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Behavioral/Systems/Cognitive

A Novel Postsynaptic Mechanism for Heterosynaptic Sharingof Short-Term Plasticity

Kathryn J. Reissner,1 Lu Pu,1 Joanna H. Schaffhausen,3 Heather D. Boyle,1 Ian F. Smith,1 Ian Parker,1

and Thomas J. Carew1,2

1Department of Neurobiology and Behavior and 2Center for the Neurobiology of Learning and Memory, University of California, Irvine, Irvine, California92697, and 3ABC News Medical Unit, Needham, Massachusetts 02494

Postsynaptic release of Ca 2� from intracellular stores is an important means of cellular signaling that mediates numerous forms ofsynaptic plasticity. Previous studies have identified a postsynaptic intracellular Ca 2� requirement for a form of short-term plasticity,post-tetanic potentiation (PTP) at sensory neuron (SN)–motor neuron synapses in Aplysia. Here, we show that postsynaptic IP3-mediated Ca 2� release in response to a presynaptic tetanus in an SN that induces PTP can confer transient plasticity onto a neighboringSN synapse receiving subthreshold activation. This heterosynaptic sharing of plasticity represents a dynamic, short-term synapticenhancement of synaptic inputs onto a common postsynaptic target. Heterosynaptic sharing is blocked by postsynaptic disruption ofCa 2�- and IP3-mediated signaling, and, conversely, it is mimicked by postsynaptic injection of nonhydrolyzable IP3 , and by photolysis ofcaged IP3 in the MN. The molecular mechanism for heterosynaptic sharing involves metabotropic glutamate receptors and Homer-dependent interactions, indicating that Homer can facilitate the integration of Ca 2�-dependent plasticity at neighboring postsynapticsites and provides a postsynaptic mechanism for the spread of plasticity induced by presynaptic activation. Our results support a modelin which postsynaptic summation of IP3 signals from suprathreshold and subthreshold inputs results in molecular coincidence detectionthat gives rise to a novel form of heterosynaptic plasticity.

IntroductionSynaptic plasticity exists in a wide range of temporal domains.Activity-dependent short-term plasticity, lasting on the order ofseconds to minutes, includes paired-pulse facilitation, paired-pulse depression, and post-tetanic potentiation (PTP). Whereaslong-lasting forms of plasticity, such as long-term potentiation(LTP) and long-term depression (LTD), are generally consideredto provide a means of information storage in neurons, theseshort-term forms of plasticity are critical for dynamic, moment-to-moment adjustments of synaptic strength during online pro-cessing of neural information. At most synapses, PTP is typicallythought to be mediated solely by presynaptic mechanisms(Zucker and Regehr, 2002). However, a major component of PTPat sensory neuron–motor neuron (SN-MN) synapses in Aplysiarequires postsynaptic Ca 2� (Bao et al., 1997; Schaffhausen et al.,2001; Jin and Hawkins, 2003). The fact that postsynaptic Ca 2�

contributes significantly to PTP at SN-MN synapses in Aplysialed us to propose that the induction of PTP at one synapse mighttransiently facilitate induction of plasticity at neighboringsynapses onto the same MN via shared postsynaptic Ca 2�-dependent mechanisms (Schaffhausen et al., 2001). Here, we

show that heterosynaptic sharing of PTP can indeed occur whenan input sufficient for PTP induction (20 Hz activation of an SN)is paired with a subthreshold input to a neighboring SN (acti-vated at 7 Hz). The mechanism for heterosynaptic sharing isdependent upon postsynaptic Ca 2�- and Homer-mediated in-teractions. Heterosynaptic sharing is blocked by postsynapticinjection of BAPTA, as well as inhibitors of intracellular Ca 2�

release, including heparin, thapsigargin, and caffeine. Moreover,heterosynaptic sharing is facilitated by postsynaptic injection ofnonhydrolyzable IP3 and by photoactivated caged IP3. Finally,heterosynaptic sharing is blocked by disruption of Homer-mediated protein interactions following postsynaptic injection ofa synthetic peptide containing the Homer binding sequence ofthe Aplysia IP3 receptor (IP3R).

These findings provide evidence for a novel form of short-term heterosynaptic plasticity that arises from postsynapticinteractions between presynaptic inputs, and indicates thatactivity-dependent spread of plasticity is not limited to thelong-term time domains. Moreover, this unique form of plastic-ity provides a mechanism for dynamic, temporally restricted am-plification of synaptic inputs onto a common postsynaptic target.

Materials and MethodsGeneral methods. Wild-caught Aplysia californica (120 –250 g) were ob-tained commercially (Marinus). Animals were anesthetized by injectionof isotonic MgCl2, and isolated ganglia were pinned and desheathed indishes containing 1:1 MgCl2/artificial seawater (ASW) (containing, inmM: 460 NaCl, 55 MgCl2, 11 CaCl2 10 KCl, and 10 Tris, pH 7.6).

Protein quantification was performed using the BCA Protein Assay(Pierce). Western blots were performed by electrophoresis using the

Received Sept. 25, 2009; revised March 16, 2010; accepted April 12, 2010.This work was supported by National Institutes of Health Grant R01 MH14-10183 and National Science Founda-

tion Grant IOB-0444762 (T.J.C.). We thank Kelsey Martin and Wayne Sossin for kind gifts of Aplysia SN and CNSlibraries. We thank Thomas Fischer, John Guzowski, Michael Sutton, Robert Zucker, and members of the Carewlaboratory for constructive comments on an earlier version of the manuscript.

Correspondence should be addressed to Thomas J. Carew at the above address. E-mail: [email protected]:10.1523/JNEUROSCI.4767-09.2010

Copyright © 2010 the authors 0270-6474/10/308797-10$15.00/0

The Journal of Neuroscience, June 30, 2010 • 30(26):8797– 8806 • 8797

Invitrogen NuPAGE gel system. Blotting con-ditions were varied depending on primary an-tibody used. Blocking was performed in 3%Blotto in TBS with 0.1% Tween 20, and blotswere probed overnight in primary antibody inblocking solution, followed by 1 h at roomtemperature in HRP-conjugated secondary an-tibody at 1:2500. Blots were developed usingthe ECL Western Blotting Analysis System byGE Healthcare.

Cloning. For use in degenerate reverse tran-scriptase (RT)-PCR, whole RNA was preparedfrom pleural and pedal ganglia using Trizol Re-agent (Invitrogen). Initial forward and reversedegenerate Homer primer sequences wereCAYGTNTTYCANATHGAYCC and CCAYT-GNCCRAAMTTYTG, respectively. SubsequentRT-PCR was performed using a separatedegenerate forward primer (ATGGGNGAR-CARTTYATHTT) and a gene-specific reverseprimer from within the initially identified se-quence. PCR was then performed on Aplysiasensory neuron or CNS libraries [generouslyprovided by Kelsey Martin (University of Cal-ifornia, Los Angeles, CA) and Wayne Sossin(McGill University, Montreal, Canada), re-spectively] with different combinations of for-ward gene-specific primers and T3 or T7 vectorprimers. Typically, PCR products were clonedinto the Invitrogen pCR2.1 sequencing vectorand submitted for DNA sequencing (LagunaScientific). This approach yielded a total of 353aa. While a termination codon was not identi-fied within the library screen, alignment ofApHomer with Drosophila and vertebrateHomers indicated that the sequence obtainedlikely represented �95% of the coding region ofApHomer. The translation start site was con-firmed using 5� rapid amplification of cDNAends with the GeneRacer kit from Invitrogen, us-ing pleural/pedal RNA as starting material.

Cloning of the IP3 receptor in Aplysia wasperformed by degenerate RT-PCR from pleu-ral/pedal ganglia and PCR of an Aplysia CNSlibrary using gene-specific and vector primers,as described for ApHomer. Initial degenerateforward and reverse primers were CCICC-NAARAARTTYMG and AAWTTYTCYTGY-TCNGCRTG. DNA sequence obtained fromthe initial fragment of Aplysia cDNA was usedto make gene-specific primers, which werethen coupled to vector primers for use in am-plification from the Aplysia CNS library. Forexpression in bacteria, the first 258 aa ofApIP3R were cloned into the pENTR vectorfrom Invitrogen. Following recombination into the pDEST15 glutathi-one S-transferase (GST) expression vector, recombinant protein was ex-pressed in BL21(DE3)Star cells. Bacterial pellets were resuspended inlysis buffer (PBS � 0.5% Triton X-100, 10% glycerol, 10 mM imidizole) andsonicated. Soluble protein was recovered by centrifugation and coupled toglutathione agarose (Pierce). Constructs for the expression of recombinantApHomer were prepared using the EcoRI and NotI sites in the pGEX4T-3vector (GE Healthcare), containing either amino acids 1–335 or 163–335.Following expression, bacterial cell pellets were resuspended and lysed bysonication in a smiliar fashion. The accuracy of all constructs was confirmedby DNA sequencing (Laguna Scientific, Cogenics).

Tissue distribution and ApHomer expression. Regulation of Homer ex-pression was investigated using four separate methods: injury, in vivo5-HT, KCl depolarization, and tailshock. To look at the effect of injury,

the animal was anesthetized, and the pleural and pedal ganglia wereimmediately excised into Trizol for RNA preparation. One hour later, theother half was removed and processed identically. For in vivo 5-HT treat-ment, animals were placed in 6 L of ASW or 6 L of ASW containing 250�M 5-HT for 1.5 h at 18°C. After 1.5 h, animals were either taken for tissueor moved to ASW. Tailshock was performed as previously described(Sharma et al., 2003). Five shocks were given to one side of the tail with a10 min intershock interval. The unshocked side served as a control to thatanimal. Pleural and pedal ganglia were dissected into Trizol. In all cases,reverse transcription was performed using 3.0 �g of total RNA, and 1.0 �lof cDNA was used in the PCR. Cycle number was optimized for eachprimer set, to ensure amplification was kept within the linear range forcomparison between control and experimental groups. In all cases, his-tone h4 was used as a negative control, and the immediate early gene

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Figure 1. Induction of PTP can be shared with a neighboring synapse receiving subthreshold activation. A, Inset, Experimentaldesign: two presynaptic SNs are identified that synapse onto the same postsynaptic MN. The 20 and 7 Hz trains are deliveredintracellularly to the two SNs, while postsynaptic EPSPs are recorded in the MN. Representative traces are shown from synapsesreceiving 7 Hz alone (A1, top left), 20 Hz alone (A1, bottom left), or 7 Hz paired with 20 Hz (A2, top and bottom, respectively).B, Histogram of results indicating that an SN receiving a 7 Hz train acquires the ability to induce heterosynaptic sharing, but onlywhen paired with a 20 Hz train to a neighboring SN. Previously, we have shown that small synapses (�6.2 mV) exhibit anenhanced form of PTP, which is more robust than the magnitude of PTP observed across synapses of all sizes (�160 vs 285%)(Schaffhausen et al., 2001). Here, we observed a similar result, in which 20 Hz PTP varied from �200%, when tested among smalland large synapses alike, to �300%, when experiments were restricted to small synapses (compare Fig. 1 B and Figs. 1C, 4 A, and7C). Interestingly, the magnitude of the shared 7 Hz PTP is consistently �200%, regardless of the degree of potentiation at the“donor” SN. *p � 0.05, n � 4 –7 per group. C, Top, schematic diagram of experimental protocol. Pretests of both SNs are taken�100 ms apart, 1 min before the onset of the overlapping 7 and 20 Hz trains (each 2 s in duration). Post-test measurements aretypically made 10 s after the offset of the trains (e.g., as in 2 B). For time course experiments, a single post-test measurement wasmade for each synapse at 1 s, 1 min, or 10 min after the overlapping trains. As shown in C (bottom), while 20 Hz PTP remainssignificantly above baseline at 10 min, the shared 7 Hz PTP is no longer significantly above baseline by 1 min (n � 4 –7 per group).D, Sharing of PTP requires that the 7 and 20 Hz trains must occur within a narrow time window.

8798 • J. Neurosci., June 30, 2010 • 30(26):8797– 8806 Reissner et al. • Heterosynaptic Sharing of Short-Term Plasticity

c/ebp was used as a positive control. c/ebp has previously been demon-strated to be rapidly transcribed following injury and in vivo 5-HT expo-sure (Alberini et al., 1994). RT-PCR was also used to assess tissuedistribution, similarly as described for investigation of experience-dependent expression of ApHomer.

Antibody preparation and detection of protein interactions. Frozen ratbrains were obtained from Pel Freez Biologicals. Metabotropic glutamatereceptor 1 (mGluR1) antibody was from BD Biosciences, and IP3R anti-body was from Affinity BioReagents. Recombinant ApHomer proteinswere coupled to 0.1 ml of packed glutathione-agarose overnight at 4°C.As described by Tu et al. (1998), rat cerebellar extracts were prepared bysonication in PBS with 1% Triton X-100 (PBST) plus protease inhibitors,followed by centrifugation at 15,000 � g for 10 min. Glutathione beadswere washed three times with 0.3 ml of PBST plus protease inhibitors,and 40 mg of cerebellar extract was allowed to bind for 4 h at 4°C. Beadswere again washed once with PBS followed by two additional washes withPBST, and bound material was removed by boiling in 100 �l of SDS-PAGE sample buffer. Equal amounts of eluted material were separatedby gel electrophoresis for Western blotting with antibodies againstmGluR1a and type 1 IP3R.

In the case of recombinant GST-ApIP3R, soluble proteins were simi-larly coupled to glutathione-agarose overnight at 4°C. Ganglia from fourto six animals were homogenized with a glass-Teflon, motor-drivenhomogenizer into 1.0 –1.2 ml of PBST plus a 1:100 Sigma mammalianprotease inhibitor mixture, 3 mM Na orthovanadate, and 30 mM NaF,and were centrifuged for 10 min at 10,000 � g. A small amount ofsupernatant was saved for use as input material and then was dividedequally for addition to agarose-coupled recombinant proteins. Toinhibit the Homer–IP3R interaction, a synthetic peptide correspond-ing to Aplysia IP3R amino acids 41–52 (GDLNNPPKKFRD; mutantpeptide GDLNNPLKKRRD) was synthesized by Invitrogen. Peptidewas added to Aplysia CNS extract before addition of the extract toglutathione-coupled recombinant protein.

Following binding on a rotator for 4 h at 4°C, unbound material wasremoved by three washes of PBST plus inhibitors. Bound material waseluted by addition of SDS-PAGE sample buffer and boiling for 5 min,followed by analysis by Western blot for ApHomer. A custom affinity-purified ApHomer antibody was prepared by Covance Research Prod-ucts against the ApHomer peptide,-LQDSQRQLQDGRSSRDQEHRE(amino acids 324 –344). The synthetic peptide was conjugated to KLHand used as immunogen for antibody generation in rabbits.

Electrophysiology. For electrophysiology, pleural and pedal gangliawere desheathed in 1:1 isotonic MgCl2/ASW to expose the tail sensoryand motor neuron clusters. MgCl2 was removed by perfusion of thepreparation with ASW for at least 15 min. Intracellular recording wasperformed using glass microelectrodes containing 3 M KCl with electroderesistance of 8 –12 M�. Motor neurons were hyperpolarized to approx-imately 70 mV. The resting potential and input resistance of the SN andMN were monitored throughout each experiment. Baseline EPSP ampli-tudes were determined by injection of current to elicit a single test spikein the SN in each of the two presynaptic SNs (�200 ms apart). Oneminute later, a tetanizing stimulus (generated by individual currentpulses [10 nA] at 20 or 7 Hz for 2 s) was applied to the SNs. A posttestEPSP was measured 10 s after the offset of the stimulus train. The 200 mM

BAPTA (tetrapotassium salt, Invitrogen), 25 �M heparin (Calbiochem),and 125 �M IP3 were dissolved in 3 M KCl, 10 mM HEPES, and 3 mM FastGreen dye (Fisher Scientific). Drugs were injected into the MN with twoto four pulses (20 ms, 20 – 40 psi) from a picospritzer (General Valve),and at least 20 min were allowed for drugs to take effect before pretestvalues were obtained. For caffeine experiments, caffeine (5 mM, Sigma)was dissolved in ASW and perfused over the ganglion through the exper-iment. Thapsigargin (5 �M, Sigma) was prepared in 0.1% DMSO andcontinually perfused as in the case of caffeine.

For mGluR1-blocking experiments, LY367385 (300 �M) and S-MCPG(500 �M) (Tocris Bioscience) were prepared in 110 mM NaOH and di-luted 1:250 in ASW immediately before each experiment. In vehicle con-trol experiments, 200 �l of 110 mM NaOH was added to 50 ml of ASW(1:250) and perfused in a similar manner as drug-treated preparations.Following removal of MgCl2 by perfusion, drug or vehicle was bath ap-

plied for at least 30 min, at a flow rate of 0.5–1 ml/min, before thecommencement of recording.

Synthetic ApIP3R peptide (active and mutant) was prepared at a con-centration of 1.0 mM in 0.5 M KOAc, 10 mM TrisCl, pH 7.5, and 15 mM

Fast Green dye (�1.0%). Peptides were injected into the MN by 28 –32pulses, 10 – 40 ms each, at 20 psi. The number and duration of pulses wasdetermined depending upon the resistance of injection electrode and thesize of the cell. Following injection, cells were continually perfused inASW for at least 45 min before recording from MN. Monosynaptic EP-SPs were measured and PTP performed as described above. Short-termfacilitation induced by a single pulse of 5-HT was also assessed followingthree pretests taken at 15 min intervals. Ten minutes after the last pretest,50 �M 5-HT was perfused for 5 min, and a single post-test was performedimmediately.

Input resistance and excitability of the injected MN were monitored inall experiments, and any preparations that indicated possible cellulardamage were discarded before onset of the stimulus train (�10% ofcases).

Cell culture. Cell culture was performed based on a modified protocolas described previously (Gruenbaum and Carew, 1999; Lyles et al., 2006).Briefly, pleural and abdominal ganglia from 80 to 100 g of Aplysia werefirst incubated in 10 mg/ml Protease type IX (Sigma) at 34.5°C for 2 h and15 min and 2 h and 10 min, respectively. The ganglia were thendesheathed. SNs were isolated from the pleural ganglia, and LFS MNswere isolated from the abdominal ganglia. Identified SNs or MNs werethen plated in culture dishes. The sensory neuron processes werecarefully manipulated to contact the motor neurons. Culture dishes

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Figure 2. Heterosynaptic sharing requires postsynaptic intracellular calcium. A, Degree ofsharing (control, open bar, n � 7) is significantly blocked in the presence of Ca 2� inhibitorsBAPTA (n � 8), caffeine (n � 5), heparin (n � 7), and thapsigargin (n � 5). While 25 �M

heparin is sufficient to block sharing, that concentration is without effect on PTP (black bar, n �6). In contrast, 100 �M heparin results in significant, attenuation of PTP (n � 4). *p � 0.05,within-group comparison. B, Injection of nondegradable IP3 into an MN has no effect on base-line synaptic transmission from an unstimulated SN (0 Hz, open bar), but when paired with a 7Hz train delivered to the SN results in significant PTP (n � 7 per group).

Reissner et al. • Heterosynaptic Sharing of Short-Term Plasticity J. Neurosci., June 30, 2010 • 30(26):8797– 8806 • 8799

were precoated with poly-L-lysine (0.5 mg/ml).Culture medium consisted of 50% Aplysia he-molymph and 50% L-15 medium supple-mented with salts (NaCl, 260 mM; CaCl2, 10mM; KCl, 4.6 mM; MgSO4, 25 mM; MgCl2, 28mM; NaHCO3, 2 mM; HEPES, 15 mM) andD-glucose (34.6 mM). Additionally, glutamine(2 mM), penicillin (100 U/ml), and streptomy-cin (100 U/ml) were also included in the cul-ture medium. Cultured cells were left on themicroscope stage overnight and then trans-ferred to an 18°C incubator. After 4 d, Ca 2�

imaging was performed on MNs, and synapticstrengths between the SN-MN cocultures weremeasured. Electrophysiological recordingswere performed similarly to those in the intactCNS.

Photolysis of caged IP3 and calcium imaging inMN neurites. Cultured MNs were injected with1 mM caged IP3 (Invitrogen) and 10 mM cell-impermeable Fluo-4 Ca 2� indicator dye (In-vitrogen) using a picospritzer, similar to themicroinjection procedure described in the in-tact ganglia. To aid visualization of calcium re-lease in MN neurites, cells were pretreated witha high-K � (100 mM), high-calcium (55 mM),and low-magnesium (11 mM) ASW for 5 min(Malkinson and Spira, 2010). This condition-ing depolarization loads intracellular calciumstores (Garaschuk et al., 1997; Hong and Ross,2007). Imaging was accomplished using acustom-built microscope system based aroundan Olympus IX 50 microscope equipped withan Olympus 60� oil objective lens (numericalaperture, 1.45), using excitation by a solid-state488 nm laser (Spectra Physics sapphire) andimaging emitted fluorescence at � � 510 nmusing a Cascade 650 electron-multiplied CCDcamera (Roper Scientific).

Fluorescence signals are expressed as ratios(F/F0 or F/ F0) of the fluorescence (F) at eachpixel relative to the mean resting fluorescence(F0) before stimulation. IP3 was photoreleasedfrom a caged precursor by delivering flashes ofUV light (350 – 400 nm) focused uniformlythroughout the field of view. Image data werestreamed to computer memory and then stored on disc for offline anal-ysis using the MetaMorph software package (Molecular Dynamics).

Photolysis of caged IP3 and electrophysiology in SN-MN coculture. Theelectrophysiological studies using SN-MN cocultures were performed ona different experimental setup than that used to visualize Ca 2� transientsin neurites (as described above). Following microinjection with caged IP3

and fluo-4, cultured cells were again subject to a conditioning depolar-ization by treatment with a high K � (100 mM) ASW for 5 min to loadintracellular Ca 2� stores and then subject to a 2 s UV flash. This condi-tioning depolarization pretreatment, followed by washout, had no effecton subsequent baseline synaptic transmission. Flashes of UV light werederived from a Zeiss Xite 120 metal halide light source via a UV filter setin a Zeiss Axiovert 200 inverted microscope (Leissring et al., 1999) andwere delivered to the MN soma and proximal neurites.

EPSPs of sensory–motor synapses before and after (1) 20 Hz stimuli,(2) 7 Hz stimuli, (3) 7 Hz stimuli paired with the UV flash, (4) UV flashalone, and (5) no stimulation were then compared.

Data analysis and statistics. Postsynaptic EPSP amplitudes were mea-sured as the peak voltage of the EPSP. In the case of 5-HT-inducedfacilitation, baseline EPSP was determined based on the average of threepretests in ASW, and only EPSPs with pretests within 20% of the meanwere used for further analysis. In the case of PTP, a single pretest wasperformed as described previously (Schaffhausen et al., 2001). All EPSP

amplitudes are expressed as a percentage of the baseline mean. Two-tailed t tests were used for data analysis. All data are shown as �SEM.

ResultsPTP can be shared at SN-MN synapsesTo directly test the hypothesis that convergent inputs might re-sult in postsynaptic sharing of PTP, we first asked whether pre-synaptic activation of an SN at a level that is ineffective inproducing PTP might gain the ability to induce PTP when pairedwith effective activation of a neighboring presynaptic SN. Intactganglia (abdominal or pleural-pedal, in separate experiments)were excised, and neurons were impaled for intracellular record-ing. By eliciting a single presynaptic action potential and record-ing the evoked postsynaptic EPSP, two presynaptic SNs wereidentified that made synapses onto a common MN. At least 15min after identification of convergent synapses, each SN was ac-tivated with a train (2 s in duration) of either 7 Hz (which pro-duces no PTP) or 20 Hz (which induces PTP) (Fig. 1, inset).EPSPs were then measured 10 s after the offset of the trains. While7 Hz alone produced no PTP, when paired with a 20 Hz train tothe other SN, the 7 Hz train induced a robust enhancement ofsynaptic strength (Fig. 1A,B). Additional experiments with no

Figure 3. Photolysis of caged IP3 induces Ca 2� release in MN neurites. A, Monochrome image of resting fluo-4 fluorescence inneurites from a motor neuron. A, Circled white areas indicate regions of interest from which intensity profiles were generated overtime (shown at right). Traces depict local Ca 2� transients from each region evoked by photorelease of IP3 using a UV flash(indicated by the arrow). B, Pseudocolored Ca 2� images (F/F0) before (top left) and at different times after (1a, 2b, and 3c)photorelease of IP3. Images are taken from the same experiment as in A and were captured at times indicated by the correspondingletters by the traces. Arrows indicate the neuritic regions where Ca 2� transients were captured. Each image is an average of eightconsecutive frames acquired at an exposure time of 200 ms.


activation of one SN and 20 Hz activationof the other SN revealed that nonactivatedsynapses do not passively inherit this en-hancement (Fig. 1B), indicating that the 7Hz train likely generates a subthresholdpostsynaptic signal that summates withthe signal initiated by the 20 Hz train.Thus, the temporal registry of trains fromboth cells is required for the “sharing” ofplasticity between synapses.

We next examined the time course ofheterosynaptic sharing and found that itdecayed faster than PTP induced by the 20Hz train (Fig. 1C). By 1 min after simulta-neous activation, the EPSP associated withthe 7 Hz train was no longer significantlydifferent from baseline, while the corre-sponding 20 Hz EPSP was still significantlyelevated at 10 min. Next, to determine thetemporal constraints on induction of het-erosynaptic sharing, we varied the time be-tween 7 and 20 Hz activation of the two SNs.Sharing was observed when the onset oftrains was separated by �3 s (Fig. 1D) andwas independent of the order of activa-tion, indicating that the two SNs need notbe coactive but that their activation mustoccur within a restricted time window.The symmetrical and relatively broad (3 s)

permissive time window for heterosynaptic sharing contrastswith previously described forms of associative plasticity such asspike timing-dependent plasticity and input timing-dependentplasticity, which have much more restricted temporal windows(in the range of milliseconds), and even for associative LTD in thecerebellum, in which pairing of inputs can occur on the order ofhundreds of milliseconds (Sjostrom and Nelson, 2002; Wang etal., 2003; Dan and Poo, 2004; Dudman et al., 2007; Sarkisov andWang, 2008). This dissociation of temporal requirements sug-gests that heterosynaptic sharing represents a unique form ofsynaptic interaction whereby activity at one set of inputs canchange the gain for local information flow at other synaptic sites.

Heterosynaptic sharing requires postsynaptic Ca 2�

To examine the postsynaptic Ca 2� dependency of heterosynapticsharing, we injected the Ca 2� chelator BAPTA into the MN, and20 and 7 Hz trains of activation were delivered to two SNs. Incontrast to the control condition, synaptic enhancement in the 7Hz SN was entirely blocked (Fig. 2A). We and others have previ-ously shown that NMDA receptor activation is not required forinduction of PTP at the SN-MN synapse (Schaffhausen et al.,2001; Jin and Hawkins, 2003). Moreover, Jin and Hawkins (2003)have previously shown that PTP in SN-MN cultures is dependenton intracellular Ca 2� release, but not on voltage-gated Ca 2�

influx or extracellular Ca2� concentration. Thus, we next examinedthe contribution of Ca2� from intracellular stores. Perfusion of caf-feine (which causes Ca2� store depletion via the ryanodine recep-tor) and thapsigargin (a sarco/endoplasmic reticulum Ca2�-ATPase pump antagonist), as well as postsynaptic injection ofheparin (an IP3R antagonist), also resulted in a total block ofheterosynaptic sharing (Fig. 2A). Interestingly, although injec-tion of heparin (25 �M) completely blocked heterosynaptic shar-ing, this concentration had no effect on PTP. However, when weinjected a higher concentration of heparin (100 �M) we observed

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Figure 4. Sharing-like potentiation is induced by pairing 7 Hz activation with photolysis of caged IP3. SN-MN pairs are cocul-tured. A 20 or 7 Hz train is delivered extracellularly to the SN, while EPSPs are recorded in the MN. The MN is microinjected withcaged IP3 and receives a UV flash (Fig. 3). A, Representative EPSPs are shown from SN-MN synapses before and after a 20 Hz train(top), a 7 Hz train alone (middle), or a 7 Hz train paired with a photolysis flash (bottom). B, Summary histograms indicating that 7Hz activation paired with the photolysis of caged IP3 in the postsynaptic MN acquires the ability to induce potentiation comparableto a 20 Hz train. In contrast, 7 Hz activation alone or photolysis alone exhibits synaptic decrement comparable to unstimulatedSN-MN pairs. n � 4 – 8 in each group.

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a significant attenuation of PTP (Fig. 2A),which is consistent with previous observa-tions by Jin and Hawkins (2003), who alsoobserved a significant attenuation of PTPin cultured SN-MNs by injecting this con-centration of heparin into the MNs. Theremaining PTP in the heparin (100 �M)experiments (Fig. 2A) is likely due to thepresynaptic contribution to PTP at thesesynapses (Schaffhausen et al., 2001).

While perfusion of thapsigargin andcaffeine in the bath could affect both pre-synaptic and postsynaptic Ca 2�, the blockobserved by these reagents, taken togetherwith the block observed following injec-tion of heparin into the MN, are consis-tent with a possible role of IP3-mediatedpostsynaptic plasticity. To directly testthis idea, we injected nondegradable IP3

into the MN. In this case, a 7 Hz SN teta-nus alone induced significant PTP (Fig.2B). Collectively, these results suggestthat effective (20 Hz) SN activation gener-ates IP3-dependent release of Ca 2� frominternal stores in the MN, which facilitatesthe induction of sharing at the neighbor-ing (7 Hz) synapse.

Photolysis of caged IP3 induces Ca 2�

transients in the MN neuritesAs demonstrated above, nondegradableIP3–7 Hz pairing induces PTP (Fig. 2 B).One potential mechanism that mayaccount for this observation involves IP3-mediated Ca 2� release. To test this hy-pothesis, MNs were injected with Fluo-4and caged IP3. As shown in Figure 3A,B,repetitive, localized Ca 2� transients inneurites were evoked by photorelease ofIP3 following a UV flash. These observations are representative ofresults in four cells, obtained at 16 locations up to 500 �m awayfrom the cell body. These localized Ca 2� transients likely arisefrom clusters of IP3Rs and are reminiscent of puff activity asreported in mammalian cells (Smith et al., 2009).

Heterosynaptic sharing is mimicked by pairing 7 Hzactivation and photolysis of caged IP3

To further elucidate the mechanisms of heterosynaptic sharing,we used the photolysis of caged IP3 to mimic the in situ genera-tion of an IP3 signal induced by 20 Hz stimuli. We predicted thatif IP3-mediated intracellular Ca 2� release is a critical componentof the mechanism for sharing, then the transient Ca 2� releaseinduced by the photoactivation of caged IP3 in the MN, whenpaired with a train of subthreshold 7 Hz tetanus in the SN, shouldmimic facilitation induced by heterosynaptic sharing. In SN-MNcocultures, MNs were injected with caged IP3 and the calciumindicator Fluo-4. A UV flash, which liberates free IP3 and inducescalcium transients in the MN neurites (Fig. 3) was delivered to theMNs. We then examined the synaptic strengths at the SN-MNsynapses before and after (1) a 20 Hz train, (2) a 7 Hz train, (3) a7 Hz train paired with the photolysis of caged IP3, (4) photolysisalone, and (5) no stimulation (No Stim) (Fig. 4). We observedthat the SN-MN synapses were significantly potentiated by 20 Hz

stimuli alone (Fig. 4A,B). The significant potentiation exhibitedfollowing 20 Hz stimulation is all the more striking when com-pared with the No Stim control (Fig. 4B), which exhibited syn-aptic decrement typically observed in SN-MN cultured synapses(Eliot et al., 1994; Bao et al., 1997; Jin and Hawkins, 2003). Thus,the 20 Hz-induced potentiation is superimposed on a progres-sively decrementing baseline [a similar result was previously ob-served by Jin and Hawkins (2003)]. SN-MNs exposed to either 7Hz alone or photolysis alone also exhibited synaptic decrementthat was comparable to the No Stim condition (Fig. 4B). In con-trast, the pairing of 7 Hz stimuli and photolysis of caged IP3

induced significant synaptic potentiation (Fig. 4A,B). The facil-itation induced by pairing 7 Hz with IP3 photolysis was signifi-cantly greater than that induced by the 7 Hz stimuli alone and wasnot significantly different from the facilitation induced by the 20Hz stimuli alone (Fig. 4B). These data support the hypothesisthat the postsynaptic intracellular Ca 2� transients induced by IP3

receptor activation are critically involved in heterosynapticsharing.

Molecular mechanisms of heterosynaptic sharing requirepostsynaptic Homer-mediated protein interactionsWe next explored the postsynaptic molecular mechanisms un-derlying sharing. Previous work by Jin and Hawkins (2003)indicated that mGluRs are required for PTP in cell culture.

- - - MGEQPI F T CQAHV F HI DPK T KR T WI T A 27Drosophila Homer- - - MGEQPI F ST R AHV F QI DPN T KK NWV PT 27Mouse Homer 1bMANS GEQPI F ST K AHV F QI DAGT KK DWL PA 30Aplysia Homer

SMKA VNV SF F YDS SRN L YR I I S VEGT KA VI 57Drosophila HomerSKHA VT V SYF YDS T RN VYR I I S L DGSKA I I 57Mouse Homer 1bSKAA VNV SYY YDS T RN T YR I I S VDGSKA I V 60Aplysia Homer

NST I T PN MT F T QT SQK F GQWSD VRA NT V YG 87Drosophila HomerNST I T PN MT F T KT SQK F GQWAD SRA NT V YG 87Mouse Homer 1bNST I T PN MT F T KT SQK F GQWSD PRA NT V YG 90Aplysia Homer

L GF A SEA EL T KF V EKF QEV KEA T KN AMK SA 117Drosophila HomerL GF S SEH HL S KF A EKF QEF KEA ARL AKE KS 117Mouse Homer 1bL GF S SET EL S KF I EKF KEI KEL T RQAAQL K 120Aplysia Homer

NGSN AVT PT T SAN T SP I SGRAV GSMQND NT 147Drosophila HomerQE- - - - - - - - - - - - - - - - - - - - - - - - - - - - 119Mouse Homer 1bQG- D - - - - - - - - - - - - VN- - - - G- - - - - - - 126Aplysia Homer

A I DP HT V EPP NMS NT N T QN ANP DSP SHK L L 177Drosophila HomerKMEL T ST PSQESA GGD L QS P- - - - - - - - - L 140Mouse Homer 1bAVDN HAP SPQMGR QHL HQR T N- - - - - - - - - 147Aplysia Homer

NT SD VKA DI GSAT PSP QPT SGV T GGGGV T I 207Drosophila HomerT PES I N- - - GT DD - - - ERT PDV T QN S- - - - 160Mouse Homer 1bSL SS I QS DSGNL R - - - ERR NSF T QGG- - - - 170Aplysia Homer

SSGGSI V GMH T GP GAGAT A EQQL KY ENE RL 237Drosophila Homer- - - - - - - - - - - - - - - - - - E PRA EPT QN- - - 169Mouse Homer 1bSNG- - - - - - H P- - - AN APT EAQL KY END RL 191Aplysia Homer

KMAL AQS CAN AKK WEI EL A T L K NNN I RL T S 267Drosophila HomerAL PF PHS SAI SKH WEA EL A T L K GNN AKL T A 199Mouse Homer 1bKL AL AQS SAN AKK WEV EL QT L K NNN ARL T A 221Aplysia Homer

AL QE ST A NVD EWK RQL HT Y KEE NI R L KR DM 297Drosophila HomerAL L E ST A NVK QWK QQL AAY QEE AER L HK RV 229Mouse Homer 1bAL QE ST A NVE EWK KQL AAY KEE SAR L KK RL 251Aplysia Homer

EQL C VGGGVV AAA GGGAT E DEL RRE VAT L K 327Drosophila HomerT EL E CVS SQA NAV HSH KT E L NQT VQEL E ET 259Mouse Homer 1bL DQE KGGGGE SHMVAQL T D QI E RI R L DN HN 281Aplysia Homer

ART E QL QKEL MQQEL E - L K S- - - - - - - - - A 347Drosophila HomerL KVK EEE I ER L KQEI D NAR EL QEQR DSL T Q 289Mouse Homer 1bKQEE I KR L NS QL QEVT - AR DT QN- - ST L QA 308Aplysia Homer

NI SL REK SND QT L AKL SEV NGV F AK HL S EL 377Drosophila HomerKL QE VEI RNK DL E GQL SDL EQR L EK SQN EQ 319Mouse Homer 1bRI QA L EE ENH SL N L RL QDS QRQL QD GRS SR 338Aplysia Homer

YGVQKDMESL I QI AKC T 394Drosophila HomerEAF R SNL KT L L EI L DGKI F EL T EL R DNL AK 349Mouse Homer 1bDQEH REL L SL QEA L GGRI 356Aplysia Homer

394Drosophila HomerL L EC S 354Mouse Homer 1b 356Aplysia Homer

B

C

D

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H4

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ApHomer

M. Homer 1b

EVH1 CC

63% 35%

75% 38%

HomerH4

1 2 3 4 5 6

ApSAP

A

Figure 6. Identification of a Homer ortholog expressed in Aplysia. A, Amino acid sequence of ApHomer is shown in alignmentwith Drosophila Homer and mouse Homer 1b. Identical amino acids are shaded in red. The EVH1 region is bracketed in blue. ThecDNA sequence for ApHomer is available under GenBank accession number EU999995. B, Schematic comparison of Aplysia Homerwith mouse and Drosophila Homers. Alignment within the EVH1 domain shows a particularly high degree of sequence identityamong Homer family members. The percentages shown indicate the degree of sequence identity within that region betweenAplysia and Drosophila Homer, or Aplysia and mouse Homer 1b. C, Semiquantitative RT-PCR indicates expression of Homer messagein a variety of tissues. CDNA was prepared from buccal mass (lane 1), CNS (lane 2), heart (lane 3), muscle (lane 4), and sperm sac(lane 5). Lane 6 was amplified from water as a template. ApSAP was amplified as a CNS-enriched control, and histone H4 wasamplified as a ubiquitously expressed control. D, Semiquantitative RT-PCR of RNA from pleural and pedal ganglia. Lane 1, Watercontrol template; lane 2, cDNA prepared from RNA taken from an ASW-treated animal; compare with lane 3, cDNA prepared froman in vivo 5-HT-treated animal; lane 4, cDNA prepared from RNA taken immediately from an anesthetized animal; lane 5, cDNAprepared from the other half of the same animal, 1 h later. No change is observed in amplification of histone H4 or Homer, whileC/EBP levels are robustly increased. Similar results were observed following tailshock and KCl (100 mM) depolarization (data notshown).


Thus, we examined the effects on monosynaptic PTP in pleu-ral/pedal ganglia of two type I mGluR inhibitors, LY367385and S-MCPG. While control synapses demonstrated robustPTP, both LY367385 and S-MCPG caused a significant attenua-tion of PTP, with no effect on baseline synaptic transmission (Fig.5A,B). The remaining PTP observed in the presence of inhibitors islikely due to the presynaptic component of PTP at these synapses(Schaffhausen et al., 2001).

Given the mGluR/Ca 2� dependency and IP3 involvement insharing, an attractive molecular candidate for mediating PTP andheterosynaptic sharing was Homer, a well described protein inthe vertebrate postsynaptic density. Homer proteins mediate aseries of interactions with other proteins including mGluRs aswell as IP3 receptors (Tu et al., 1998; Worley et al., 2007). More-over, Homer proteins have been demonstrated both to couplemGluR signaling with intracellular Ca 2� release in vertebrateneurons (Tu et al., 1998) and to localize IP3Rs within dendriticsubregions, allowing for synaptically localized IP3-mediatedCa 2� release (Sala et al., 2001, 2005). However, there was noavailable evidence to indicate that a Homer ortholog is ex-pressed in Aplysia, or whether it might play a role in the spreadof plasticity.

Thus, to directly explore the possible involvement of Homerin sharing, we first cloned Aplysia Homer (ApHomer) using acombination of degenerate RT-PCR and library screening (Fig.6A,B). ApHomer shares the domain organization of otherHomer proteins, including an N-terminal EVH1 domain and aC-terminal coiled-coil region. In vertebrates, Homer has beenpredominantly studied in the nervous system but is also ex-pressed in tissues outside the CNS, as observed for ApHomer(Fig. 6C). In comparison, the gene for Aplysia MAGUK proteinApSAP is expressed predominantly within the CNS (Reissner etal., 2008). Because Homer was first identified as an activity-dependent immediate early gene (Brakeman et al., 1997; Kato etal., 1997), we used semiquantitative RT-PCR to assess messagelevels in response to injury, in vivo 5-HT, KCl depolarization, ortailshock (which induces behavioral sensitization). None of thesetreatments induced a significant change in the cDNA levels ofhistone H4 or Homer but did induce a substantial increase in apositive control, C/EBP (Fig. 6D). These findings thus indicatethe lack of an activity-dependent splice variant of Homer andsuggest that there is no change in the amount of the constitutivelyexpressed splice form of Homer following experience. These re-sults are consistent with findings in Drosophila, where there ap-pears to be only one gene and one transcript for Homer (Diaganaet al., 2002). However, we did identify an alternative splice variantat the extreme 5� end of the mRNA, which would translate intoproteins differing among the first six amino acids (supplementalFig. 1, available at www.jneurosci.org as supplemental material).

A prerequisite for our hypothesis that ApHomer has a mech-anistic role in heterosynaptic sharing is that Homer-mediatedinteractions observed in vertebrates, particularly with mGluRsand IP3Rs, should be conserved in Aplysia. We found that recom-binant ApHomer could successfully coprecipitate rat mGluR1�in a GST pull-down experiment (Fig. 7). However, a low signal-to-noise ratio result made it difficult to interpret pull-down re-sults for IP3R (Fig. 7, bottom right). To directly examine apossible interaction between ApHomer and Aplysia IP3R, we de-termined the coding sequence for the initial 154 aa of ApIP3R. AcDNA for Aplysia IP3R has also been reported, matching thesequences we obtained (Cummins et al., 2007). The consensussequence for Homer binding is PXXFr (Tu et al., 1998), and in ratIP3R it is found at amino acid positions 49 –54. This sequence is

100% conserved between Aplysia and other vertebrates and in-vertebrates (Fig. 8A).

The first 256 aa of ApIP3R were expressed as a GST fusionprotein for a pull-down assay, as described above. A custom an-tibody was generated against ApHomer amino acids 324 –344,which resulted in immunoreactivity against multiple bands inAplysia CNS extract (Fig. 8B, lane 1). However, Western blotfollowing the GST pull-down assay indicated that, while the anti-ApHomer antibody recognizes a complex series of bands in CNSextract used as input, only a single band of �47 kDa is observed insamples eluted from beads coupled to GST-ApIP3R (Fig. 8B,lanes 3– 6). This is in agreement with the �45–50 kDa size ofmammalian and Drosophila Homers and the expected size ofApHomer. Moreover, inclusion of a 12 amino acid peptidematching the Homer recognition sequence within ApIP3R com-peted away this interaction in a concentration-dependent man-ner, whereas a mutant peptide had no effect (Fig. 8B).

The synthetic peptide used to inhibit the ApIP3R–ApHomerinteraction in vitro now provided a powerful experimental tool todirectly explore Homer-mediated IP3 activation in sharing. Wepressure injected MNs with either the ApIP3R peptide or mutantpeptide, in which two critical amino acids in the Homer-bindingsequence were substituted. One hour postinjection, two SNs wereactivated with either 20 or 7 Hz to explore PTP and sharing (Fig.8C). Following injection of the mutant peptide, 20 Hz activationinduced significant PTP, and simultaneous 7 Hz activation in-duced significant heterosynaptic sharing. In contrast, followinginjection of the ApIP3R peptide into the MN, the 20 Hz PTP wasreduced, indicating a partial block by the active peptide, which is

163-335

(GST only)GST 1

2

3

GST(Truncated)

GST 1-335

(Full-length)

163-335

GST

GST

GST 1-335

-

Pull-down

Fusion protein(bait)

*-

*

Figure 7. ApHomer coprecipitates rat mGluR 1�. Top, GST (1) or GST fusion proteins witheither amino acids 163–335 (2) or the first 335 aa of ApHomer (3) were bacterially expressedand purified for a GST pull-down with rat cerebellar extract. Bottom, Western blot for mGluR1�and IP3R of pull-downs. Lane numbers represent the matching number beside constructs usedas bait. In, input. Full-length GST–ApHomer coprecipitates mGluR1� (Western blot: left panel,upper portion, lane 3), whereas GST alone (lane 1) and a GST fusion protein containing theC-terminal portion of ApHomer (lane 2) do not. In contrast, an interaction between ApHomerand rat IP3R could not be conclusively demonstrated due to a nonspecific signal observed be-tween all constructs and IP3R. Top, Left and right, shows Western blot for mGluR1 and IP3R,whereas bottom, left and right, shows the same blots stripped and reprobed with an anti-GSTantibody. The presence of GST or GST fusion proteins is indicated by arrowheads at left.


consistent with our previous observationthat 20 Hz PTP has both presynaptic andpostsynaptic components (Fig. 5) (seealso, Schaffhausen et al., 2001). Impor-tantly, heterosynaptic sharing induced bysimultaneous activation with 7 Hz wascompletely blocked. No effect was ob-served on baseline synaptic transmission.Finally, the attenuation of PTP and blockof sharing by the peptide was restricted toshort-term, activity-dependent plasticityat the SN-MN synapse. Short-term facili-tation induced by a single pulse of 5-HTwas unaffected by the peptide (supple-mental Fig. 2, available at www.jneurosci.org as supplemental material).

DiscussionOur results elucidate a series of molecularsteps that provide a mechanism throughwhich short-term plasticity may be sharedbetween two neighboring synapses. Collec-tively, our findings support the hypothesisthat low-level (e.g., 7 Hz) activation of anSN alone leads to postsynaptic release ofIP3, which is subthreshold for the induc-tion of PTP, but can summate with IP3

liberated by high-level activation (e.g., 20Hz) of another presynaptic input (Fig. 9).The idea that IP3 summation can result inmolecular integration has been previouslysuggested by Parker and colleagues, whofound that caged IP3 responses can sum-mate at distances up to 20 �m (Parker,1989; Parker and Miledi, 1989; Parker andIvorra, 1992). SN-MN synapses in Aplysiacommingle with intersynaptic distances in many cases wellwithin this 20 �m range (Marinesco and Carew, 2002), support-ing the idea that subthreshold IP3 released in the postsynapticneuron by low-level activation may summate with additional IP3

from high-level input to generate a form of molecular coinci-dence detection that induces a sufficient Ca 2� signal to give riseto heterosynaptic sharing.

In the long-term time domain (e.g., LTP and LTD), Ca 2�-dependent heterosynaptic expansion of synaptic plasticity hasbeen observed in other systems (Nishiyama et al., 2000; Royerand Pare, 2003). In addition, communication between proximalhippocampal CA1 synapses has been revealed by facilitation ofLTP at a minimally activated synapse following LTP induction ata nearby synapse (Harvey and Svoboda, 2007). However, theheterosynaptic amplification of hippocampal LTP is not depen-dent on intracellular Ca 2�, whereas the heterosynaptic sharingwe observe requires a postsynaptic Ca 2� response coupled toHomer- and IP3R-dependent protein–protein interactions.

The current study raises two important questions regardingCa 2� signaling. First, how might postsynaptic Ca 2� mediate theinduction of PTP at strongly activated synapses, and second, howmight postsynaptic Ca 2� contribute to heterosynaptic sharing atweakly activated synapses? Considering the first question, onecandidate mechanism for the induction of PTP at strongly acti-vated synaptic sites is CaMKII, since it is well established thatCa 2�-dependent kinases can be critical mediators of synapticplasticity. For example, CaMKII regulates the conductance of

AMPA receptors during LTP by phosphorylation of GluR1 atSer 831 and also may indirectly mediate insertion of AMPA recep-tors into the membrane by phosphorylation of interacting mol-ecules that govern membrane insertion (for reviews, see Bredtand Nicoll, 2003; Malenka, 2003; Boehm and Malinow, 2005).Consistent with this possibility, Jin and Hawkins (2003) havefound that postsynaptic CaMKII is required for PTP in culturedSN-MN synapses in Aplysia, supporting the hypothesis thatCa 2�-activated CaMKII may contribute to a postsynaptic mech-anism for the induction of PTP we observe at strongly activatedcentral synapses. Considering the second question, how mightCa 2� contribute to sharing? It is possible that the spread of IP3

from strongly activated synapses to neighboring weakly activatedones could induce Ca 2� release at the weakly activated synapsesand thus engage the same CaMKII-mediated signaling events atthose neighboring sites as are responsible for the induction ofPTP at the “donor” synapse. However, we cannot exclude thepossibility that the postsynaptic mechanisms contributing to theinduction of PTP at strongly activated synapses could differ fromthose required for heterosynaptic sharing. What we can say, how-ever, is that both classes of plasticity require postsynaptic Ca 2�.Other Ca 2� targets beyond CaMKII may contribute both to theinduction of PTP and to heterosynaptic sharing as well. For ex-ample, it has recently been shown that Ca 2�-dependent Ras ac-tivation occurs rapidly within individual spines (Harvey et al.,2008) and is thus a candidate for PTP induction at the stronglyactivated site. Moreover, activated Ras can spread to neighboring

20 Hz 7 Hz 20 Hz 7 Hz

Mutant peptide Blocking peptide

p<.05

A

C

*

* *

EP

SP

(% o

f bas

elin

e)

- - - - - - MATNFVHI GDI VSLYAEGT VNGFL 24Aplysia IP3RMSE- - - MSASFLHFGDI VSLYAEGSVNGFI 27Starfish IP3RMSD- - - KMSSFLHI GDI CSLYAEGSTNGFI 27Mouse IP3RMGDNI I GSASFLHLGDI VSLYAEGSVCGFL 30Drosophila IP3R

STLGLVDDRSVVQPDAGDL NNPPKKFRDCL 54Aplysia IP3RSTLGLVDDRCVVQPDFGDL NNPPKKFRDCL 57Starfish IP3RSTLGL VDDRCVVQPEAGDL NNPPKKFRDCL 57Mouse IP3RSTLGLVDDRT VVCPEAGDL SCPPKKFRDCL 60Drosophila IP3R

GGGGDDDDLL NNNNNNNNPPPPPPKKKKKKFFFFRRRRDDCC

B

Figure 8. Interaction between ApHomer and ApIP3R is required for PTP sharing. A, Alignment of the first 54 aa of ApIP3R withstarfish, mouse, and Drosophila. Conserved amino acids are shaded. A synthetic peptide including the Homer-binding consensussequence is boxed in blue. B, GST pull-down in which GST–ApIP3R was used to isolate ApHomer from Aplysia CNS extract. Lane 1,Input; lane 2, pull-down with GST as bait; lane 3, ApIP3R pull-down without any peptide included; lane 4, pull-down includinglow-concentration peptide; lane 5, pull-down including high-concentration peptide; lane 6, pull-down including high-concentration mutant peptide. Blots for ApHomer (top) were stripped and reprobed for GST (bottom). C, IP3R peptide (n � 6) ormutant peptide (n � 5) was injected into an MN before induction of PTP and sharing. The mutant peptide has no effect, whereasthe blocking peptide completely prevents sharing. *p � 0.05, within-group comparison.


spines (Harvey et al., 2008), making it also an attractive candidatefor mediating signaling contributing to PTP sharing. Thus, anumber of interactive Ca 2�-dependent postsynaptic signalingevents may contribute both to the initial induction of PTP as wellthe sharing of PTP that we observe.

Our data also indicate that ApHomer-mediated protein inter-actions are critical for heterosynaptic sharing, since the syntheticpeptide that interrupts Homer–IP3R interactions completelyblocks sharing. Interestingly, injection of the ApIP3R peptide intothe MN also tends to reduce the level of PTP (although not to alevel that reaches statistical significance), suggesting that, as in thecase of heparin, blockade of the signaling pathway betweenHomer and intracellular Ca 2� release is more readily impaired inthe case of heterosynaptic sharing compared with PTP. Anotherdifference between heterosynaptic sharing and PTP is their time

course. Sharing is significantly attenuated after 60 s, whereas PTPlasts �10 min. Thus, although not conclusive, the differentialsensitivity of heterosynaptic sharing and PTP to both heparinconcentration and the ApIP3R peptide, taken together with theirdifferential time courses, suggests that these two forms of synap-tic plasticity are not mechanistically identical.

What are the mechanisms by which disruption of Homer-mediated interactions might inhibit induction of PTP and het-erosynaptic sharing? An obvious candidate is postsynapticCa 2� release at the activated synapse, localized by a protein scaf-fold linking type I mGluRs, Homer, and IP3Rs. However, sincethe conserved Homer binding sequence is present in additionalHomer ligands, these interactions are also likely to be disruptedby the synthetic peptide as well. For example, a number of Homerligands containing the PXXFr sequence have been identified, in-cluding the Ryanodine receptor Shank, Dynamin III, PI3 kinaseenhancer, and transient receptor potential cation channels (Tu etal., 1998; Feng et al., 2002; Rong et al., 2003; Yuan et al., 2003; Luet al., 2007). These interactions serve to regulate release of Ca 2�

from intracellular stores, activation of PI3 kinase, maturation ofdendritic spines, as well as the localization of synaptic AMPAreceptors and modulation of synaptic strength (Sala et al., 2001;Rong et al., 2003; Sala et al., 2005; Lu et al., 2007). Although ourresults support a model in which mGluR–Homer–IP3R interac-tions mediate a local Ca 2� signal near activated synapses, whichmay travel to nearby synapses as part of the mechanism of sharing(Fig. 9), we cannot rule out other Homer-dependent mechanisticcomponents to heterosynaptic sharing.

Finally, an intriguing question arises from the present work.What is the functional significance of heterosynaptic sharing?The SN-MN synapses examined in this study mediate the mono-synaptic component of the tail-elicited tail withdrawal reflex inAplysia, in which a tactile stimulus to the tail gives rise to thewithdrawal of the tail from the site of stimulation. Because thereis an approximate topographic arrangement of SN-mediated tac-tile input from the tail skin onto the tail MNs (Walters et al.,2004), the sharing of plasticity we observe could allow the animalto be transiently more responsive to a weak stimulus applied to aneighboring area on the tail where a strong stimulus recentlyoccurred. This enhancement of sensitivity within the net recep-tive field of the reflex would be constrained to a restricted timewindow commensurate with the duration of the net plasticityinduced at neighboring SN presynaptic inputs to the tail MNs. Inthis fashion, the functional tactile receptive field of the reflexwould be dynamically and rapidly expanded following a focalstrong stimulus to the tail, with a time course sufficient for theanimal to make an integrated, adaptive behavioral response.

In principle, one could expand this general notion to any sitein a neural network where there is convergent presynaptic inputonto a common postsynaptic target. At these convergent sites,heterosynaptic sharing of short-term plasticity could provide apotential mechanism for the time-limited rapid and dynamicexpansion of the functional “receptive field” of a postsynapticneuron. From this perspective, inputs whose activation are belowthreshold for the induction of plasticity can, by virtue of temporalcoincidence with other suprathreshold inputs, subsequently ac-quire plasticity and thus more effectively drive the postsynaptictarget. In this fashion, the net enhanced synaptic input onto thepostsynaptic neuron is dynamically expanded within a con-strained time course dictated by the duration of the compositeplasticity at those presynaptic sites. This general mechanismcould allow for a novel form of integration in a neural network byproviding a means of rapid and transient sharing of plasticity

Figure 9. Molecular model for sharing of short-term synaptic plasticity. Model for molecularcoincidence detection: dotted line represents IP3 threshold for intracellular Ca 2� release suffi-cient to induce PTP. Abscissa indicates Ca 2�/IP3 spread within a neurite. A, 20 Hz delivered toan SN is sufficient for local threshold IP3 generation (blue), thus leading to intracellular Ca 2�

release (green). B, However, 7 Hz alone does not achieve the IP3 threshold required for Ca 2�

release. C, In contrast, when neighboring synapses are coactivated at 7 and 20 Hz within anarrow temporal window, summation of local IP3 signals surpass the threshold for Ca 2� re-lease and thus give rise to potentiation of the 7 Hz SN synapse, resulting in sharing of synapticenhancement.


arising from temporally correlated inputs to a common postsyn-aptic target.

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