (creatinine sulfate complex, Sigma) were dissolved in ASW.
Data analysis
To examine the effects of KN-62 on the efficacy and 5-HT-induced changes of synaptic connections, excitability, or spike duration, two t-tests were performed. First, a t-test was performed between the pre-5-HT EPSPs in the inhibitor and the control groups. The values for each group were obtained by normalizing the average value of the three trials in the pre-5-HT period to the average value of the three trials in the baseline period (Fig. 1). Second, a t-test was performed between the post-5-HT EPSPs in the inhibitor and the control groups. The values for each group were obtained by normalizing the average of the three trials in the post-5-HT period to the average value of the three trials in the pre-5-HT period (Fig. 1). A P value of <0.05 (two-tailed) was considered significant.
 |
RESULTS |
KN-62 increased the amplitude of EPSPs and attenuated 5-HT-induced facilitation
To examine the role of CaMKII in sensorimotor synaptic transmission in Aplysia, single action potentials were elicited repeatedly in sensory neurons before and after the application of 10 µM KN-62. Control experiments were identical except that KN-04 was used instead of KN-62. The role of CaMKII in facilitation was tested by comparing the effects of 5-HT in the presence of KN-62 with the effects of 5-HT in controls (Fig. 1). Figure 2A illustrates a typical result. In the control experiments, repeated stimulation (ISI = 5 min) of the sensory neuron led to synaptic depression. Application of 5-HT led to synaptic facilitation (Fig. 2A1). Figure 2A2 illustrates the effects of KN-62. Application of KN-62 by itself increased the amplitude of EPSP. In addition,KN-62 attenuated the facilitation produced by 5-HT. Average data are presented in Fig. 2A3, and further analyses of the data are illustrated in Fig. 2, B and C. The mean amplitude of the three control EPSPs during the pre-5-HT period was 80.9 ± 3.9% of the baseline (mean ± SE, n = 23; Fig. 2B). In contrast, the mean amplitude of the three KN-62 EPSPs during the pre-5-HT period was 108.8 ± 4.6% of the baseline (n = 12). The KN-62-induced increase in the amplitudes of EPSPs was statistically significant (t33 = 4.39, two tailed P < 0.001). In the post-5-HT period, the mean amplitude of the control EPSPs was 159.9 ± 12.6% of the pre-5-HT period, whereas the mean amplitude of theKN-62 EPSPs was 91.7 ± 7.0% of the pre-5-HT period (Fig. 2C). This difference was statistically significant(t33 = 3.74, two tailed P < 0.007). These results suggested that CaMKII may have a dual role in regulating transmitter release and 5-HT-induced facilitation.

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| FIG. 2.
KN-62 (10 µM) by itself increased amplitude of EPSPs and attenuated 5-HT-induced synaptic facilitation. A1 and A2: typical results illustrating action potentials elicited in sensory neurons (SN, bottoms) and EPSPs produced in motor neurons (MN, tops) during baseline (t = 0 min), 5 min after treatment (t = 15 min), 15 min after treatment (just before application of 5-HT, t = 25 min), and 5 min after 5-HT application (t = 30 min). A3: summary data from 35 experiments: 23 controls and 12 KN-62. B: summary data illustrate that KN-62 significantly enhanced amplitudes of EPSPs. C: summary data illustrate that KN-62 significantly attenuated 5-HT-induced facilitation. Summary data in this and subsequent figures show means ± SE.
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The effects of KN-62 did not appear to be due to differences in the initial state of the synaptic connections or a generalized effect on the motor neuron. The average amplitudes of the three baseline EPSPs (i.e., 0, 5, and 10 min) were 7.1 ± 1.0 mV in the control group and 7.3 ± 1.0 mV in the KN-62 group (t33 = 0.16). The average input resistance of the motor neurons in baseline period was 10.2 ± 0.7 M
in the control group and 11.3 ± 1.5 M
in theKN-62 group (t33 = 0.74).
We also examined the effects of a lower concentration of KN-62 (1 µM). The lower concentration of KN-62 produced an increase in the pre-5-HT EPSPs and attenuated 5-HT-induced facilitation. However, these effects were not statistically significant, indicating that concentrations of KN-62 >1 µM were required to significantly affect the sensorimotor synapses in Aplysia.
Because 5-HT increases the level of mRNA for CaM (Eskin et al. 1993
; Zhang et al. 1995
; Zwartjes et al. 1991
), we also examined the effects of inhibitors of CaM (25 µM calmidazolium and 50 µM TFP) on the amplitudes of EPSPs. Calmidazolium and TFP had effects on the pre-5-HT EPSPsand 5-HT-induced facilitation similar to, but weaker than, KN-62. Calmidazolium and TFP increased the amplitude of the pre-5-HT EPSP (control, 78.2 ± 9.6%, n = 6 vs.calmidazolium, 86.8 ± 4.1%, n = 7; and control, 85.2 ± 14.7%, n = 5 vs. TFP, 109.5 ± 20.4%, n = 4). These effects were not statistically significant, however. In addition, both inhibitors appeared to attenuate 5-HT-induced facilitation (control 292.5 ± 86.0% vs. calmidazolium 147.0 ± 11.6% and control 216.0 ± 65.0% vs. TFP 98.3 ± 9.4%), but again the effects were not statistically significant.
KN-62 did not affect the excitability of sensory neurons nor did it affect 5-HT-induced enhancement of excitability
The effects of KN-62 on synaptic efficacy and on 5-HT-induced facilitation may be due to an action of KN-62 on membrane currents in sensory neurons. To investigate this possibility, we examined the effects of KN-62 on excitability and spike broadening in isolated somata of sensory neurons. KN-62 (10 µM) by itself had no effect on the excitability of sensory neurons (Fig. 3, A1 and A2). Figure 3B illustrates the mean changes in excitability (control, 113.7 ± 4.9% versus KN-62, 127.5 ± 7.6%). These effects were not statistically significant (t17 = 1.55). After application of 5-HT, there was an increase in excitability in both the control and KN-62 groups. Figure 3C illustrates the mean changes in excitability produced by 5-HT (5-HT, 322.3 ± 62.5% versus 5-HT + KN-62, 248.2 ± 21.5%). There was also no statistically significant difference between these two groups (t17 = 1.07). These results indicate that the attenuation of 5-HT-induced synaptic facilitation by KN-62 (Fig. 2C) was probably not due to block of the membrane current(s) that contribute to the regulation of excitability. The 5-HT-induced enhancement of excitability in Aplysia is believed to be due to PKA-mediated closure of S-K+ channels (Baxter and Byrne 1990a
; Klein et al. 1982
; Pollock et al. 1985
; Siegelbaum et al. 1982
). Thus these results suggest that CaMKII does not affect the S-K+ channel of sensory neurons nor 5-HT-induced modulation of these channels.

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| FIG. 3.
KN-62 (10 µM) did not change excitability of sensory neuron nor did it affect 5-HT-induced enhancement of excitability. A1: typical results illustrating excitability of a sensory neuron in control saline (baseline), after application of KN-62 and after application of 5-HT to bath, which still contained KN-62. A2: summary data from 10 control [(N-{1-[N-methyl-p-(5-isoquinolinesulfonyl ) benzyl ] 2 - ( 4 - phenylpiper azine)ethyl}-5-isoquinolinesulfonamide) (KN-04)] and 9 KN-62 experiments. B: summary data for control and KN-62 groups during pre-5-HT period. C: 5-HT-induced enhancement of excitability in presence (KN-62 + 5-HT) and absence (control + 5-HT) of KN-62. There was no significant difference between KN-62 and control groups in Band C.
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KN-62 slightly increased spike duration of sensory neurons but did not block 5-HT-induced spike broadening
The 5-HT-induced spike broadening is believed to be due to the modulation of both a transient voltage-dependent K+ channel (Ik,v) and the S-K+ current (Baxter and Byrne 1989
, 1990a
; Hochner and Kandel 1992
; Klein et al. 1982
; Pollock et al. 1985
; Siegelbaum et al. 1982
; Sugita et al. 1994
). Application of KN-62 (10 µM) led to a small broadening of the action potential but did not affect 5-HT-induced spike broadening (Fig. 4A2). Average data from 10 experiments in control (KN-04) and 10 experiments in KN-62 are illustrated in Fig. 4A3. During the pre-5-HT period, the mean spike duration was102.0 ± 1.7% in the control group and 107.3 ± 1.9% in the KN-62 group (Fig. 4B). The difference was small but statistically significant (t18 = 2.10, two tailed P < 0.05). This difference was in the same direction as the effect of KN-62 on EPSPs (Fig. 2B), so the KN-62-induced increase in synaptic efficacy may be due to its effect on spike duration. In the presence of 5-HT, the mean duration of spikes in the control group was 157.8 ± 16.5% of the pre-5-HT period and the mean duration in the KN-62 group was 172.8 ± 25.1% of the pre-5-HT period(Fig. 4C). The difference was not statistically significant(t18 = 0.50). This result suggests that KN-62 had no effect on the membrane currents contributing to 5-HT-induced spike broadening.

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| FIG. 4.
KN-62 (10 µM) slightly increased spike duration but had no effect on 5-HT-induced spike broadening. A1: a control experiment. KN-04 (10 µM) did not affect either spike duration or 5-HT-induced spike broadening. A2: a KN-62 experiment. KN-62 slightly increased duration of action potential, but did not affect 5-HT-induced spike broadening. A3: summary data, from 10 control (KN-04) and 10 KN-62 experiments. B: summary data revealed a significant difference between KN-62 and control groups during pre-5-HT period. C: summary data revealed no significant difference between 5-HT-induced broadening inpresence (KN-62 + 5-HT) and absence (control +5-HT) of KN-62.
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DISCUSSION |
An increasing body of evidence indicates that CaMKII is an important kinase involved in synaptic plasticity in both invertebrate and vertebrates (see Byrne et al. 1993
; Lisman 1994
for reviews; see also Chapman et al. 1995
; Malenka et al. 1989
; Malinow et al. 1989
; Silva et al. 1992
; Wang et al. 1994
). The present finding that a CaMKII inhibitor, KN-62, attenuated the facilitation produced by 5-HT supports the hypothesis that CaMKII is involved in synaptic plasticity, in general, and in heterosynaptic facilitation of the Aplysia sensorimotor synapse in particular. Thus in addition to the previously described roles of PKA and PKC in facilitation, CaMKII also may be involved.
Specificity of KN-62
The evidence for the involvement of CaMKII in synaptic transmission and its plasticity is based on the use ofKN-62, which is considered a specific inhibitor of CaMKII. KN-62 does not affect PKA, PKC, myosin light chain kinase, and Ca2+/CaM-dependent phosphodiesterase in rat, even at a concentration of 100 µM (Ishikawa et al. 1990
). It is not known whether KN-62 affects Ca2+/CaM-dependent adenylyl cyclase, however. Nor have biochemical studies been performed to examine its specificity in Aplysia. Indirect evidence indicates that KN-62 does not affect PKA and PKC in Aplysia, however. For example, the 5-HT-induced increases in excitability of the sensory neurons are known to be predominately mediated by PKA (Baxter and Byrne 1990a
; Klein et al. 1986
). An activator of PKC can increase excitability (Sugita et al. 1992
), but this effect appears due, at least in part, to a PKC-induced activation of cAMP levels (S. Sugita and J. H. Byrne, unpublished observation). In addition, the 5-HT-induced increases in spike duration are mediated mainly by the combined actions of PKA and PKC (Baxter and Byrne 1990a
; Braha et al. 1993
; Castellucci et al. 1982
; Sugita et al. 1992
). These actions also are not affected by KN-62. Although additional studies are needed, the present results raise the intriguing possibility that CaMKII plays a dual role in regulating synaptic efficacy and 5-HT-induced short-term facilitation in Aplysia.
Interrelationships among kinases
CaMKII, PKA, and PKC have at least one common action in that they all seem to participate in 5-HT-induced short-term facilitation of the sensorimotor connections (Table 1). Two mechanisms are believed to contribute to the facilitation. One mechanism for facilitation is broadening of the sensory neuron spike, whereas a second one is via spike-duration-independent (SDI) processes (see Byrne and Kandel 1996
for review). PKA and PKC seem to engage both spike-duration dependent and SDI processes whereasCaMKII does not. Although KN-62 increased the spike duration slightly, CaMKII does not appear to be necessary for 5-HT-induced spike broadening. CaMKII also does not appear to be necessary for the enhancement of excitability produced by 5-HT. These results suggest that CaMKII may play a role exclusively in SDI processes.
We do not know how CaMKII is activated in response to 5-HT. One possibility is that a receptor to 5-HT is linked to the CaMKII. For example, the sensory neurons appear to have at least two receptors for 5-HT, one coupled to cAMP and the other coupled to diacylglycerol (DAG) (Li et al. 1995
; Mercer et al. 1991
). However, our observation that inhibitors of CaMKII and CaM have similar effects suggests that CaMKII is activated via CaM in Aplysia. Therefore, the most likely possibility is that CaMKII is activated through an increase in the level of intracellular Ca2+, triggered either by the inositol trisphosphate (IP3)-dependent Ca2+ release from intracellular stores (Fink et al. 1988
; Sawada et al. 1989
; Scholz et al. 1988
) or by a second messenger-dependent modulation of membrane Ca2+ channels (Braha et al. 1993
; Kuno and Gardner 1987
).
The present study focused on the involvement of CaMKII in short-term facilitation. Serotonin also leads to a long-term (24-h) enhancement of the sensorimotor connection (Bailey et al. 1992
; Emptage and Carew 1993
; Montarolo et al. 1986
; Zhang et al. 1997
), and this facilitation is associated with enhanced excitability as well as structural changes in the sensory neurons (Bailey et al. 1992
; Dale et al. 1987
; Glanzman et al. 1990
; Wu et al. 1995
). Considerable evidence indicates a critical role for the cAMP/PKA cascade in this process (Bartsch et al. 1995
; Bergold et al. 1990
; Dash et al. 1990
; O'Leary et al. 1995
; Schacher et al. 1988
; Scholz and Byrne 1988
; Wu et al. 1995
). Recently, we found that KN-62 did not affect either the initiation or the maintenance of long-term facilitation (Zhang et al. 1995
), providing further support for the hypothesis that at least some of the actions of the kinases are segregated.
Dual actions of CaMKII
The effects of KN-62 were complex and cannot be readily explained by monotonic actions at a single site. Rather, the effects may be explained by assuming that CaMKII has an inhibitory effect on release at low levels of activation (i.e., in the absence of 5-HT or at low levels of 5-HT) and a facilitatory effect on release at higher levels of 5-HT. [A ceiling effect is unlikely, because the difference between the groups after the treatment with 5-HT in the presence and the absence of KN-62 is still significant when the EPSP amplitudes were normalized to the baseline (P < 0.05, Fig. 2A3), instead of to the pre-5-HT group (Fig. 2C)]. In addition, the degree of broadening produced by KN-62 was small compared with that produced by 5-HT (Fig. 4A3). Thus we speculate that higher levels of CaMKII (e.g., in the presence of 5-HT) facilitate transmitter release and these facilitatory effects override the inhibitory effects at low levels of CaMKII (e.g., in the absence of 5-HT). It is unlikely that a site of these facilitatory effects is the action potential, because KN-62 did not attenuate 5-HT-induced spike broadening (Fig. 4C). A second possible site for the facilitatory effects of CaMKII on release is the release mechanism itself.
Increasing evidence suggests dual roles of CaMKII in synaptic transmission. In the CA1 region of the hippocampus of mice heterozygous for a targeted mutation of
-CaMKII, paired pulse facilitation (PPF) is attenuated, but posttetanic facilitation (PTP) is enhanced (Chapman et al. 1995
). These results suggest that CaMKII has a facilitatory role in one form of synaptic plasticity (PPF) but an inhibitory role in another form (PTP). In addition, excitatory synaptic currents were enhanced but PPF and PTP were reduced in transformed strains of Drosophila that express a specific inhibitor of CaM kinase (Wang et al. 1994
). Our observation that a CaMKII inhibitor enhanced the amplitude of the EPSP and attenuated the 5-HT-induced facilitation also suggests a dual role for CaMKII in synaptic transmission.
The mechanisms for these dual roles of CaMKII are not clear. One possibility is that CaMKII plays different roles at different subcellular loci. For example, in Aplysia 5-HT leads to a translocation of a subunit of Ca2+-calmodulin-dependent kinase from the membrane-cytoskeleton complex to the cytoplasm (Saitoh and Schwartz 1983
, 1985
). When bound to the membrane-cytoskeleton complex, CaMK may have tonic effects on the membrane currents that contribute to repolarization of the action potential. A block of CaMK at this locus would broaden the spike and enhance synaptic transmission. After treatment with 5-HT, CaMK is released from the membrane-cytoskeleton complex to the cytoplasm (Saitoh and Schwartz 1983
, 1985
). The free CaMK in the cytoplasm may have a facilitatory effect on synaptic transmission similar to that in the giant synapse of squid (Llinas et al. 1985
). A block of CaMK at this locus would inhibit5-HT-induced facilitation. An alternative hypothesis is that the complex effects of CaM and CaMKII inhibitors arise from cross-talk among the multiple second messenger systems implicated in the plasticity at the sensorimotor synapse (e.g., Byrne et al. 1993
). These interactions may occur at multiple levels, ranging from the receptors to substrate proteins.