And Long-Term Effects of Serotonin, Small Cardioactive Peptide, and Tetanic Stimulation on Sensorimotor Synapses of Aplysia in Culture

The Journal of Neuroscience, October 1990, fO(10): 32863294

Selective Short- and Long-Term Effects of Serotonin, Small Cardioactive Peptide, and Tetanic Stimulation on Sensorimotor Synapses of Aplysia in Culture

Samuel Schacher,’ PierGiorgio Montarolo,l** and Eric Ft. Kande11m2 ‘Center for Neurobiology and Behavior, and *New York State Psychiatric Institute, Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, New York. New York 10032

Synapses between the sensory and motor cells of Aplysia to hours (Pinsker et al., 1970; Carew et al., 1971; Frost et al., can be enhanced by heterosynaptic or homosynaptic stim- 1985; Marcus et al., 1988) whereasrepeated stimuli produce ulation. We have used the isolated sensorimotor synapse of behavioral sensitization lasting days to weeks (Pinsker et al., Aplysia in cell culture to explore short- and long-term het- 1973; Frost et al., 1985). Studies on the cellular correlates of erosynaptic facilitation produced by 2 facilitatory transmit- thesebehavioral changesindicate that all are accompaniedby ters and compared these to homosynaptic facilitation pro- an increasein the efficacy of the sensoryneuron synapses(Cas- duced by posttetanic potentiation. We found that brief tellucci et al., 1970; Castellucci and Kandel, 1976; Hawkins et application of 5-HT or small cardioactive peptide (SCP) al., 1983; Walters and Byrne, 1983; Frost et al., 1985). Several evokes comparable short-lasting enhancement of nonde- lines of evidence suggestthat this facilitation of sensoryneuron pressed sensorimotor synapses. The effect evoked by SCP synapsesis mediated by the activation of heterosynaptic path- diverges from that of 5-HT when the sensorimotor synapse ways and the releaseof the facilitatory neurotransmitter 5-HT. is first depressed by low-frequency homosynaptic stimula- Sensitizing stimuli increasethe firing rate of identified seroto- tion. Whereas 5-HT facilitates sensorimotor synapses nergic cells in the cerebral ganglion, which evokes short-lasting whether or not they are depressed, SCP has little or no effect facilitation of sensorimotorsynapses (Hawkins, 1989; Mackey on synapses that have been depressed by more than 75%. et al., 1989). Bath application of 5-HT can simulatethe cellular The 2 transmitters also differ in producing long-term facili- effectsevoked by sensitizingstimuli (Brunelli et al., 1976; Cas- tation. Whereas repeated applications of 5-HT evoke long- tellucci and Kandel, 1976; Klein and Kandel, 1978; Klein et term facilitation of the synapses, SCP applications do not. al., 1986). The key role of 5-HT in mediating the effects of To determine whether these failures to facilitate could be sensitizingstimuli is also supportedby pharmacologicalstudies. overcome by increasing levels of CAMP, we applied SCP in Depletion of 5-HT with injections of the neurotoxin 5,7-dihy- the presence of phosphodiesterase inhibitors, which result- droxytryptamine reduces significantly behavioral dishabitua- ed in SCP evoking both short- and long-term changes com- tion of the reflex (Glanzman et al., 1989). In addition to its role parable to that of 5-HT. Homosynaptic facilitation by post- in short-term changes,5-HT may also play an important role tetanic potentiation differed from heterosynaptic facilitation in the long-term enhancementof this synapsethat accompanies in that tetanic stimulation failed to evoke long-lasting changes long-term sensitization. Repeated applications of 5-HT evoke in the synapse. These results support recent findings that long-lasting enhancement of sensorimotor synapsesreconsti- 5-HT is a critical neuromodulator in behavioral sensitization tuted in dissociatedcell culture (Montarolo et al., 1986; Dale and dishabituation and suggest that critical levels of CAMP et al., 1988), including the structural changesin the sensory may be required for long- and short-term facilitation of de- neurons (Glanzman et al., 1990) found in the intact nervous pressed synapses. In addition, the selective action of 5-HT system of long-term sensitizedanimals (Bailey and Chen, 1983, in evoking long-term facilitation suggests that long-term 1988a, b). change of this identified synapse may require the activity of Other neuromodulators besides5-HT, however, can also en- specific neuromodulatory pathways. hance the synapsesbetween sensory and motor cells and may be involved in aspectsof either short- or long-term dishabitua- The gill and siphon withdrawal reflex of Aplysia can undergo tion, sensitization,or associativeconditioning of the reflex. Sen- short- and long-term forms of nonassociative and associative sitizing stimuli that activate the serotonergiccells also activate behavioral enhancement.A singlenoxious stimulus to the body the identified L29 cells in the abdominal ganglion. The L29 surfacecan evoke dishabituation or sensitizationlasting minutes cells, whose transmitter is not known, can directly facilitate sensorimotorsynapses both in vivo and in vitro (Hawkins et al., Received Mar. 6, 1990; revised May 18, 1990; accepted May 23, 1990. 1981; Hawkins and Schacher,1989). In addition, the small car- This work was supported by NIH grant GM 32099 (S.S. and P.G.M.) and by dioactive peptides (SCP, and SCP,) can produce short-lasting the Howard Hughes Medical Institute (P.G.M. and E.R.K.). We thank L. Eliot, D. Glanzman, and R. Hawkins for their comments and R. Woolley for his technical changeswhen applied directly to the intact abdominal ganglion assistance. (Abrams et al., 1984). Becauseboth 5-HT and SCP enhance Correspondence should be addressed to Samuel Schacher, Ph.D., Center for Neurobiology and Behavior, Columbia University College of Physicians and Sur- levels of CAMP in sensorycells (Bemier et al., 1982; Abrams geons, 722 West 168th Street, New York, NY 10032. et al., 1984; Occor and Byrne, 1985), and becauseincreased Copyright 0 1990 Society for Neuroscience 0270-6474/90/103286-09$03.00/O levels of CAMP accompany short- and long-term sensitization The Journal of Neuroscience, October 1990, 70(10) 3287

(Bemier et al., 1982; Scholz and Byrne, 1988) and appear to be 5 ~1from a 50 ~1Hamilton syringe with its tip placed upstream (about critical for producing short- and long-term facilitation (Brunelli 150 pm) and above (about 300 pm) the cells. In pilot experiments, we first determined the concentrations of each neuromodulator that gave et al., 1976; Castellucci et al., 1980; Schacher et al., 1988; Braha a maximal level of facilitation at 1 min after treatment. We used trans- et al., 1990), one might expect both transmitters to have similar mitter concentrations of 5, 25, 50 and 100 KM (data not shown). The 5 effects. ~1 of perfusion medium contained final concenirations of either 50 PM To determine whether these 2 neuromodulators evoke similar 5-HT (&ma). 50 UMSCP, or SCP. (MolecularProbes). 50 UM SCP, short- and long-term enhancement of sensory neuron synapses, plus&Pi, or’j0 ,h SCP,plus 25 r;i-HT, andwas pre&ed;ust prio; to each application. The 2 forms of SCP evoked similar short- and long- we examined the consequences of brief bath applications of term changes. We therefore used SCP, in all the experiments. 5-HT or SCP on the sensorimotor synapse reconstituted in cell Short-term plasticity. Cultures consisted of one sensory cell and L7. culture. We found that both 5-HT and SCP evoked short-term To measure facilitation of nondepressed synapses, the initial amplitude facilitation when applied to synapses that were not depressed. of the EPSPwas measured by a singlestimulus to eachsensory cell 10 min before application of control solution, SCP or 5-HT, or a 2 set Unlike 5-HT, however, SCP failed to evoke short-term facili- tetanusto the sensorycell. The EPSPamplitude was measured 1, 5, tation when the synapse was first depressed by low-frequency and 10 min after treatment. Both transmitters evoked a transient de- homosynaptic stimulation of the sensory cell. In addition, SCP Dolarization of 3-10 mV in L7 (when hvDeroolarized 30 mV below its also failed to evoke long-term facilitation with repeated appli- resting potential) lasting 10-20 $ec. -- - cations. However, the lack of responses to SCP can be overcome To examine facilitation of depressed synapses, sensory cells were stimulated at 30-set intervals starting 10 min after measuring the initial by applying the transmitter in the presence of phosphodiesterase EPSP amplitude. Transmitter or control applications, or tetanus, were inhibitors such as isobutyl methyl xanthine (IBMX). These re- given when the EPSP was depressed to 20-25% of the initial amplitude sults are consistent with the idea that of the transmitters capable (10-20 stimuli). IBMX (100 PM; Sigma) was added to the perfusion of producing short-term facilitation, 5-HT may be of particular medium for some cultures immediately after the test stimulus and was present during the entire stimulation procedure. The rate of homosynap- importance in mediating long-term synaptic facilitation asso- tic depression was not altered by the presence of IBMX. Following ciated with behavioral sensitization of the gill-withdrawal reflex treatments, cells were given up to 5 additional stimuli at 30 set intervals as well as for short-term dishabituation. In addition, the failure to determinethe degreeof facilitation. of homosynaptic facilitation to evoke long-lasting changes in Long-term facilitation. Cultures contained 1 or 2 sensory cells and the synapse parallel those found for long-term synaptic depres- one L7. When both sensory cells formed synapses, the sum of their EPSPson each day wasused to determinepercent change in EPSP sion (Montarolo et al., 1988) and suggest that long-term mod- amplitude. Beginning 10 min after measuring the initial EFSP ampli- ulation of this synapse may require the actions of specific neu- tude. I-2-set huffs of 5-HT or SCP were aDDlied 4 times at 20-min romodulators. Some of these results have been reported in intervals, or sensory cells were given a 2-set tetanus 4 times at 20-min abstract form (Schacher and Montarolo, 1988). intervals. Some cultures were perfused with 100 PM IBMX after measuring the initial EPSP. After the last treatment, cultures were rinsed with culture medium, placed back into the incubator, and the EPSP Materials and Methods amplitude reexamined 24 hr later. A comparison ofthe resting potentials Dissociated cell culture. The cell culturetechniques and mediaused to of L7 recordedon the secondday to thosemeasured on the first showed isolateand maintain identified neurons of Aplysia have beendescribed no significant difference with treatments (mean change of -4 to -7% (Schacherand Proshansky,1983; Schacher, 1985; Rayport and Scha- for all control and experimental groups). cher, 1986).Culture medium for maintainingthe cells,and perfusion Analysis ofdata. All data in the text and figures are presented as mean mediumused during electrophysiologicalrecording and experimental percent change + SEM (unless specified) in the EPSP measured in L7 procedureswere supplemented with penicillin(50 units/ml)and strep- normalized to the pre-treatment baseline. The baseline in Figure 3 is iomycin (50 &ml). Eachdish containedsiphon sensory neurons iso- normalized to the average of the last 2 depressed EPSPs before treat- latedfrom abdominalganelia of adultanimals (SO-1 20 gsn)cocultured ment.This baseline is CornDared to the averageof the 2 EPSPsmeasured with a singlegill and sip&n motor cell L7 (Fig. 1) isoiatid from ab- after the various treatmenis. For the results-shown in Figures 2 and 3, dominalganglia of juvenile animals(l-3 gm). All animalswere raised a 2-factor analysis of variance (repeated measures) was used to test for in the laboratoryat the HowardHughes Medical Institute mariculture overall significance. For Figure 2, multiple t-tests (1 -tailed) for planned facility. Cultureswere maintained for 4-6 d at 18”. comparisons corrected for the 2-factor interaction error were used to Electrophysiology and synaptic potential measurements. The stimu- test for significance of treatment means to the control mean (pre-hoc) lation andrecording techniques have been described (Dale et al., 1988; at the various time points. These tests were used because the results Montaroloet al., 1988).The motor cell L7 wasimpaled intracellularly matched the predicted changes. The Dunnett’s t-tests (2-tailed) for mul- with a glassmicroelectrode (15-25 mQ)containing 2.0 M KC1and held tiple comparisons of experimental means to the control mean (post- at a potentialof - 30 mV belowthe restinglevels (range of -45 to - 58 hoc) were used in Figure 3, since the results did not match the predicted mv). EPSPswere evoked in L7 by stimulatingeach sensory cell with a changes.A 1-factor analysis of varianceand Dunnett’st-tests were used brief (0.2-0.5msec) depolarizing pulse using an extracellularelectrode in Figure4 to measuresignificance of the long-termchanges produced filledwith perfusionmedium. The initial EPSPamplitudes ranged from by the various treatments. As indicated in the text, either paired or 5 to 40 mV. To producehomosynaptic depression, sensory cells were unpaired t-tests were used for the remaining comparisons. givena singlestimulus every 30 sec.Homosynaptic (posttetanic potentiation) facilitation wasproduced when sensory cells were stimulated Results tetanicallyat 5, 10or 20 Hz for 2 set, or 20 Hz for 4 set with the same extracellularelectrode. Stimulus strength was increased by lO-20%above Both 5-HT and SCP evoke short-term facilitation of thresholdto insurea 1:1 correspond&cebetween sensory cell stimulus nondepressedsynapses, but only 5-HT facilitates andevoked EPSPs in L7. In nilot exnerimentswe found that the 20 Hz depressedsynapses tetanusfor 2 set evokedthe largestfacilitation measured1 min after Previous studieson the intact nervous system of Aplysia dem- the tetanus(data not shown).During the recording,cultures were per- fusedat a rate of 1.5 ml/min (bath volume, 3.0 ml) at 18-19” with onstrated that synapsesbetween sensoryand motor cellscan be perfusionmedium, a 1:1 mixture of Leibovitz-15 (Flow Laboratories) facilitated by application of 5-HT (Brnnelli et al., 1976) or SCP and artificial seawater (Instant Ocean)supplemented with 5% sterile (Abrams et al., 1984). Recently, examination of the facilitation filteredAplysia hemolymph,pH 7.6. Resultswere stored on a 4-channel evoked by 5-HT indicated that at least 2 distinct processescan taperecorder (Hewlett-Packard), and recordswere made with a Gould penrecorder. be activated by 5-HT. The contribution of each processto syn- Application of neuromodulators. Neurotransmittermodulators were apseenhancement depends on the prior stimulus history of the appliedto cellsduring continuous perfusion by a brief (l-2 set)pti of sensorycell, that is, whether or not the synapseis first depressed 3288 Schacher et al. l Heterosynaptic Versus Homosynaptic Facilitation in Aplysia

Figure 1. Phase-contrastlight micro- graphof 5-d-oldculture containing motor cell (L7) and 2 sensorycells (SNl andSN2). The structureemerging from the cell body of L7 and lying between the 2 sensoryneuron cell bodies is the initial segmentof the axon of L7 that wasattached to L7 at platingof the cells. The other neuriteshave regenerated duringthe 5 d of culture.Scale bar, 40 urn. by homosynaptic stimulation (Gin&h and Byrne, 1985; Hoch- motor synapsesthat were depressedby at least 75% from their ner et al., 1986a, b). We first compared the facilitation evoked initial level with low frequency stimulation of the sensorycell by 5-HT to that of SCP on sensorimotorsynapses that had not (Fig. 3); a situation that is comparable to that occurring in the undergoneany depressionby low frequency stimulation of the intact animal with dishabituation. A l-2 setapplication of 5-HT sensorycells (Fig. 2). We then compared the effects of the 2 evoked a significant facilitation of 338% + 45 (N = 9, t = 4.718, transmitters after the synapsehad first beendepressed by homo- p < 0.01; Dunnett’s t-test, 2-tailed) compared to controls, 96% synaptic stimulation (Fig. 3). * 2 (N = 5). By contrast, SCP application evoked little facili- Following a singletest stimulusto the sensorycell, a brief l-2 tation of 108% + 13 (N = 10) which was not significantly dif- set application of either 5-HT (N = 5) or SCP (N = 5) evoked ferent from control (t = 0.236). Preliminary evidence suggests a significant enhancementin the amplitude of the EPSP (F3,,6 that the failure of SCP to evoke facilitation under these con- = 15.405,p < 0.001 for treatment; Fps4,= 7.196, p < 0.001 for ditions is not due to an activation of some inhibitory process time-treatment interaction). This heterosynapticfacilitation was when sensorimotor synapsesare depressedby homosynaptic short-lived (Fig. 2A). Both 5-HT and SCP facilitated the EPSP stimulation. Simultaneousapplication of SCP (50 PM) and 5-HT at 1 min after treatment; 131% + 8 for 5-HT(t,, = 9.061, p < (25 PM) produced a facilitation of 387% f 29 (N = 3). O.OOl),and 125%+ 2forSCP(t,, = 7.959,~ < O.OOl)compared The failure of SCP to facilitate depressedsensorimotor syn- to control level of 86% f 5 (N = 5). After 10 min, both 5-HT apsesmay result from its inability to increaseCAMP to a critical and SCP treatments werenot different than controls (td8= 1.714, level within the sensory cell terminals necessaryfor initiating p > 0.05 and t,, = 1.632, p > 0.1, respectively). the transmitter mobilization processrequired to facilitate a de- We next examined the effects of 5-HT and SCP on sensori- pressedsynapse (Abrams et al., 1984; Braha et al., 1990). To The Journal of Neuroscience, October 1990, IO(10) 3289

600 -10 min 0 lmin 5min IOmin B ~------Figure 2. Short-term facilitation of a nondepressedsynapse by 5-HT, SCP, and tetanus. A, Each point is the mean percent change + SEM in the amplitude of the EPSP normalized to the initial amplitude (- 10 min) for each culture (1 sensorimotor connection). Note that the amplitude of the EPSP in control cultures decreased with each stimulus, which is characteristic of homosynaptic depression. A 2-factor overall analysis of variance indicated a difference with treatment (see Materials and Methods and Results). A comparison of the means at each time point indicated that all 3 treatments were significantly different than control 1 min after treat- 5-HT E -Jy ---- z; ments, and not significantly different than control at 10 min (see Results). B, Examples of facilitation evoked by 5-HT, SCP, and tetanus compared to IOmV the depression evoked with control 25msec stimulation. test this possibility, we examined the facilitation of depressed reversible.One hr after washout of the IBMX and rest, the same synapses by SCP in the presence of a phosphodiesterase inhib- depressedconnections that were facilitated in the presenceof itor IBMX (Fig. 3). As previously reported (Schacher et al., IBMX (N = 3) failed to facilitate with SCP alone application 1988), IBMX alone did not affect nondepressed synapses, nor (105% rf: 9). did it alter the rate of homosynaptic depression. The same number of stimuli to the sensory cells depressed the amplitude of 5-HT selectivity evokes long-term facilitation of sensorimotor the EPSP to below 75% of the initial value when IBMX was synapses present as non-IBMX treated cultures. Application of SCP in We had previously demonstrated(Montarolo et al., 1986; Dale the presence of IBMX (N = 6) now evoked a significant facili- et al., 1988) that repeated5 min applicationsof 5-HT produced tation of 274% + 5 1 (t = 3.197, p < 0.05; Dunnett’s t-test, a significant enhancementof the L7 EPSP amplitude when the 2-tailed). Facilitation of the EPSP amplitude of 235% + 29 (N connections were reexamined 24 hr later. We have confirmed = 3) was evoked by SCP in the presence of another phospho- theseresults using, instead of 5-min applications,the brief l-2 diesterase inhibitor (100 PM RO 20-I 724; Hoffmann-La Roche, set puffs describedabove in the experiments on short-term fa- Nutley, NJ). In addition, the effects of IBMX appear to be cilitation. The brief applications of 5-HT evoked a significant 3290 Schacher et al. l Heterosynaptic Versus Homosynaptic Facilitation in Aplysia

A 400 A 7o 1 1 60 300- i 3 ? - 6 $t 200-

B - s loo- 1 OJ Elpre post flpre post I Control SCP 3MX 5- Tetanus B 1 I COIli Tet SCP -I-HT Tetanus L7/----- e ----Jo--P-- 6 0 hl 24 hr 5-HT I-r------r\ L7~~

SCP ___----___----__--__ L7q---A -2 -/------1 2 1omn 5-HT Pre t Post 12sms Tetanus 25msec 5-HT SCP+lBMX Figure 4. 5-HT applications selectively evoke long-term facilitation. SCP A, The height of each bar is the mean percent change + SEM in the amplitudeof the EPSPevoked in L7 whenretested 24 hr aftertreatment. Figure 3. SCP fails to facilitatea synapsedepressed by 75% with Eachculture counted as 1 sample.A l-factor analysisof variancein- homosynapticdepression. A, Eachbar is the meanpercent change +- dicated a difference with treatment F3.23= 9.304; p < 0.001). A com- SEM in the averageamplitude of the first 2 EPSPsevoked in L7 mea- parisonof the means(Dunnett’s t-test; seeResults) indicated that 5-HT suredafter control or experimentaltreatments (Post) normalized for treatment significantly increased the EPSPrelative to control, while eachculture (one sensorimotor connection) to the averageof the last2 tetanus and SCP treatments were not significantly different than con- depressedEPSPs evoked in L7 beforetreatments (Pre). A 2-factoranal- trols. B, Examples of the facilitation in the amplitude of the EPSP24 ysisof variance(repeated measures) indicated a significanteffect with hr following repeated applications of 5-HT, but not following control, treatmentand treatment-testinteractions (F4,33 = 10.669,p < 0.001). tetanus, and SCP treatments. Comparisonsofthe means(Dunnett’s t-tests; see Materials and Methods and Results)indicated that 5-HT, tetanus,and SCPplus IBMX treat- mentswere significantly different from controls,but SCPalone was not. cations in the presenceof IBMX (Fig. 5A). SCP applicationsin B, Examplesof the facilitationin the amplitudeof the EPSPevoked in the presenceof IBMX (N = 6) now evoked a significant (tlo = L7 followingtetanus, application of 5-HT, and applicationof SCPin 3.3 14, p < 0.01; unpaired t-test, 2-tailed) increaseof 135% f the presenceof IBMX, but not applicationof SCPalone. 7 compared to 102% -t 6 (N = 6) for culturestreated with IBMX alone. enhancement(t = 4.468, p < 0.01; Dunnett’s t-test, 2-tailed) of 158% f 11 (N = 6) compared to 104% f 6 (iV = 6) for control Tetanic stimulation of the sensory cell evokes short- but not (Fig. 4). By contrast, repeatedapplications of SCP failed to evoke long-term facilitation a significant changein the EPSP after 24 hr; 107% f 10 (N = Previous studiescomparing the effectsof homosynapticand het- 6; t = 0.249). The failure of SCP to evoke a long-term change erosynapticmechanisms on depressionof the sensorimotorsyn- apparently wasnot due to desensitizationcaused by the repeated apseindicated that long-term inhibition of this synapsemay applications. We examined the short-term facilitation (N = 2 require the actions of specific heterosynaptic pathways. Al- cultures) evoked with each SCP application given at 20-min though sensorysynapses can be depressedfor a short duration intervals. Each application of SCP was capable of evoking a by application of either the neuropeptide FMRFamide or do- short-term facilitation (range of 118-l 28%). pamine, or by homosynaptic depression(low frequency stim- To test the idea that the failure of SCP to evoke a long-term ulation of the sensorycell), long-term depressionis evoked se- changewas due to its inability to increaseCAMP to somecritical lectively by applicationsof FMRJ?amide(Montarolo et al., 1988). level, we examined the long-term consequencesof SCP appli- In the intact nervous system, tetanic stimulation of the sensory The Journal of Neuroscience, October 1990, IO(10) 3291

Figure 5. SCP applications,but not tetanus,in the presenceof IBMX evoke long-term facilitation. The height of each bar is the mean percent change t SEM in the amolitude of the EPSP OW evoked in Ll when retested 24 hr after IBhiX treatments. Eachculture countedas 1 A2 B2 sample, AI, SCP applications in the Ohr 24hr Ohr 24hr continuous presence of IBMX signifi- ,eMxL7 ~-----~ cantly increased the EPSPcompared to treatment with IBMX alone (see Re- sults). A2, Examples of the facilitation in the EPSP evoked in Ii’ 24 hr following applications of SCP in the presence of IBMX, but not following treatment with IBMX alone. BI, Repeated tetani to the sensory cells in the presence of ------__------SCP Tet. IBMX evoked little long-term chanee ,e’Mx ~ ------~- and was not significantly-different thin I&X treatment with IBMX alone (see Re- Lb sults). B2. Bxamoles of the absence of J L7 J OmV L7J / 10mV a long-term facilitation 24 hr following -.b _I repeated tetani in the presence ofIBMX 25msec 25msec or treatment with IBMX alone. cells can also facilitate sensorimotor synapsesfor a short du- Tetanic stimulation in the presenceof IBMX did not causethe ration (Walters and Byrne, 1984, 1985; Clarke and Kandel, connections to fatigue, since the amplitude of the first EPSP 1984). We therefore compared the consequencesof this homo- evoked during the last tetanus was significantly enhanced by synaptic pathway on short- and long-term facilitation of the 123% -t 8 (N = 6; t,= 3.015, p < 0.05; paired t-test, 2-tailed) sensorimotorsynapses to thoseevoked by the neuromodulators. compared to the EPSP evoked before the first tetanus. Tetanic stimulation of the sensorycell, as 5-HT, evokes facilitation of both nondepressedand depressedsynapses (Figs. Discussion 2, 3). One min after tetanus was given to a sensory cell that Multiple modulatory pathways produce short-termfacilitation received only a single stimulus, the EPSP amplitude was sig- of sensorimotorsynapses of Aplysia nificantly enhancedto 126% f 7 (N = 5; ta8= 8.163, p < 0.00 1) Our study confirms previous work indicating that 5-HT plays comparedto controls (Fig. 24). The facilitation was short-lived: a critical role in facilitating the synapsesbetween the sensory by 10 min, treated cells were no longer significantly different and motor cells, either when the synapsesare not depressedor from controls (t,, = 1.632, p > 0.1). Tetanic stimulation also when they are depressedas is the casein behavioral sensitization evoked a significant facilitation of depressedsynapses (Fig. 3) or dishabituation, respectively. At least 2 processesinitiated by to 264% f 22 (N = 8; t = 3.207, p < 0.05; Dunnett’s t-test, 5-HT contribute to the increase in transmitter releaseat sen- 2-tailed) compared to controls (Fig. 3A). sorimotor synapsesduring short-term facilitation. One process Although tetanus can evoke short-term enhancement, re- involves broadening of the action potential in the sensorycell peated tetanic stimulation failed to evoke a significant long- through closure of a serotonin-sensitivepotassium channel by term change(Fig. 4). The EPSP amplitudes reexamined 24 hr CAMP-dependent phosphorylation leading to the increase in after repeated tetanic stimulation were not significantly in- calcium entry (Castellucci et al., 1982; Klein et al., 1982; Sie- creased,107% f 6 (N = 6; t = 0.235; Dunnett’s t-test, 2-tailed) gelbaumet al., 1982). This processcontributes as significantly compared to controls (Fig. 4A). The failure to evoke a long- when the synapseis not depressedas would occur with sensi- lasting changewas not due to synapsefatigue during the tetani. tization of the reflex (Hochner et al., 1986a, b). Broadening of The amplitude of the EPSP evoked with the first stimulus of the action potential is not sufficient however, to enhancea syn- the final tetanuswas increasedsignificantly by 119% f 5 (N = apsethat has been depressedwith homosynaptic depression.A 4; t, = 3.265, p < 0.05; paired t-test, 2-tailed) compared to the secondprocess, independent of spike broadening, is required to EPSP amplitude measuredbefore tetanic stimulation. In ad- mobilize transmitter release(Gingrich and Byrne, 1985; Hoch- dition, treatment with IBMX, which resulted in long-term fa- ner et al., 1986a, b, Dale and Kandel, 1990). Recent evidence cilitation with SCP applications, dud not alter the long-term suggeststhat this second processcan be initiated either by in- consequencesof tetanic stimulation (Fig. 5B). Repeatedtetanic creasedlevels of CAMP or by activation of C-kinase (Braha et stimulation in the presenceof IBMX evoked little long-term al., 1990). The role of 5-HT in this secondprocess is supported changein the amplitude of the EPSP, 103% f 9 (N = 6), com- by the findings that application of 5-HT is known to increase pared to IBMX alone cultures, 101% + 4 (N = 6; t,, = 0.202). levels of CAMP (Bemier et al., 1982; Occor and Byrne, 1985) 3292 Schacher et al. l Heterosynaptic Versus Homosynaptic Facilitation in Aplysia and CAMP-dependent phosphorylation (Sweatt and Kandel, In both cases, the increase in intracellular calcium map activate 1989), and can also translocate C-kinase from the inactive sol- calcium-dependent processes affecting cytoskeletal elements or uble form to the active membrane-bound form (Saktor and other substrate proteins involved with the transmitter release Schwartz, 1990). process or mobilization (Llinas et al., 1985; Rasmussen and The ability of SCP to facilitate nondepressed sensorimotor Means, 1989; Kennedy, 1990). synapses is consistent with the ability of SCP to increase levels of CAMP in sensory cells, thereby closing serotonin-sensitive Specific modulatory pathway evokes long-term facilitation of potassium channels as well as broadening the action potentials sensorimotor synapses of Aplysia (Abrams et al., 1984; Occor and Byrne, 1985). The failure of Although a single brief application of 5-HT or SCP evokes com- SCP, however, to significantly enhance depressed synapses in parable short-term enhancement of sensorimotor connections, culture or in the intact nervous system (Pieroni and Byrne, 1989) only repeated brief applications of 5-HT produce a long-term indicates that SCP fails to activate the second process involving change. These findings parallel results reported for long-term transmitter mobilization and thus does not by itself contribute depression of this synapse produced by repeated application of to facilitation associated with dishabituation. Because SCP re- the neuropeptide FMRFamide or dopamine (Montarolo et al., ceptors and the associated signal transduction system are dis- 1988). A single application of either inhibitory neurotransmitter tinct from that of 5-HT (Abrams et al., 1984; Occor and Byrne, evoked comparable short-term changes in the synapse, but only 1986), there are at least 2 explanations for the failure of SCP to FMRFamide produced a long-term change. The failure to pro- facilitate depressed sensorimotor synapses. First, the distribu- duce a long-term change with repeated tetanic stimulation, where tion of SCP receptors on sensory cells may differ from that of a single tetanus evokes short-lasting facilitation, also parallels 5-HT such that it fails to enhance sufficiently CAMP levels with- the inability to produce long-term depression with repeated low- in the terminals of the sensory cells. Isolated growth cones of frequency stimulation of the sensory cells (Montarolo et al., sensory cells contain receptors for 5-HT and the appropriate 1988). Thus, short-lasting changes in this synapse may be evoked intracellular machinery for signal transduction. (Belardetti et by activation of different heterosynaptic and homosynaptic al., 1986). If fewer SCP receptors are present at the terminals, pathways, but long-term facilitation or depression may require SCP might fail to facilitate because high levels of CAMP in the specifically 5-HT or FMRFamide, respectively. terminals may be required for transmitter mobilization (Braha The failure of SCP to evoke a long-term change, unless it was et al., 1990). When SCP is now applied in the presence of phos- applied in the presence of the phosphodiesterase inhibitor IBMX, phodiesterase inhibitors, or when applied together with 5-HT, indicates that repeated SCP applications alone fail to induce a CAMP levels within the terminals could reach the levels required change in the cells that is necessary for the long-term facilitation. for transmitter mobilization. Previous studies on the intact nervous system and on the in A second explanation for the failure of SCP to facilitate de- vitro synapse suggest that CAMP, persistant CAMP-dependent pressed synapses is that unlike 5-HT (Saktor and Schwartz, phosphorylation of substrate proteins, persistent activation of 1990), SCP may not activate the C-kinase system in sensory A-kinase, and CAMP induction of gene expression are critical cells. In support of the role of C-kinase in facilitating depressed for the expression of long-term facilitation and other changes synapses are the findings that phorbol esters can mimic the associated with long-term behavioral sensitization (Greenberg actions of 5-HT in facilitating depressed synapses. Moreover, et al., 1987; Schacher et al., 1988; Sholz and Byrne, 1988; Sweatt the kinase inhibitor H7, which in Aplysia appears to be more and Kandel, 1989; Dash et al., 1990). Thus, although SCP can selective for C-kinase (Conn et al., 1989), becomes progressively enhance levels of CAMP sufficiently to evoke a short-lasting more effective in blocking 5-HT facilitation as the sensorimotor facilitation of nondepressed synapses, it does not sufficiently synapse is depressed to lower levels (Braha et al., 1990). The enhance CAMP levels and the CAMP-dependent processes re- ability of 5-HT to facilitate a depressed synapse may therefore quired to evoke a long-term change. A critical level of CAMP involve the coactivation of 2 second-messenger systems: the elevation may also underlie long-term facilitation of crayfish A-kinase and C-kinase systems. The synergistic effects on syn- neuromuscular junctions (Dixon and Atwood, 1989). This crit- apse plasticity of activating multiple second-messenger systems ical level can be achieved by 5-HT alone, or when the actions have also been implicated in long-term potentiation in the hip- of SCP on CAMP levels are enhanced by the presence of IBMX. pocampus (Lynch and Baudry, 1984; Malenka et al., 1986,1989; This difference between 5-HT and SCP may arise from an in- Manilow et al., 1989; Williams et al., 1989), and associative trinsic difference in the transduction of the repeated receptor conditioning in Aplysia (Hawkins et al, 1983; Walters and Byrne, activation with the CAMP cascade. Alternatively, SCP may fail 1983, 1985; Occor et al., 1985) and Hermissenda (Acosta-Ur- to activate sufficiently the A-kinase system for the long-term quidi et al., 1984; Farley and Auerbach, 1986). In contrast, SCP change because unlike 5-HT, it may not activate the C-kinase might not be able to facilitate depressed synapses because it system. According to this latter scheme, the ability of repeated activates only 1 of the 2 second-messenger systems. 5-HT applications to evoke repeated and simultaneous acti- Our results also confirm previous work on posttetanic poten- vation of both the A-kinase and the C-kinase systems may result tiation of sensory neuron synapses in the intact nervous system in a sufficient enhancement of CAMP levels and/or A-kinase of Aplysia (Walters and Byrne, 1984, 1985; Clarke and Kandel, activity that is necessary for long-term facilitation. With ap- 1984). Posttetanic potentiation is thought to be mediated in part propriate pharmacological tools, it may be possible to test these by the increase in intracellular calcium within the terminals, alternative mechanisms. thereby facilitating the release of transmitter with subsequent action potentials (Katz and Miledi, 1968; Rahamimoff, 1968; References Kretz et al., 1982; Connor et al., 1986). Our results further Abrams TW, Castellucci VF, Camardo JS, Kandel ER, Lloyd P (1984) indicate that this increase in calcium can serve as another mech- Two endogenous neuropeptides modulate the gill- and siphon-with- anism in facilitating both nondepressed and depressed synapses. drawal reflex in Apfysiu by presynaptic facilitation involving CAMP- The Journal of Neuroscience, October 1990, lO(10) 3293

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