H. Byrne Diasinou Fioravante, Rong-Yu Liu, Anne K. Netek, Leonard ...

98:3568-3580, 2007. First published Oct 3, 2007; doi:10.1152/jn.00604.2007 J NeurophysiolH. Byrne Diasinou Fioravante, Rong-Yu Liu, Anne K. Netek, Leonard J. Cleary and John

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Synapsin Regulates Basal Synaptic Strength, Synaptic Depression,and Serotonin-Induced Facilitation of Sensorimotor Synapses in Aplysia

Diasinou Fioravante, Rong-Yu Liu, Anne K. Netek, Leonard J. Cleary, and John H. ByrneDepartment of Neurobiology and Anatomy, W.M. Keck Center for the Neurobiology of Learning and Memory, The University of TexasMedical School at Houston, Texas

Submitted 28 May 2007; accepted in final form 27 September 2007

Fioravante D, Liu R-Y, Netek AK, Cleary LJ, Byrne JH. Synapsinregulates basal synaptic strength, synaptic depression, and serotonin-induced facilitation of sensorimotor synapses in Aplysia. J Neuro-physiol 98: 3568–3580, 2007. First published October 3, 2007;doi:10.1152/jn.00604.2007. Synapsin is a synaptic vesicle-associatedprotein implicated in the regulation of vesicle trafficking and trans-mitter release, but its role in heterosynaptic plasticity remains elusive.Moreover, contradictory results have obscured the contribution ofsynapsin to homosynaptic plasticity. We previously reported that theneuromodulator serotonin (5-HT) led to the phosphorylation andredistribution of Aplysia synapsin, suggesting that synapsin may be agood candidate for the regulation of vesicle mobilization underlyingthe short-term synaptic plasticity induced by 5-HT. This study exam-ined the role of synapsin in homosynaptic and heterosynaptic plastic-ity. Overexpression of synapsin reduced basal transmission and en-hanced homosynaptic depression. Although synapsin did not affectspontaneous recovery from depression, it potentiated 5-HT–induceddedepression. Computational analysis showed that the effects ofsynapsin on plasticity could be adequately simulated by altering therate of Ca2�-dependent vesicle mobilization, supporting the involve-ment of synapsin not only in homosynaptic but also in heterosynapticforms of plasticity by regulating vesicle mobilization.

I N T R O D U C T I O N

Heterosynaptic plasticity has received considerable attentionas a means to induce and maintain learning-related synapticmodifications. Modulatory neurotransmitters have been postu-lated to provide the attentional and motivational state forinformation processing and memory formation (Bailey et al.2000; Walters and Byrne 1983). However, the cellular andmolecular mechanisms mediating the effects of most ofthese modulators on synaptic plasticity and learning remainunclear. A well-established system for the study of het-erosynaptic modulation of synaptic transmission and plas-ticity is the Aplysia sensorimotor synapse. In this system,serotonin (5-HT) engages several second messenger cascades,including cAMP/protein kinase A (PKA), to induce both short-and long-term facilitation of synaptic transmission (Byrne andKandel 1996).

Extensive studies of 5-HT–induced plasticity have impli-cated broadening of the action potential as a mechanismmediating short-term synaptic facilitation (STF) (Eliot et al.1993; Hochner and Kandel 1992; Hochner et al. 1986a). Othermechanisms, independent of spike duration, were also requiredfor STF, especially when transmission was depressed before

5-HT treatment (Braha et al. 1990; Ghirardi et al. 1992;Gingrich and Byrne 1985; Goldsmith and Abrams 1992; Hoch-ner et al. 1986b; Sugita et al. 1997). Even though regulation ofsynaptic vesicle availability for release was proposed to beinvolved in STF (Byrne and Kandel 1996; Gingrich and Byrne1985; Gingrich et al. 1988), little is known about the molecularmechanisms underlying vesicle trafficking at Aplysia sensori-motor synapses.

Synapsin is a synaptic vesicle-associated protein suggestedto be a central element in the organization and regulation ofvesicles in nerve terminals (Chi et al. 2001; Li et al. 1995;Rosahl et al. 1993, 1995). In mammals, three genes code forfive synapsin isoforms (Sudhof 2004). Synapsin reversiblyassociates with synaptic vesicles, actin, microtubules, andother synapsins in a phosphorylation state-dependent fashion(Benfenati et al. 1989, 1992; Greengard et al. 1993; Huttneret al. 1983; Matsubara et al. 1996). Synapsin is a major,although not exclusive (Hell et al. 1995; Hirling and Scheller1996; Lonart et al. 2003; Nagy et al. 2004; Ono et al. 1998;Sharma et al. 2003; Thakur et al. 2004), synaptic target forPKA (Hosaka et al. 1999; Menegon et al. 2006), mitogen-activated protein kinase (MAPK) (Jovanovic et al. 1996; Yama-gata et al. 2002), and Ca2� calmodulin-dependent proteinkinase II (CAM kinase II) (Chi et al. 2001, 2003; Llinas et al.1991). These properties of synapsin make it an attractivecandidate for the regulation of synaptic vesicle availability,which may be important for short-term synaptic depression andfacilitation (Angers et al. 2002; Humeau et al. 2001; Rosahl et al.1993; Turner et al. 1999).

Several studies have implicated synapsin in homosynapticplasticity, albeit with conflicting results. Imaging studies ofsynapsin knockouts suggest that synapsin I is necessary forsynaptic mobilization and exocytosis induced by high-fre-quency stimulation of hippocampal cells (Chi et al. 2003; Ryanet al. 1996). However, recent findings in synapses of thalamo-cortical cells from synapsin I/II knockouts suggested thatsynapsin is not essential for sustained, high-rate transmission(Kielland et al. 2006). Moreover, in hippocampal slices fromsynapsin I knockouts, paired-pulse facilitation (PPF) was en-hanced (Rosahl et al. 1993), whereas in double synapsin I/IIknockouts, no effect on PPF was observed (Rosahl et al. 1995).In addition, modulating levels of synapsin I dramatically af-fected posttetanic potentiation (PTP) at cholinergic synapses inAplysia (Humeau et al. 2001) but not at the C1-M2 synapse inHelix (Fiumara et al. 2007) or in hippocampal slices (Rosahl

Address for reprint requests and other correspondence: J. H. Byrne, Dept. ofNeurobiology and Anatomy, W. M. Keck Ctr. for the Neurobiology ofLearning and Memory, The Univ. of Texas Medical School at Houston, POBox 20708, Houston, TX 77225 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Neurophysiol 98: 3568–3580, 2007.First published October 3, 2007; doi:10.1152/jn.00604.2007.

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et al. 1993). Therefore a complete understanding of the role ofsynapsin in homosynaptic plasticity remains elusive. Some ofthis uncertainty could be attributed to gene redundancy, non-specific effects of synapsin deletion on synaptic integrity,developmental effects, and homeostatic compensation (Chinet al. 1995; Powell 2006). Other factors that may contribute tothe apparent contradiction involve synapse-specific propertiesand differences in stimulation frequencies. Furthermore, littleis known about the role of synapsin in heterosynaptic plastic-ity. Spillane et al. (1995) found that forskolin-induced plastic-ity was not affected in synapsin I/II knockout mice, whereasMenegon et al. (2006) recently showed that the effect offorskolin on the recovery of transmission from depressionrequired synapsin phosphorylation. Other examples of het-erosynaptic modulation have not been examined.

In this study, we directly investigated the role of Aplysiasynapsin in basal neurotransmitter release and in two forms ofsynaptic plasticity. The Aplysia sensory-motor synapse in cul-ture is an excellent system to study these processes, becausetransmission at this synapse has been thoroughly characterized(Byrne and Kandel 1996). Moreover, a single gene codes forsynapsin in Aplysia (Angers et al. 2002). Therefore compen-satory gene regulation is not expected to confuse interpretationof the experimental results.

M E T H O D S

Plasmid construction

The sequence for Aplysia synapsin (apSyn) was obtained by PCRusing a pGEX-apSyn vector (Angers et al. 2002) as template. Thetermination codon was mutated, and the purified PCR product wasligated into pNEX� expression vector upstream of the hemagglutinin(HA) tag sequence, using standard procedures (Maniatis et al. 1989).Automated sequencing (Seqwright, Houston, TX) of the plasmid wasperformed to confirm that the sequence was error-free and insertedproperly into the vector. In pilot experiments, we also attempted toexpress a synapsin-EGFP construct for visualizing the expressedprotein. However, for reasons that are not understood, the EGFPchimera required �12 days to be expressed as opposed to 2 days forsynapsin-HA. Therefore we used the synapsin-HA vector for thisstudy.

Cultures and injections

Sensorimotor neuron co-cultures were prepared as described pre-viously (Angers et al. 2002; Rayport and Schacher 1986). On day 3,sensory neurons were injected with 0.5 �g/�l pNEX�-apSyn-HA orpNEX�-HA (empty vector, control) in injection solution (0.1% Alexa488-dextran, 100 mM KCl). A pNEX3-EGFP expression vector (0.2�g/�l) was co-injected to allow for assessment of injection efficiency.Co-cultures were returned to the incubator for 4 days, at which timeexperiments were performed. In order to confirm vector expression,co-cultures were processed for immunofluorescence analysis at theend of electrophysiological experiments. Specifically, immediatelyafter the end of stimulation, co-cultures were rinsed with 5 volumes50% isotonic L15-50% ASW and allowed to rest for a few hoursbefore fixation. This rest period was necessary because 5-HT is knownto induce synapsin dispersion, which lasts �2 h (Angers et al. 2002).

Electrophysiology

Recordings were made in 50% isotonic L15-50% artificial seawater(ASW). Sensory neurons were stimulated extracellularly with a patchelectrode filled with 50% ASW�50% isotonic L15, and postsynaptic

responses (excitatory postsynaptic potentials; EPSPs) were monitoredintracellularly with sharp electrodes of 12- to 15-M� resistance, filledwith 3 M KAc. The resting membrane potential of motor neurons wascurrent clamped at �90 mV. Responses were recorded using anAxoclamp-2B amplifier and pCLAMP 8.2 software. The stimulationprotocol consisted of a train of 10 stimuli at 1 Hz, at the end of which5-HT (50 �M final concentration) or ASW (vehicle; control) wasapplied with a pipette. A 5-min rest period followed, at the end ofwhich an additional stimulus was delivered to test for the extent ofspontaneous recovery (in the ASW-treated group) or dedepression (inthe 5HT-treated group). Off-line analysis of the magnitude of excita-tory postsynaptic potentials (EPSPs) was performed using pCLAMP8.2. EPSP kinetics were analyzed using the Statistics function ofClampfit 10.0, an analysis package that is part of pCLAMP. Rise timeand rising slope were measured from 10 to 90% of peak. Decay timeand decay slope were measured from 90 to 20% of peak.

Immunofluorescence

For fixation, 4% paraformaldehyde in 30% sucrose-PBS was ap-plied to the cultures for 30 min. After incubation with blockingmedium [Superblock medium (Promega) supplemented with 5% nor-mal goat serum and 0.2% Triton-X 100] for 30 min, anti-apSyn(1:500), anti-apVAMP (1:500), and/or anti-HA (1:100; Abcam, Cam-bridge, MA) primary antibodies were added overnight. After three15-min rinses with PBS, co-cultures were incubated with goat anti-rabbit and/or anti-mouse secondary antibodies coupled to Alexa568 (Invitrogen, Carlsbad, CA) and Cy5 (Jackson Immunoresearch,West Grove, PA), respectively. Anti-fade mounting medium (Invitro-gen) was used for mounting of coverslips. Confocal images werecollected using a 40� water-immersion lens (NA 0.8) of an Olympusupright microscope coupled to a Biorad 1024 MP confocal system.Excitation wavelengths of 488, 568, and 633 nm were used insequential mode and z-stacks of 0.5-�m step size and/or single opticalsections were collected. For imaging of isolated sensory neurons inFigs. 2, D and E, and 7, a 60� oil immersion lens (NA 1.4) was used.In experiments where two groups of cultures were compared, settingsfor image acquisition were kept the same. Off-line analysis wasperformed using Metamorph imaging system (Molecular DevicesCorp., Sunnyvale, CA) and custom MATLAB (Mathworks) software.Signal colocalization was assessed using ImageJ 1.34n software(National Institutes of Health) or Adobe Photoshop CS2. Varicositieswere defined as swellings located along neurites, at branch points, andat neurite terminals (Bailey and Chen 1988), with a diameter �1.5times the diameter of the attached neurites (Bailey et al. 1979). Totalneurite length for each image field was determined by the summedlength of lines traced along neurites. For the analysis presented in Fig.2, varicosities were confirmed as sites of vesicle accumulation byexamining VAMP staining in overlaid images. Varicosities werecounted only if VAMP staining was increased compared with theattached neurites.

FM4-64 dye uptake experiments

FM4-64 (8.5 �M final concentration; Molecular Probes) was usedto label vesicle pools in control and synapsin-overexpressing senso-rimotor co-cultures that co-expressed EGFP. Loading of vesicles wasachieved through KCl stimulation (5 min, 100 mM) in the presence ofFM4-64. KCl was washed out with 4 volumes modified L15 �FM4-64, and cultures were allowed to recover for 20 min. The dyewas washed out with 5 volumes modified L15, and cultures wereimaged immediately using the Biorad confocal system describedabove. The blue and green lines of a Kr/Ar laser were used sequen-tially at the minimal required power for excitation of EGFP andFM4-64, respectively. Imaging was restricted to the initial segment ofthe motor neuron axon contacted by sensory neuron processes (Glanz-man et al. 1990), and z-stacks of 5 �m of optical sections at 0.5-�m

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step size were collected. Analysis was performed off-line using theMetamorph software.

Pharmacology

5-HT (Sigma-Aldrich, St. Louis, MO) was applied to cultures atthe indicated concentrations using a pipetteman. The PKA inhib-itor KT5720 (Calbiochem) was bath-applied at a final concentrationof 10 �M in 0.075% final DMSO concentration.

Computer simulations

To mathematically model the dynamics of transmitter mobilizationand release from sensory neuron terminals, we used a model of thesensorimotor synapse that was previously developed (Gingrich andByrne 1985, 1987) and confirmed by experimental results (Sugitaet al. 1997). This model was based on a system of coupled ordinarydifferential equations. The equations described the action potential-evoked calcium influx (FCa), which stimulated release of transmitterfrom a release pool, and the mobilization of transmitter from a feedingpool to replenish the release pool. Repetitive stimulation of themodeled terminal led to gradual depletion of the releasable transmitterand depression of transmitter release to steady state. Recovery oftransmitter release from depression occurred spontaneously with timeafter the end of stimulation. Decline of release to steady state withrepetitive stimulation and the spontaneous recovery after the end ofstimulation were accomplished through the process of transmittermobilization from the feeding pool. Mobilization of transmitter couldbe driven by a concentration gradient between the release and feedingpools (FVD), by elevation of cAMP in response to 5-HT treatment(FcAMP), or by the concentration of calcium in the cytosol. Calcium-dependent mobilization of transmitter, in turn, could take place in twodifferent ways, which varied in their sensitivity to calcium concen-tration and in their rate constants (fast: FF; slow: FS). The fastmobilization was primarily responsible for sustaining steady-staterelease during high frequencies of stimulation (i.e., 0.3–1 Hz),whereas the slow mobilization was primarily responsible for sustain-ing release at moderate frequencies of stimulation (0.03–0.3 Hz).Low-frequency release (0.01 Hz) was sustained by the concentration-driven mobilization.

The free parameters of the model by Gingrich and Byrne (1985)had been originally selected to match the empirically observed dy-namics of the sensorimotor synapse of the abdominal ganglion (Byrne1982). Because these dynamics are somewhat different from thedynamics of sensorimotor synapses in culture, we had to modify theparameters of the original model to fit the rates of synaptic depressionand spontaneous recovery that were observed in cultures, undercontrol conditions (see Fig. 4), as well as the effects of 5-HT ondedepression. The new parameters resulted from empirical observa-tions and allowed the model to fit the data obtained from controlcultures. The differences in the parameters between the original modelof Gingrich and Byrne and the one used in this study are presumablycaused by minor differences in synaptic morphology between culturesand ganglia. Despite these differences, results obtained from culturescan be generalized to ganglia, as previously shown (Dumitriu et al.2006; Ghirardi et al. 1992; Martin et al. 1997; Sharma et al. 2006;Sugita et al. 1992).

The time-course of cAMP concentration (in �M), which waselevated during 5-HT application, was calculated using the followingequation: 80,000 � (1 � e�t/40)2 � (e�t/19 � 0.0049 � e�t/1,000). ThecAMP dynamics of the model matched what was previously reportedfor 5-HT–treated Aplysia sensory neurons (Bacskai et al. 1993). Thescaling factor of 80,000 was necessary to achieve the experimentallyobserved maximal concentration of cAMP (5,000 �M) (Bacskai et al.1993). Optimization of the free model parameters to fit the experi-mental data were performed with the parameter optimization softwarePEST (freely available at http://www.sspa.com).

To simulate the control depression, recovery, and dedepression datafrom cultured synapses, parameter optimization with PEST indicatedthat the release rate constant (KR), the fast mobilization rate constant(KF), and the volume of the feeding pool (VF) were critical parametersto the fitting of the control depression kinetics. In addition, the rateconstants of calcium diffusion (KD) and of concentration-gradient–driven mobilization (KVD) were critical for the recovery from depres-sion. Finally, the cAMP-driven mobilization rate constant of storedtransmitter (KFC) was also adjusted to reproduce the empiricallyobserved synaptic dedepression by 5-HT (Fig. 5). Because slowmobilization (FS) did not contribute to this stimulation frequency, theslow rate constant (KS) was fixed to 0.0 in order to enhance theefficiency of the optimization. The final parameter changes fromGingrich and Byrne (1985, 1987) for the cultured synapse dynamics(in ASW and 5-HT) were as follows: KD, �70%; KR, �128%; KF,�103%; KVD, �50%; VF, �65%; KFC, �16%. After the repara-meterization process was complete, the error (average squared differ-ence) between experimental and simulated EPSP peak amplitudes hadbeen minimized to 2.65 (from 63.21 without reparameterization). Thesimulated effects of synapsin overexpression (see RESULTS) were notspecific to the new set of free parameter values that we used to fit thesynaptic dynamics of cultured synapses: when the original modelparameters (Gingrich and Byrne 1985, 1987) were used, the simulatedoverexpression of synapsin had similar effects on synaptic depressionand recovery as did the new, reparameterized model (data not shown).This observation suggests that the predicted effects of synapsinoverexpression do not depend on the specific dynamics of culturedsynapses.

R E S U L T S

Intracellular localization of overexpressed synapsin

To assess the role of Aplysia synapsin in homo- and het-erosynaptic plasticity, we overexpressed full-length wild-typesynapsin fused to a HA tag in sensory neurons co-cultured withmotor neurons. This approach allowed us to increase the intra-cellular concentration of synapsin, enhancing its functions. Incontrast to previous studies where mammalian synapsin wasintroduced in invertebrate systems (Dearborn et al. 1998; Hu-meau et al. 2001; Llinas et al. 1985), we overexpressed theAplysia synapsin in Aplysia sensory neurons, minimizing thedifferences from endogenous synapsin. The extent of overex-pression and the subcellular distribution of synapsin-HA weredetermined qualitatively using immunofluorescence (Angerset al. 2002). Sensory neurons were co-injected with a plasmidexpressing enhanced green fluorescent protein (EGFP) to allowfor visualization of sensory neuron processes and to control forthe efficiency of expression. Cells were analyzed if theyexpressed EGFP and if levels of HA expression were greaterthan background.

Injection of the synapsin-HA construct resulted in increasedlevels of synapsin compared with injection of empty vector(control; Fig. 1A). The subcellular distribution of synapsin-HAappeared punctate throughout the sensory neuron processes,and thus resembled the distribution of endogenous synapsin(cf. Fig. 1, A and B). Co-localization studies using the anti-synapsin and anti-HA antibodies indicated that the HA tag canserve as an effective marker of the overexpressed protein (Fig.1G). Using the anti-HA antibody, we found that synapsin-HAlocalized to presynaptic varicosities (Fig. 1F), defined as swell-ings along sensory neuron processes labeled with EGFP (Fig.1D). These varicosities stained positive for the synaptic vesiclemarker vesicle-associated membrane protein (VAMP) (Fig. 2,

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A and B), suggesting that they constituted putative sites ofsynaptic contacts. Collectively, these results show that synap-sin-HA was expressed in sensory neurons and localized atputative synapses, where it could affect synaptic transmissionand plasticity.

Synapsin regulates the functional size, but not the number,of vesicle clusters

Because synapsin interacts with phospholipids and varioussynaptic molecules (Hilfiker et al. 1999) and is implicated in theformation and maintenance of synaptic structures (Chin et al.1995; Ferreira et al. 1995; Lu et al. 1996), we examined thepossibility that synapsin overexpression may affect the number ofpresynaptic vesicle clusters. To this end, we monitored the num-ber of VAMP-containing varicosities per 100 �m neurite incontrol (Fig. 2D) and synapsin-overexpressing (Fig. 2E) sensoryneurons. VAMP is an integral synaptic vesicle protein that hasbeen used extensively as a marker of vesicle pools (Angers et al.2002; Ryan 2006) and whose levels are affected by manipulationsthat perturb vesicle clusters (Gitler et al. 2004; Rosahl et al. 1995).We found no significant effect of synapsin overexpression on thenumber of VAMP-containing varicosities (P � 0.79; Fig. 2F),suggesting that our manipulation did not affect the number ofpresynaptic vesicle clusters. These results, along with previousobservations that increasing synapsin levels does not have anydeleterious effects on synaptic integrity (Fiumara et al. 2001;Humeau et al. 2001; Menegon et al. 2006), suggest that theoverexpression approach is suitable for studying synapsin func-tion.

Even though the results of the VAMP immunofluorescenceexperiment would argue against an effect of synapsin-HA on thetotal vesicle complement, synapsin overexpression may still affectthe functional number of vesicles involved in neurotransmitterrelease. To study this hypothesis, we performed live-cell imagingexperiments using the lipophilic dye FM4-64 (Fernandez-Alfonsoand Ryan 2004; Gaffield and Betz 2006) (Fig. 3). Briefly, co-cultures that co-expressed EGFP and either synapsin-HA or con-trol were stimulated with KCl (100 mM, 5 min), which is knownto induce Ca2�-dependent release of vesicles (Kavalali et al.1999; Kuromi and Kidokoro 2003; Pyle et al. 1999) in thepresence of FM4-64. After the end of stimulation, co-cultureswere allowed to recover for 20 min before dye washout andimaging. The number of FM4-64 puncta that localized in presyn-aptic varicosities of control or synapsin-HA-overexpressing co-cultures was determined and normalized to 100 �m of sensoryneuron process. Comparison between control and synapsin-over-expressing co-cultures revealed a significant decrease in the num-ber of FM4-64 puncta in synapsin-overexpressing co-cultures(means � SE no. FM4-64 puncta/100 �m neurite: control: 1.96 �0.2, n � 4; synapsin-HA: 0.44 � 0.09, n � 4; t6 � 6.9, P �0.001). Because synapsin overexpression does not lead to mor-phological loss of synapses (see VAMP data above), these resultssuggested that synapsin regulates the functional size of vesicleclusters. Synapsin has not been implicated in the regulation ofendocytosis (Hilfiker et al. 1999; Ryan et al. 1996); therefore thisimpairment in dye uptake is probably caused by impaired vesiclemobilization and/or release. In control co-cultures, not all vari-cosities took up FM4-64 (Fig. 3, A–C). This observation could beexplained by technical limitations related to the loading protocol

A B

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F G H

FIG. 1. Synapsin overexpression in Aplysia sensorimotorcocultures. A and B: confocal images of co-cultures with asensory neuron (sn) injected either with empty expressionvector (control-HA) (A) or vector containing the synapsinsequence fused to the hemagglutinin tag (synapsin-HA) (B).Synapsin was detected with an anti-Aplysia synapsin antibody.A: in control cultures, synapsin distribution appears punctatealong neurites, as described previously (Angers et al. 2002).These puncta localize predominantly in varicosities, the pre-sumed sites of presynaptic specializations (Bailey and Chen1989). mn, postsynaptic motor neuron. B: levels of synapsinwere increased in a sensory neuron injected with pNEX�-apSyn-HA compared with control, showing successful expres-sion of the construct. C: synapsin-HA localization along sen-sory neuron processes, as indicated by immunostaining with ananti-HA antibody. The HA signal appears punctate throughoutthe sensory neuron processes. D: same sensory neuron ex-pressed EGFP, allowing for visualization of presynaptic vari-cosities. E: total synapsin (endogenous and overexpressed)staining using an anti-synapsin antibody. F: overlay of C and Dindicated that synapsin-HA localized in EGFP-filled varicosi-ties, showing that synapsin-HA distributed throughout the sen-sory neuron. G: overlay of C and E indicated that synapsin-HAco-localized with endogenous synapsin. Note endogenous syn-apsin puncta (marked by arrowheads) that did not co-localizewith synapsin-HA. H, overlay of D and E showed that thelabeled puncta in G did not contain EGFP. Therefore thesepuncta must belong to the motor neuron. Scale bar: 50 �m forA and B; 25 �m for C–H.




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and signal detection or by the existence of presynaptic silentsynapses (Kim et al. 2003).

Synapsin regulates basal synaptic strength andactivity-dependent synaptic depression

The interactions of synapsin with synaptic vesicles, actinand other cytoskeletal elements are thought to tether vesicles in

a “feeding pool” (Pieribone et al. 1995) (also referred to as“reserve pool”; Zucker and Regehr 2002), which is morpho-logically and functionally distinct from the “readily releasable”pool (RRP) of vesicles docked on the presynaptic active zone(Rosenmund and Stevens 1996; Schikorski and Stevens 2001).We used the term “feeding pool” as opposed to “reserve pool”to be consistent with the terminology used by Gingrich andByrne (1985), whose computational model was used in thisstudy. The feeding pool and RRP are thought to be in dynamicequilibrium (Gingrich and Byrne 1985; Murthy and Stevens1999; Rizzoli and Betz 2004, but see Gitler et al. 2004), withvesicles from the feeding pool being recruited during periodsof prolonged synaptic activity to sustain release (Brodin et al.1997; Neher 1998; Rizzoli and Betz 2004; Zucker and Regehr2002). If these hypotheses are correct and synapsin tetherssynaptic vesicles, overexpression of synapsin would be ex-pected to sequester vesicles in an enlarged feeding pool,perhaps at the expense of the readily releasable pool, thusimpeding their mobilization. This hypothesis is further sup-ported by our FM4-64 experiment, which showed an apparentimpairment of vesicle mobilization and/or fusion in synapsin-overexpressing cells (Fig. 3).

To further study the role of synapsin in synaptic transmis-sion and plasticity, we performed electrophysiological experi-ments using sensorimotor co-cultures that co-expressed EGFPand either synapsin-HA or control. To induce synaptic depres-sion, sensory neurons were stimulated with a 10-s train ofelectrical stimuli delivered at 1 Hz. This frequency was se-lected to challenge the release machinery, because it leads tosignificant homosynaptic depression (Byrne 1982) and partlydepletes synaptic vesicle pools (Armitage and Siegelbaum1998; Gingrich and Byrne 1985; Royer et al. 2000; Zhao andKlein 2004). Compared with empty vector-injected controls,sensory neurons expressing synapsin-HA displayed signifi-cantly reduced basal synaptic strength, assessed by the ampli-tude of the first EPSP in the train (Fig. 4, A and C; P � 0.05).This result could be explained by synapsin trapping vesicles inthe feeding pool, effectively reducing the size of the RRP. Thishypothesis would also be in agreement with the reducedFM4-64 loading of presynaptic boutons in synapsin-HA neu-rons.

To determine the specificity of the effect of synapsin over-expression on transmission, we analyzed the kinetics ofpostsynaptic responses from control and synapsin-overexpress-ing cultures. The two groups did not differ significantly in therise time of the first EPSP during the 1-Hz train (means � SE:control: 5.5 � 0.2 ms, n � 15; synapsin-HA: 5.51 � 0.19 ms,n � 13; t26 � 0.01, P � 0.99), suggesting that the duration oftransmitter release is probably not affected by synapsin over-expression (Gingrich et al. 1988; Hochner et al. 1986a,b). Thedecay kinetics and the half-maximal width of the responseswere also not significantly affected by synapsin manipulation[decay time (ms): control: 379.23 � 71.32, n � 8; synapsin-HA: 258.87 � 33.75, n � 10; t16 � 1.63, P � 0.12; decayslope (mV/ms): control: �0.04 � 0.01, n � 8; synapsin-HA:�0.04 � 0.01, n � 10; t16 � 0.11, P � 0.91; half-maximalwidth (ms): control: 46.22 � 2.29, n � 13; synapsin-HA:48.34 � 5.59, n � 12; t23 � 0.36, P � 0.72]. In some cases,the decay phase of the EPSP was very slow, and the decaykinetics could not be measured. These results suggest thatsynapsin overexpression probably did not have postsynaptic

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FIG. 2. Synapsin-HA co-localizes with VAMP and does not affect thenumber of presynaptic vesicle clusters. A: confocal section of EGFP-filledprocesses of a sensory neuron that was co-cultured with a motor neuron andco-expressed synapsin-HA. After 4 days of expression, the co-culture wasfixed and processed for immunofluorescence using anti-VAMP (B) and anti-HA (C) antibodies. Arrowheads point to presynaptic varicosities containingVAMP, a synaptic marker, and synapsin-HA. Note arrow pointing to a VAMPpunctum that did not co-localize with EGFP and therefore was probably in themotor neuron. This punctum did not co-localize with HA, supporting thespecificity of the anti-HA antibody. D–F: synapsin overexpression did notsignificantly affect the number of VAMP-containing presynaptic varicosities.D and E: sensory neurons expressing EGFP and immunostained for VAMP.Arrowheads point to VAMP puncta in EGFP-filled presynaptic varicosities ofa control-HA sensory neuron (D) or of a synapsin-HA-expressing sensoryneuron (E). F: synapsin overexpression did not affect the number of VAMP-containing presynaptic varicosities (means � SE number of varicosities/100�m neurite: control: 1.08 � 0.2; synapsin-HA: 1.16 � 0.23; t14 � 0.27, P �0.79). Scale bar: 50 �m.




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effects (Johnston and Wu 1995). In agreement with the effectof synapsin overexpression on basal synaptic strength (Fig.4C), there was a significant decrease in the rising slope of thefirst EPSP (control: 4.65 � 0.54 mV/ms, n � 15; synapsin-HA:3.15 � 0.56 mV/ms, n � 13; t26 � 1.93, P � 0.03). A decreasein the rising slope of the EPSP would be expected if the size ofthe releasable pool was reduced (Gingrich et al. 1988). Theseresults suggest that synapsin predominantly regulates the rateof transmitter release.

Synapsin-HA–expressing neurons exhibited more pro-nounced depression than controls (Fig. 4B). The depressionratio between the second and the first EPSP was significantlysmaller in the synapsin-HA group (P � 0.01; Fig. 4D). More-

over, the steady-state transmission was significantly decreasedin the synapsin-HA group (P � 0.001; Fig. 4E), expressed asthe amplitude of the steady state EPSP (average of last 3 EPSPsin the train) normalized to the first EPSP. These results wereconsistent with those obtained in other systems in which apresynaptic increase in synapsin concentration inhibited re-lease (Hackett et al. 1990; Lin et al. 1990; Llinas et al. 1985,1991; Nichols et al. 1992; but see Humeau et al. 2001). Thisinhibition of release could be explained by assuming thatincreased intraterminal concentration of synapsin tetheredmore vesicles away from the RRP and impaired their avail-ability and mobilization, resulting in reduced synaptic efficacyand increased depression.

A B C

D E F

FIG. 3. Synapsin negatively regulates loading of synaptic vesicleswith a lipophilic dye. A: control sensorimotor co-culture expressingEGFP presynaptically. B: stimulation of the same co-culture withKCl-induced uptake of FM4-64. C: FM4-64 puncta localized predom-inantly at presynaptic varicosities (arrowheads). D: different co-culture, injected with pNEX�-apSyn-HA, also expressing EGFP pre-synaptically. E: stimulation of the co-culture in D with KCl-induceduptake of FM4-64. F: fewer FM4-64 puncta were observed in pre-synaptic varicosities (arrowheads), suggesting that vesicle mobiliza-tion and/or fusion was impaired. Scale bar: 75 �m.

FIG. 4. Overexpression of synapsin de-creases basal synaptic strength and enhancessynaptic depression. A: representative tracesof excitatory postsynaptic potentials (EPSPs)from control or synapsin-HA-expressing co-cultures during a train of 10 stimuli at 1 Hz.B: cumulative data from control or synapsin-HA–expressing co-cultures showed that over-expression of synapsin enhanced homosynap-tic depression. C: overexpression of synapsinsignificantly reduced basal synaptic strength(mean amplitude � SE: control: 29.1 � 2.4mV, n � 19; synapsin: 20.9 � 2.6 mV, n � 16;t33 � 2.3, P � 0.05). D: synapsin overexpres-sion significantly enhanced depression startingas early as the second stimulus [mean percentratio (2nd/1st EPSP) � SE: control: 40.6 �2.89, n � 11; synapsin: 27.6 � 2.95, n � 15;t24 � 3.07, P � 0 0.01]. E: steady-state de-pression was also significantly enhanced insynapsin-HA–expressing neurons [mean per-cent steady state � SE: control: 37.78 � 2.5,n � 11; synapsin: 25.23 � 1.8, n � 14; t23 �4.11, P � 0.001]. Successful expression ofsynapsin-HA was confirmed immunocyto-chemically in all sensory neurons.




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Overexpression of synapsin enhances5-HT–induced dedepression

We next examined the potential involvement of synapsin inheterosynaptic plasticity and, in particular, in 5-HT–inducedfacilitation of previously depressed transmission (dedepres-sion), a form of heterosynaptic plasticity. 5-HT, which inducesfacilitation of release at the sensorimotor synapse, activateskinases that phosphorylate synapsin causing its subcellularredistribution (Angers et al. 2002). This redistribution could beexplained by the dissociation of synapsin from synaptic vesi-cles, which occurs in response to 5-HT (Fioravante and Byrne,unpublished observations). Thus vesicles would become avail-able for release, enhancing synaptic transmission. If overex-pression of synapsin resulted in increased mobilization, wewould predict that, after 5-HT, the synapsin-overexpressingsensory neurons would show enhanced dedepression comparedwith controls.

5-HT was applied immediately after the 10th EPSP andremained in the bath for 5 min, a protocol typically used toinduce STF (Bartsch et al. 1998, 1995; Martin et al. 1997;Ormond et al. 2004; Phares et al. 2003; Sugita et al. 1997). Atthe end of the incubation period, a single stimulus was deliv-ered to assess the magnitude of dedepression (Fig. 5A). In all

co-cultures, 5-HT induced dedepression of the 11th EPSP inrelation to the steady state of the previously induced depression(P � 0.001). However, the synapsin-HA group displayedsignificantly more dedepression than control (P � 0.05; Fig.5B), suggesting that more vesicles were mobilized for releaseafter 5-HT. This result was not caused by a “ceiling” effect inthe control group because EPSPs can have values larger thanthe average amplitude of the control group, which was 38.6 �5.0 (SE) mV (Fioravante and Byrne, unpublished observa-tions).

Overexpression of synapsin does not affect spontaneousrecovery of release

The potentiating effect of synapsin overexpression on 5-HT–induced dedepression could be partly attributed to increasedrecovery of transmission, mediated by accumulation of intra-cellular calcium with repetitive activity. In that case, synapsinoverexpression would be expected to affect spontaneous re-covery of transmission. To address this question, we examinedthe recovery of transmission from depression in a subset ofcontrol and synapsin-overexpressing co-cultures after treat-ment with ASW (vehicle control) in lieu of 5-HT (Fig. 5C). Nodifference was observed between the two groups (P � 0.55;Fig. 5D), suggesting that the effect of synapsin overexpressionon 5-HT–induced dedepression was not due to a covert effecton spontaneous recovery.

PKA-dependent redistribution of synapsin-HA

The enhanced dedepression observed in the synapsin-HAgroup could be explained by increased availability of vesiclesto be mobilized from the feeding pool by 5-HT. For thisincreased availability to occur, inhibitory constraints such assynapsin tethering of vesicles must be removed. In the case ofendogenous synapsin, 5-HT induces its phosphorylation, dis-sociation from synaptic vesicles, and subcellular redistribution(Angers et al. 2002). Does synapsin-HA also redistribute after5-HT? To answer this question, synapsin-HA–expressing neuronswere treated with 5-HT or ASW (control) for 5 min, fixed, andprocessed with an anti-HA antibody for immunofluorescenceanalysis. After control treatment, synapsin-HA staining appearedpunctate throughout sensory neuron processes (Fig. 6, B and C),as observed previously (Figs. 1 and 2). However, fewer syn-apsin-HA puncta could be observed after 5-HT treatment (Fig.6E). High-magnification confocal images of sensory neuronprocesses revealed that synapsin-HA displayed a more diffusestaining pattern along the processes of sensory neurons (Fig.6F), which could result from its dissociation from synapticvesicles.

We previously reported that the 5-HT–induced redistributionof endogenous synapsin depends critically on PKA (Angerset al. 2002). To examine whether the overexpressed chimericsynapsin-HA is subjected to similar regulatory control byPKA, we performed immunofluorescence experiments usingan anti-HA primary antibody and an inhibitor of the PKAcascade (Fig. 7). We predicted that inhibition of PKA wouldblock 5-HT–induced redistribution of synapsin-HA. Briefly,sensory neurons were pretreated with the PKA inhibitorKT5720 or vehicle (DMSO) for 1 h, followed by 5-minexposure to 5-HT or ASW (control). Cells were fixed imme-

FIG. 5. Overexpression of synapsin potentiates 5-HT–induced dedepres-sion of synaptic transmission without affecting spontaneous recovery.A: representative traces of the 10th (last EPSP in the 1-Hz train) and 11th (1stEPSP after 5-min treatment with 5-HT) postsynaptic responses in control andsynapsin-overexpressing co-cultures. B: cumulative data showing potentiatingeffect of synapsin overexpression on 5-HT–induced dedepression (percentdedepression � SE: control: 351.8 � 39.9, n � 7; synapsin: 591.9 � 95.5, n �8; t13 � 2.20, P � 0.05). C: representative traces of the 10th (last EPSP in the1-Hz train) and 11th (1st EPSP after 5-min rest period) postsynaptic responsesin control and synapsin-overexpressing co-cultures. D: cumulative data show-ing that synapsin overexpression did not significantly alter the magnitude ofspontaneous recovery of transmission in vehicle-treated [artificial seawater(ASW)] co-cultures (percent recovery � SE: control: 205.4 � 24.0, n � 4;synapsin: 245.9 � 59.4, n � 4; t6 � 0.63, P � 0.55).




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diately after the end of 5-HT treatment and were processed forimmunofluorescence analysis. One-way ANOVA followed bya Tukey post hoc test (Zar 1999) showed that KT5720 blockedthe 5-HT–induced redistribution of synapsin-HA (P � 0.05)without significantly affecting the basal number of synap-sin-HA puncta (DMSO: 7.81 � 0.89 puncta/100 �m neurite,n � 5; KT5720: 5.67 � 0.75 puncta/100 �m neurite, n � 6;q � 3.06, P � 0.05), suggesting that the overexpressedchimera is regulated by PKA in the same way as the endoge-nous protein. Because PKA is at least partly involved in5-HT–induced dedepression (Dumitriu et al. 2006; Ghirardiet al. 1992), these results suggest that 5-HT–activated kinases,including PKA, phosphorylate and promote the dispersion ofboth endogenous and overexpressed synapsin-HA. This pro-cess could increase vesicle mobilization, potentiating synapticfacilitation.

Mathematical model of the Aplysia sensorimotor synapsesupports the role of synapsin in vesicle mobilization

The experimental results presented above support a role forsynapsin in the regulation of vesicle availability and traffick-ing. To identify cellular processes that could be regulated bysynapsin and could result in the observed synaptic physiology,

we adopted a computational approach. Specifically, we used apreviously developed computational model of synaptic trans-mission in Aplysia (Gingrich and Byrne 1987) to fit theexperimental data and identify processes that could potentiallymediate the observed physiological effects.

The model (Fig. 8) consisted of a set of coupled ordinarydifferential equations that described activity-induced influx ofcalcium, regulation of intracellular calcium concentration, stor-age, trafficking, and release of synaptic vesicles. The effects of5-HT were mediated in the model through recruitment of anadditional vesicle mobilization process, which was cAMP-depen-dent. Using the parameterization software PEST, the model pa-rameters were optimized to fit the data of both the control andsynapsin-HA groups (see METHODS for details). The decrease inbasal synaptic strength, observed in the experimental data ofthe synapsin-HA group, could be reproduced in the model bydecreasing the volume of the release pool (VR) by 33%. Therewas a commensurate increase in the volume of the feeding poolby 3.3% to hold the total number of vesicles constant. With therelease and storage vesicle pools clamped to their new opti-mized values, the effect of synapsin on enhancing the rate ofactivity-induced synaptic depression was best reproduced by a63% reduction in the rate constant of the fast vesicle mobili-zation process (KF). Because of this reduction in KF and,consequently, in the rate at which vesicles were mobilizedfrom the feeding pool to the release pool, at the end of thestimulus train, the feeding pool was larger in the synapsin-HAgroup than control. On application of 5-HT, the cAMP mobi-lization process (FcAMP) acted on an enlarged feeding pool,thus leading to enhanced dedepression.

An alternative way to reproduce the synapsin-induced de-crease of basal synaptic strength was by reducing the releaserate constant (KR) by 54%, a manipulation that would presum-ably reflect a change in release probability. However, whensimulations were run with this reduced value of release rateconstant, the error between experimental and simulated EPSPpeak amplitudes increased more than six times (from 2.65 to17.12), suggesting that the effect of synapsin on basal releaseis probably caused by a change in the volume of the releasepool and not the probability of release. Collectively, the resultsof the computational simulations suggest that overexpressionof synapsin leads to a considerable reduction in the number ofreadily releasable vesicles and in the rate at which they arereplenished with activity, as well as a relatively small increasein the number of stored vesicles.

D I S C U S S I O N

In mammals, three genes have been identified thus far thatcode for five synapsin isoforms (Kao et al. 1999). Even thoughsynapsin I and II isoforms present some structural and func-tional differences, they are thought to be functionally redun-dant. Synapsin III, on the other hand, seems to play distinctregulatory roles in synaptic transmission and development(Feng et al. 2002; Ferreira et al. 2000). Numerous studies haveexamined the role of synapsin in transmission and plasticity(Chi et al. 2001, 2003; Gitler et al. 2004; Li et al. 1995; Rosahlet al. 1993, 1995; Ryan et al. 1996; Spillane et al. 1995).However, none of these studies investigated the potential involve-ment of synapsin in short-term heterosynaptic plasticity.

FIG. 6. Subcellular redistribution of synapsin-HA in sensorimotor co-cul-tures after 5-HT. A: EGFP-expressing sensory neuron (sn) co-cultured with amotor neuron (mn). B: sensory neuron co-expresses synapsin-HA, visualizedusing an anti-HA antibody. C: high-magnification, pseudocolored image illus-trating punctate distribution of synapsin-HA along processes under basalconditions. D: a different sensory neuron in co-culture expressing both EGFPand synapsin-HA was treated with a brief pulse of 5-HT and fixed immediatelyafter the end of treatment. E: after 5-HT, fewer synapsin-HA puncta weredetected along the sensory neuron processes. F: high-magnification, pseudo-colored image showing dissipation of synapsin-HA puncta, suggesting thatsynapsin-HA redistributes after 5-HT, as does endogenous synapsin (Angerset al. 2002). Scale bar: 50 �m for A, B, D, and E; 10 �m for C and F.




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To delineate the role of synapsin in heterosynaptic facilita-tion, we took advantage of the glutamatergic sensorimotorsynapse in Aplysia, which exhibits homosynaptic plasticity andis heterosynaptically regulated by 5-HT (Antzoulatos and Byrne2004; Byrne and Kandel 1996). A single gene codes forAplysia synapsin, which shares many important similaritieswith its mammalian homologues in terms of domain arrange-ment, sequence conservation of the central C domain, and highsequence homology of the N-terminal PKA phosphorylationsite (Angers et al. 2002). We found that overexpression ofsynapsin in sensory neurons negatively regulated vesicularuptake of FM4-64 dye (Fig. 3), impaired basal synapticstrength, and increased synaptic depression (Fig. 4). Moreover,synapsin overexpression potentiated 5-HT–induced recoveryfrom depression without affecting spontaneous recovery oftransmission (Fig. 5). Mathematical simulations indicated thatthe effects of synapsin on release and plasticity could beaccounted for by a change in the size of the feeding and readilyreleasable pools and a decrease in Ca2�-dependent vesiclemobilization (Fig. 8).

In resting synaptic terminals, synapsin associates predomi-nantly with vesicles clustered away from the RRP (Bloom et al.2003; Pieribone et al. 1995), where it could regulate vesicleavailability and mobilization. In our study, overexpressed syn-apsin localized in presynaptic varicosities containing the syn-aptic vesicle marker VAMP (Fig. 2, A–C), suggesting thatsynapsin-HA was positioned appropriately with the potential toaffect vesicle trafficking. Overexpression of synapsin did notseem to affect the number of presynaptic vesicle clusters (Fig.2, D–F), but it did decrease their functional size, indicated bydecreased vesicular uptake of a lipophilic dye (Fig. 3). Thetransient expression of synapsin-HA over a period of 4 dayscould have resulted in progressive trapping of vesicles in thefeeding pool, effectively reducing the size of the RRP. Thisreduction in RRP could explain the reduced FM4-64 uptake insynapsin-HA neurons (Fig. 3). Moreover, in our simulations,this reduction could adequately explain the effect of synapsinon basal synaptic strength (Fig. 8). Alternatively, based on asimple binomial model of release, the effect on the first EPSP

could arise from a decrease in release probability. However,such a change would be expected to result in less depressionduring the 1-Hz train caused by a surplus of vesicles, whichwas not observed in our study. Indeed, reducing the releaseprobability in our simulations increased the error in fitting theexperimental data.

The effect of synapsin on basal transmission is controversial.In some systems, interference with synapsin did not affectbasal synaptic strength (Gitler et al. 2004; Humeau et al. 2001;Li et al. 1995; Menegon et al. 2006; Rosahl et al. 1995; Ryanet al. 1996), whereas in others, synapsin manipulation inhibitedbasal release (Hilfiker et al. 1998; Llinas et al. 1985, 1991).This discrepancy could be attributed to synapse-specific dif-ferences (Brodin et al. 1997; Gitler et al. 2004), such as initialrelease probability and level of tonic activation of variouskinases. The latter possibility is particularly interesting becausethe biochemical properties of synapsin are modulated by phos-phorylation (Bahler and Greengard 1987; Bonanomi et al.2005; Chi et al. 2001, 2003; Hilfiker et al. 1999; Jovanovicet al. 1996). PKA-mediated phosphorylation of synapsin reg-ulates its association with synaptic vesicles and its subcellulardistribution in several systems (Angers et al. 2002; Fiumaraet al. 2004; Hosaka et al. 1999; Menegon et al. 2006) (also seeFig. 7). Moreover, in Aplysia basal phosphorylation of syn-apsin by ERK MAPK is required for the proper localization ofsynapsin on vesicles (Angers et al. 2002). If basal levels ofkinase activity are synapse-specific (which seems to be the caseat least for PKA, Hilfiker et al. 2001), experimental perturba-tion of synapsin levels might be expected to have differentialeffects on basal transmission, depending on the basal phos-phorylation level of synapsin.

In contrast to the uncertainty around the involvement ofsynapsin in basal transmission, its role in sustaining releaseover a range of stimulation frequencies is better documented(Hilfiker et al. 1999). In general, genetic deletion (Gitler et al.2004; Rosahl et al. 1995) or antibody-mediated neutralization(Humeau et al. 2001; Pieribone et al. 1995) of synapsinenhances synaptic depression, as does injection of recombinantsynapsin peptides or protein (Hilfiker et al. 2005; Lin et al.

A B C

D E F

FIG. 7. The 5-HT–induced redistribution of synapsin-HArequires protein kinas A (PKA). A: synapsin-HA exhibits thecharacteristic punctate distribution along neurite processes, inthe presence of DMSO (vehicle). B: brief application of 5-HT(5 min, 1 �M) results in dispersion of synapsin-HA puncta ina different sensory neuron, probably because of the transloca-tion of synapsin-HA from vesicles to cytoplasm. C: 5-HT–induced a significant reduction in the number of synapsin-HApuncta (means � SE # puncta/100 �m neurite: DMSO: 7.81 �0.89, n � 5; DMSO � 5-HT: 2.98 � 0.39, n � 7; q � 7.12,P � 0.05, ANOVA followed by Tukey post hoc tests) (Zar1999), confirming previous observations (Angers et al. 2002).D and E: synapsin-HA puncta in a sensory neuron treated withthe PKA inhibitor KT5720 alone (D) or KT5720 � 5-HT (E).F: pretreatment with KT5720 blocked the 5-HT–induced dis-persion of synapsin-HA puncta (means � SE # puncta/100 �mneurite: KT: 5.67 � 0.75, n � 6; KT �5HT: 5.34 � 0.64, n �7, q � 0.51, P � 0.05), suggesting that PKA is required for theactions of 5-HT on both endogenous synapsin (Angers et al.2002) and synapsin-HA. Scale bar: 50 �m.




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1990; Llinas et al. 1985) (but see Dearborn et al. 1998;Humeau et al. 2001). In the triple synapsin knockout, depres-sion of excitatory transmission during trains of stimuli wasincreased threefold (Gitler et al. 2004). It should be noted thatin the study of Gitler et al., the effect of synapsin on depressionwas observed late during the 10-Hz train, suggesting thatsteady-state release is synapsin-sensitive. In our study, over-expression of synapsin also increased the extent of homosyn-aptic depression (Fig. 3E), whose mechanisms are predomi-nantly presynaptic (Antzoulatos et al. 2003; Armitage andSiegelbaum 1998), from as early as the second response. At thesensorimotor synapse, the second response was already de-

pressed by 60% under control conditions (also see Chin et al.2002). Because the stimulation frequencies with which the twosystems were challenged are different, direct comparison of thetwo studies cannot be made. The increased depression ob-served at synapsin-overexpressing synapses can be explained ifoverexpressed synapsin traps additional vesicles and preventsthem from being mobilized to the RRP, effectively decreasingthe size of the RRP and increasing the size of the feeding pool.

If the feeding pool is increased by overexpressed synapsin,one might expect that synaptic depression would actually bedecreased during synaptic stimulation. The additional synapsinmolecules would be phosphorylated, freeing the trapped vesi-cles to be mobilized for release. Interestingly, such an enhance-ment of release was observed in the crayfish (Dearborn et al.1998), and also in mice with constitutively active H-ras, whichresults in increased phospho-synapsin (Kushner et al. 2005). Inour study, synapsin overexpression increased depression, sug-gesting that calcium accumulation during the stimulus trainwas not sufficient to regulate the interactions of synapsin withvesicles. This conclusion is also supported by the observationthat synapsin does not redistribute after 1-Hz stimulation (An-gers et al. 2002).

In our study, the 5-HT–induced facilitation of moderatelydepressed transmission (dedepression) was enhanced in syn-apsin-overexpressing neurons (Fig. 5). According to our sim-ulations, this enhancement could result from enhanced mobi-lization of synaptic vesicles, which in turn stems from synap-sin-mediated enlargement of the feeding pool. Phosphorylationof synapsin by kinases such as PKA would allow vesicles totransition from the feeding to the release pool, enhancingdedepression. Indeed, results from previous studies suggestedthat PKA is involved in facilitation of moderately depressedtransmission (Dumitriu et al. 2006; Ghirardi et al. 1992).Consequently, one would predict that inhibition of PKA wouldimpair synapsin-dependent enhancement of dedepression.

In addition to PKA, protein kinase C (PKC) has also beenimplicated in facilitation of depressed synapses (Dumitriu et al.2006; Ghirardi et al. 1992; Manseau et al. 2001). Comparedwith PKA, the contribution of PKC to synaptic facilitationbecomes more important as depression becomes more pro-nounced (Ghirardi et al. 1992). Because synapsin does notseem to be significantly phosphorylated by PKC despite themultiple putative PKC phosphorylation sites (Angers et al.2002; Fiumara et al. 2004), we would predict that synapsinoverexpression would not affect facilitation of highly de-pressed synapses, which depends on PKC, but it would en-hance facilitation of nondepressed synapses, which dependspredominantly on PKA. Extending this line of reasoning,synaptic targets other than synapsin must be important forfacilitation of heavily depressed synapses. As indicated by thework of Houeland et al. (2007), SNAP-25 and its phosphory-lation by PKC is at least one other important modulator ofshort-term plasticity at the sensorimotor synapse.

The role of synapsin on recovery of transmitter release fromdepression is controversial. At hippocampal synapses, deletionof the synapsin I gene impaired recovery after 20- and 50-Hzstimulation (Li et al. 1995) but not after 10 Hz (Ryan et al.1996). At the calyx of Held, deletion of synapsin I and II didnot affect recovery after strong depolarization that depleted theRRP (Sun et al. 2006). Some of this uncertainty could beattributed to synapse-specific properties and the differences in

A

B

C D

FIG. 8. A mathematical model of transmitter mobilization and release fromsensory neuron terminals can capture observed effects of synapsin overexpres-sion. A: schematic diagram of model, which was adapted from Gingrich andByrne (1987). Text in red indicates elements modified to model effects ofsynapsin overexpression. Dashed red lines indicate changes in volumes offeeding and release pools after synapsin overexpression. B: repetitive stimu-lation of the modeled sensory neuron terminal led to homosynaptic depression(green and red lines), as was observed empirically (black and gray lines).Model could adequately reproduce observed effects of overexpressing synap-sin on rate of activity-dependent synaptic depression. C: extent to whichmodeled terminal recovered from depression shown in B after 5 min ofquiescence was very similar to the empirical observations, both for the controlcondition (left) and the synapsin overexpression condition (right). D: as withspontaneous recovery data shown in C, the mathematical model could ade-quately reproduce extent of 5-HT–induced dedepression, as well as potentia-tion of 5-HT effects by overexpression of synapsin.




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stimulation protocols, which could trigger different recoverymechanisms. At the sensorimotor synapse, synapsin did notaffect spontaneous recovery (Fig. 5). A similar observation wasmade in the crayfish after 3-Hz stimulation (Dearborn et al.1998). This result suggests that, in several systems includingAplysia, the regulation of vesicles responsible for spontaneousrecovery of release is synapsin-independent. In our model, spon-taneous replenishment of the release pool occurs through a vesicleflux driven by a concentration gradient between feeding andreadily releasable pools. We propose that a similar synapsin-insensitive process operates at the sensory-motor synapse andrestores the RRP. This hypothesis is in agreement with theunchanged endocytosis observed in synapsin I knockout mice(Ryan et al. 1996).

In summary, we showed for the first time that synapsincontributes to not only homosynaptic but also heterosynapticplasticity in Aplysia. Given the importance of heterosynapticmodulation in both the invertebrate and vertebrate nervoussystems (Bailey et al. 2000; Marder 2006), our study raises theinteresting possibility that synapsin may be an important me-diator of the actions of neuromodulators in other systems.

A C K N O W L E D G M E N T S

We thank E. Antzoulatos, D. Baxter, and M. Byrne for help with computersimulations, E. Kartikaningrum and J. Liu for preparing the cultures, H.Vishwasrao for advice on the FM4-64 experiment, N. Yadav for help withsome of the data analysis, B. K. Kaang of Seoul National University for thepNEX�-HA vector, and K. Martin of UCLA for the anti-VAMP antibody.

G R A N T S

This work was supported by National Institute of Neurological Disordersand Stroke Grants R01 NS-19895 and P01 NS-38310.

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