Neuropeptide localization in varicosities of Aplysia sensory neurons is regulated by target and neuromodulators evoking long-term synaptic plasticity

The synapses between the sensory neuron (SN) and motor neuron of Aplysia undergo long-term functional and structural modulation with appropriate behavioral training or with applications of specific neuromodulators. Expression of molecules within the presynaptic terminals may be regulated in parallel with the changes evoked by the neuromodulators. We examined with immunocytochemical methods whether the level of sensorin, the SN-specific neuropeptide, is modulated in SN varicosities by the location of interaction with the target motor cell L7 and by applications of either 5-HT that evoke long-term facilitation or FMRFamide that evoke long-term depression of Aplysia sensorimotor connections in vitro. A significantly higher proportion of SN varicosities are sensorin positive when they are in contact with the proximal axons of L7 compared to varicosities of the same SNs in contact with distal L7 neurites. Both 5-HT and FMRFamide evoked changes in the efficacy and structure of sensorimotor connections that are accompanied by changes in the frequency of sensorin-positive varicosities contacting the axons of L7. More preexisting SN varicosities are stained after 5-HT, and fewer preexisting SN varicosities are stained after FMRFamide. These results suggest that the postsynaptic target and the neuromodulators not only regulate overall structure but also regulate the level of SN neuropeptide at synaptic sites. © 1996 John Wiley & Sons, Inc.     

Protein synthesis at synapse versus cell body: Enhanced but transient expression of long‐term facilitation at isolated synapses

Protein synthesis at synaptic terminals contributes to LTP in hippocampus and to the formation of new synaptic connections by sensory neurons (SNs) of Aplysia. Here we report that after removal of the SN cell body, isolated SN synapses of Aplysia in culture express protein‐synthesis dependent long‐term facilitation (LTF) produced by 5‐HT that decays rapidly. Changes in expression of a SN‐specific neuropeptide sensorin in isolated SN varicosities parallel the changes in synaptic efficacy. At 24 h after 5‐HT the magnitude of LTF produced at isolated SN synapses was significantly greater than that produced when SN cell bodies were present. LTF was maintained at 48 h at connections with SN cell bodies, but not at isolated SN synapses. The increase in synaptic efficacy at isolated SN synapses at 24 h was blocked by the protein synthesis inhibitor anisomycin. LTF was accompanied by changes in expression of sensorin. The increase in sensorin level at isolated SN varicosities with 5‐HT was blocked by anisomycin or was reversed 48 h after 5‐HT treatment alone. The results suggest that, as is the case for initial synapse formation between SNs and L7, changes in protein synthesis at synaptic terminals may contribute directly to LTF of stable synapses. Changes in expression within the cell body provide additional contributions for long‐term maintenance of the new level of synaptic efficacy that was initiated directly by local changes in protein synthesis at or near synaptic terminals. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 275–286, 2003     

Protein synthesis at synapse versus cell body: enhanced but transient expression of long-term facilitation at isolated synapses

Protein synthesis at synaptic terminals contributes to LTP in hippocampus and to the formation of new synaptic connections by sensory neurons (SNs) of Aplysia. Here we report that after removal of the SN cell body, isolated SN synapses of Aplysia in culture express protein-synthesis dependent long-term facilitation (LTF) produced by 5-HT that decays rapidly. Changes in expression of a SN-specific neuropeptide sensorin in isolated SN varicosities parallel the changes in synaptic efficacy. At 24 h after 5-HT the magnitude of LTF produced at isolated SN synapses was significantly greater than that produced when SN cell bodies were present. LTF was maintained at 48 h at connections with SN cell bodies, but not at isolated SN synapses. The increase in synaptic efficacy at isolated SN synapses at 24 h was blocked by the protein synthesis inhibitor anisomycin. LTF was accompanied by changes in expression of sensorin. The increase in sensorin level at isolated SN varicosities with 5-HT was blocked by anisomycin or was reversed 48 h after 5-HT treatment alone. The results suggest that, as is the case for initial synapse formation between SNs and L7, changes in protein synthesis at synaptic terminals may contribute directly to LTF of stable synapses. Changes in expression within the cell body provide additional contributions for long-term maintenance of the new level of synaptic efficacy that was initiated directly by local changes in protein synthesis at or near synaptic terminals.     

An in vitro study of long-term potentiation in the carp (Cyprinus carpio L.) olfactory bulb

Long-term potentiation (LTP) of synaptic transmission is considered a cellular mechanism for neural plasticity and memory formation. Previously, we showed that in the carp olfactory bulb, LTP occurs at the dendrodendritic mitral-to-granule cell synapse following tetanic electrical stimulation applied to the olfactory tract, and suggested that it is involved in the process of olfactory memory formation. As a first step towards understanding mechanisms underlying plasticity at this synapse, we examined the effects of various drugs (glutamate and GABA receptor agonists and antagonists, noradrenaline, and drugs affecting cAMP signaling) on dendrodendritic mitral-to-granule cell synaptic transmission in an in vitro preparation. Two forms of LTP are involved: a postsynaptic form (tetanus-evoked LTP) and a presynaptic form. The postsynaptic form is evoked at the granule cell dendrite following tetanic olfactory tract stimulation and is suppressed by the NMDA receptor antagonist, D-AP5, enhanced by noradrenaline, and occluded by the metabotropic glutamate receptor agonist, trans-ACPD. The presynaptic form occurs at the mitral cell dendrite following blockade of the GABA_A receptor by picrotoxin and bicuculline, or via activation of cAMP signaling by forskolin and 8-Br-cAMP.     

Cell‐specific changes in expression of mRNAs encoding splice variants of Aplysia cell adhesion molecule accompany long‐term synaptic plasticity

Aplysia neurons express several splice variants of apCAM, a member of the Ig superfamily of cell adhesion molecules. The major transmembrane isoform is endocytosed in sensory neurons (SNs) during the early phases of long‐term facilitation (LTF) of SN synapses evoked by serotonin (5‐HT) or in the motor neuron L7 during the early phases of long‐term depression (LTD) of SN synapses evoked by Phe‐Met‐Arg‐Phe‐amide (FMRFa). We used single cell RT‐PCR to evaluate whether expression of mRNAs encoding for different apCAM isoforms in SNs and L7 is regulated during LTF produced by 5‐HT, and LTD produced by FMRFa. Single SNs and L7s express mRNAs encoding for all major isoforms, but the proportion of each isoform expressed differs for the two cells. SN expresses more mRNA encoding for GPI‐linked isoforms, while L7 expresses more mRNA encoding for the major transmembrane isoform. The neuromodulators produced significant changes in the proportional levels of mRNAs encoding for specific apCAM isoforms during the first 4 h after treatments without affecting overall levels of apCAM mRNA. 5‐HT evoked changes that exaggerated cell‐specific differences in isoform expression. FMRFa evoked changes that reduced cell‐specific differences in isoform expression. The effects of the neuromodulators on apCAM mRNA expression were not detected when cells were cultured alone or when SNs were cocultured with another motor cell that failed to induce synapse formation (L11). The results suggest that rapid cell‐specific regulation of splice variant expression may contribute to different forms of long‐term synaptic plasticity. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 152–161, 2000     

Cell-specific changes in expression of mRNAs encoding splice variants of aplysia cell adhesion molecule accompany long-term synaptic plasticity

Aplysia neurons express several splice variants of apCAM, a member of the Ig superfamily of cell adhesion molecules. The major transmembrane isoform is endocytosed in sensory neurons (SNs) during the early phases of long-term facilitation (LTF) of SN synapses evoked by serotonin (5-HT) or in the motor neuron L7 during the early phases of long-term depression (LTD) of SN synapses evoked by Phe-Met-Arg-Phe-amide (FMRFa). We used single cell RT-PCR to evaluate whether expression of mRNAs encoding for different apCAM isoforms in SNs and L7 is regulated during LTF produced by 5-HT, and LTD produced by FMRFa. Single SNs and L7s express mRNAs encoding for all major isoforms, but the proportion of each isoform expressed differs for the two cells. SN expresses more mRNA encoding for GPI-linked isoforms, while L7 expresses more mRNA encoding for the major transmembrane isoform. The neuromodulators produced significant changes in the proportional levels of mRNAs encoding for specific apCAM isoforms during the first 4 h after treatments without affecting overall levels of apCAM mRNA. 5-HT evoked changes that exaggerated cell-specific differences in isoform expression. FMRFa evoked changes that reduced cell-specific differences in isoform expression. The effects of the neuromodulators on apCAM mRNA expression were not detected when cells were cultured alone or when SNs were cocultured with another motor cell that failed to induce synapse formation (L11). The results suggest that rapid cell-specific regulation of splice variant expression may contribute to different forms of long-term synaptic plasticity.     

Changes in functional glutamate receptors on a postsynaptic neuron accompany formation and maturation of an identified synapse

N-Methyl-D-aspartate (NMDA)-type glutamate receptors play important roles at developing synapses and in activity-dependent synaptic plasticity. Recent studies in Aplysia suggest that NMDA-like receptors may contribute to some forms of plasticity of sensorimotor synapses accompanying associative learning. We examined at various times after plating neurons in culture the contribution of NMDA- and alpha-amino-3 hydroxy-5 methyl-4 isoxazole proprionic acid (AMPA)-like glutamate receptors to responses evoked in motor cell L7 either by action potentials in sensory neurons (SNs) or by focal applications of glutamate. We found that (D,L)-2-amino-5-phosphopentoic acid-sensitive receptors contributed significantly to postsynaptic responses in 1-day cultures but contributed little in the same cultures on day 4. By contrast, postsynaptic responses on day 4 increased significantly in amplitude by the addition of functional 6-cyano-7 nitroquinoxaline-2,3-dione- or 1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine hydrochloride-sensitive receptors. Receptors with NMDA-like properties are detected on day 1 only at sites on L7 apposed to SN varicosities, and are not detected on L7 cultured alone. The results indicate that changes in expression and distribution of functional receptors on L7 accompany the formation and maturation of SN synapses. Signals from the SN appear to trigger expression and clustering of functional NMDA-like receptors at sites contacted by presynaptic structures capable of transmitter release. With time, functional AMPA-like receptors are added to these sites enhancing synaptic efficacy. The results are consistent with the idea that the expression and sequential clustering of NMDA- and AMPA-type receptors may be essential for the formation and maturation of central synapses.     

The Neurexin/N-Ethylmaleimide-sensitive Factor (NSF) Interaction Regulates Short Term Synaptic Depression

Although Neurexins, which are cell adhesion molecules localized predominantly to the presynaptic terminals, are known to regulate synapse formation and synaptic transmission, their roles in the regulation of synaptic vesicle release during repetitive nerve stimulation are unknown. Here, we show that nrx mutant synapses exhibit rapid short term synaptic depression upon tetanic nerve stimulation. Moreover, we demonstrate that the intracellular region of NRX is essential for synaptic vesicle release upon tetanic nerve stimulation. Using a yeast two-hybrid screen, we find that the intracellular region of NRX interacts with N-ethylmaleimide-sensitive factor (NSF), an enzyme that mediates soluble NSF attachment protein receptor (SNARE) complex disassembly and plays an important role in synaptic vesicle release. We further map the binding sites of each molecule and demonstrate that the NRX/NSF interaction is critical for both the distribution of NSF at the presynaptic terminals and SNARE complex disassembly. Our results reveal a previously unknown role of NRX in the regulation of short term synaptic depression upon tetanic nerve stimulation and provide new mechanistic insights into the role of NRX in synaptic vesicle release.     

Rapid bidirectional modulation of mRNA expression and export accompany long‐term facilitation and depression of Aplysia synapses

Serotonin (5‐HT) and the neuropeptide Phe‐Met‐Arg‐Phe‐amide (FMRFa) modulate synaptic efficacy of sensory neurons (SNs) of Aplysia in opposite directions and for long duration. Both long‐term responses require changes in mRNA and protein synthesis. The SN‐specific neuropeptide, sensorin A, is a gene product that appears to be increased by 5‐HT and decreased by FMRFa. We examined whether changes in sensorin A mRNA levels in the cell body and neurites of SNs accompany long‐term facilitation and depression. Both 5‐HT and FMRFa evoked rapid changes in sensorin A mRNA levels in the SN cell bodies: an increase with 5‐HT and a decrease with FMRFa. Parallel changes in sensorin A mRNA levels in SN neurites were detected 2 h and 4 h later. These rapid changes in mRNA expression and net export required the presence of the appropriate target motor cell L7. The neuromodulators failed to produce changes in mRNA expression or export when SNs were cultured alone or with the inappropriate target cell L11. The changes in mRNA expression were transient because mRNA levels returned to control values 24 h after treatment, while synaptic efficacy remained altered by the respective treatments. These results indicate that two neuromodulators produce distinct, but transient, target‐dependent effects on expression and export of a cell‐specific mRNA that correlate with changes in synaptic plasticity. © 2000 John Wiley & Sons, Inc. J Neurobiol 46: 41–47, 2001     

En passant synaptic varicosities form directly from growth cones by transient cessation of growth cone advance but not of actin-based motility.

Formation of terminal synapses at sites such as the neuromuscular junction involves transformation of the motile growth cone into the nonmotile synaptic terminal. However, transformation does not need to be the mechanism when a neurite forms multiple widely spaced synaptic varicosities along a target in an en passant configuration. Synaptic varicosities could form here by specialization of the neurite after the growth cone has advanced past the site. We examined this issue by using cocultures of identified sensory (SN) and motor (L7) neurons from Aplysia. Living SNs were labeled with fluorescent dye and their neurites were observed at high resolution every few minutes growing along the axon of L7, allowing a fine-grained analysis of the behavior of the growth cone at the sites of synapse formation. All varicosities whose formation was observed indeed developed from the growth cone. Sensory varicosities were shown by electron microscopy to contain features characteristic of active zones for transmitter release within a day of their formation on the motor axon. Growth cone advance slowed or stopped transiently during varicosity formation, but the motile activity of the peripheral region of the growth cone (veils and filopodia) was maintained. These results suggest that target "stop signals" involved in the formation of synapses, at least of the en passant variety, may be of a different type from the growth inhibitory molecules, such as the collapsins, which guide axons to their targets.     

Rapid bidirectional modulation of mRNA expression and export accompany long-term facilitation and depression of Aplysia synapses

Serotonin (5-HT) and the neuropeptide Phe-Met-Arg-Phe-amide (FMRFa) modulate synaptic efficacy of sensory neurons (SNs) of Aplysia in opposite directions and for long duration. Both long-term responses require changes in mRNA and protein synthesis. The SN-specific neuropeptide, sensorin A, is a gene product that appears to be increased by 5-HT and decreased by FMRFa. We examined whether changes in sensorin A mRNA levels in the cell body and neurites of SNs accompany long-term facilitation and depression. Both 5-HT and FMRFa evoked rapid changes in sensorin A mRNA levels in the SN cell bodies: an increase with 5-HT and a decrease with FMRFa. Parallel changes in sensorin A mRNA levels in SN neurites were detected 2 h and 4 h later. These rapid changes in mRNA expression and net export required the presence of the appropriate target motor cell L7. The neuromodulators failed to produce changes in mRNA expression or export when SNs were cultured alone or with the inappropriate target cell L11. The changes in mRNA expression were transient because mRNA levels returned to control values 24 h after treatment, while synaptic efficacy remained altered by the respective treatments. These results indicate that two neuromodulators produce distinct, but transient, target-dependent effects on expression and export of a cell-specific mRNA that correlate with changes in synaptic plasticity.     

Target-dependent structural changes in sensory neurons of Aplysia accompany long-term heterosynaptic inhibition.

FMRFamide evokes both short-term and long-term inhibition of synapses between mechanosensory and motor neurons in Aplysia. We report here, using dissociated cell culture and low-light epifluorescence video microscopy, that depression lasting 24 hr of sensorimotor synapses evoked by four brief applications of FMRFamide is accompanied by a significant loss of sensory cell varicosities and neurites. These structural changes in the sensory cells require the presence of the target motor cell L7. Because the loss of structures known to contain transmitter release sites correlates significantly with the changes in the amplitude of the excitatory postsynaptic potential in L7, our results suggest that the structural changes evoked by FMRFamide reflect a loss of synaptic contacts. Thus, long-term depression parallels long-term facilitation of the sensorimotor synapse produced by serotonin in that both forms of heterosynaptic plasticity involve target-dependent modulation of the number of presynaptic varicosities.     

Calcium-dependent isoforms of protein kinase C mediate posttetanic potentiation at the calyx of Held

High-frequency stimulation leads to a transient increase in the amplitude of evoked synaptic transmission that is known as posttetanic potentiation (PTP). Here we examine the roles of the calcium-dependent protein kinase C isoforms PKCα and PKCβ in PTP at the calyx of Held synapse. In PKCα/β double knockouts, 80% of PTP is eliminated, whereas basal synaptic properties are unaffected. PKCα and PKCβ produce PTP by increasing the size of the readily releasable pool of vesicles evoked by high-frequency stimulation and by increasing the fraction of this pool released by the first stimulus. PKCα and PKCβ do not facilitate presynaptic calcium currents. The small PTP remaining in double knockouts is mediated partly by an increase in miniature excitatory postsynaptic current amplitude and partly by a mechanism involving myosin light chain kinase. These experiments establish that PKCα and PKCβ are crucial for PTP and suggest that long-lasting presynaptic calcium increases produced by tetanic stimulation may activate these isoforms to produce PTP.     

The thrombin receptor mediates functional activity‐dependent neuromuscular synapse reduction via protein kinase C activation in vitro

Activity‐dependent selective reduction of synaptic efficacy is expressed in an in vitro system involving mouse spinal cord and muscle cells. Thrombin or electrical stimulation of the innervating axons induces a decrease in neuromuscular synapse strength, and a specific thrombin inhibitor, hirudin, blocks the electrically evoked down‐regulation of synapse effectiveness. We further demonstrate that a thrombin receptor‐activating peptide (TRAP), SFLLRNPNDKYEPF, produces a decrement of synapse strength. Both TRAP and electrically evoked synapse decrement are prevented by the specific protein kinase C blocker calphostin C, and the TRAP‐evoked synapse decrement is unaffected by a specific protein kinase A blocker, H‐89. Thus, we propose that muscle activity, thrombin release, and thrombin receptor and PKC activation are initial steps in the process of the activity‐dependent synapse reduction expressed in our system. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 369–381, 1999     

Calcium transients recorded with arsenazo III in the presynaptic terminal of the squid giant synapse

Transient changes in free intracellular Ca2+ concentration were monitored in the presynaptic terminal of the giant synapse of the squid, by means of the Ca2+-sensitive dye arsenazo III. Calibration experiments showed a linear relation between the amount of Ca2+ injected by iontophoresis into the terminal, and the peak size of the arsenazo light absorbance record. A light signal could be detected on tetanic stimulation of the presynaptic axon bathed in sea water containing 45 mM Ca2+. During a 1 s tetanus the light signal rose approximately linearly, even though transmitter release declined rapidly and the light signal subsequently declined with a half-time of 2-6 s. The Ca2+ transient elicited by single nerve impulses was recorded by signal averaging, and showed a time course very much slower than the duration of transmitter release.     

Synaptic ultrastructure in nerve terminals of Drosophila larvae overexpressing the learning gene dunce

We investigated synaptic ultrastructure of individual nerve ending varicosities at the Drosophila larval neuromuscular junction in transgenic larvae overexpressing the learning gene dunce (dnc) in the nervous system. It was previously shown that cAMP is reduced to one-third normal in these larvae and that they have fewer nerve terminal varicosities and smaller junction potentials, although transmitter release from individual nerve ending varicosities is not significantly altered. We tested the hypothesis that synaptic ultrastructure is modified to compensate for possible reduced efficacy of synaptic transmission resulting from lower than normal cAMP. Synaptic size and number of presynaptic dense bodies (active zone structures) per synapse are modestly enhanced in transgenic larvae overexpressing the dnc gene product and in rutabaga (rut(1)) mutant larvae, which have reduced adenylyl cyclase activity and reduced neural cAMP. The incidence of complex synapses (possessing 2 or more presynaptic dense bodies) was not consistently different in experimental larvae compared to controls. The observations suggest that chronic reduction of cAMP levels in the nervous system of Drosophila larvae, although leading to a modest compensatory change in synaptic structure, does not markedly alter several synaptic ultrastructural parameters which are thought to influence the strength of transmitter release; thus, homeostatic mechanisms do not act to maintain normal-sized junction potentials by altering synaptic structure.     

Postsynaptic regulation of the development and long-term plasticity of Aplysia sensorimotor synapses in cell culture

The monosynaptic component of the neuronal circuit that mediates the withdrawal reflex of Aplysia californica can be reconstituted in dissociated cell culture. Study of these in vitro monosynaptic connections has yielded insights into the basic cellular mechanisms of synaptogenesis and long-term synaptic plasticity. One such insight has been that the development of the presynaptic sensory neurons is strongly regulated by the postsynaptic motor neuron. Sensory neurons which have been cocultured with a target motor neuron have more elaborate structures—characterized by neurites with more branches and varicosities—than do sensory neurons grown alone in culture or sensory neurons that have been cocultured with an inappropriate target cell. Another way in which the motor neuron regulates the development of sensory neurons is apparent when sensorimotor cocultures with two presynaptic cells are examined. In such cocultures the outgrowth from the different presynaptic cells is obviously segregated on the processes of the postsynaptic cell. By contrast, when two sensory neurons are placed into cell culture without a motor neuron, thier processes readily grow together. In addition to regulating the in vitro development of sensory neurons, the motor neuron also regulates learning-related changes in the structure of sensory neurons. Application of the endogenous facilitatory trasmitter serotonin (5-HT) causes long-term facilitation of in vitro sensorimotor synapses due in part to growth of new presynatpic varicosities. But 5-HT applied to sensory neurons alone in cultuer does not produce structural changes in these cells. More recently it has been found that sensorimotor synapses in cell culture can exhibit long-term potentiation (LTP). Like LTP of some hippocampal synapses, LTP of in vitro Aplysia syanpses is regulated by the voltage of the postsynaptic cell. Pairing high-frequency stimulation of sensory neurons with strong hyperpolarization of the motor neuron blocks the induction of LTP. Moreover, LTP of sensorimotor synapses can be induced in Hebbian fashion by pairing weak presynaptic stimulation with strong postsynaptic depolarization. These findings implicate a Habbian mechanism in classical conditioning in Aplysia. They also indicate that Hebbian LTP is a phylogenetically ancient form of synaptic plasticity. 1994 John Wiley & Sons, Inc.     

Synapse elimination from the mouse neuromuscular junction in vitro: A non-hebbian activity-dependent process

The effect of action potentials on elimination of mouse neuromuscular junctions (NMJ) was studied in a three compartment cell culture preparation. Axons from superior cervical ganglion or ventral spinal cord neurons in two lateral compartments formed multiple neuromuscular junctions with muscle cells in a central compartment. The loss of synapses over a 2–7-day period was determined by serial electrophysiological recording and a functional assay. Electrical stimulation of axons from one side compartment during this period, using 30-Hz bursts of 2-s duration, repeated at 10-s intervals, caused a significant increase in synapse elimination compared to unstimulated cultures (p< 0.001). The extent of homosynaptic and heterosynaptic elimination was comparable, i. e., of the 226 functional synapses of each type studied, 111 (49%) of the synapses that had been stimulated were eliminated, and 87 (39%) of unstimulated synapses on the same muscle cells were eliminated. Also, simultaneous bilateral stimulation caused significantly greater elimination of synapses than unilateral stimulation (p< 0.005). These observations are contrary to the Hebbian hypothesis of synaptic plasticity. A spatial effect of stimulus-induced synapse elimination was also evident following simultaneous bilateral stimulation. Prior to stimulation, most muscle cells were innervated by axons from both side compartments, but after bilateral stimulation, muscle cells were predominantly unilaterally innervated by axons from the closer compartment. These experiments suggest that synapse elimination at the NMJ is an activity-dependent process, but it does not follow Hebbian or anti-Hebbian rules of synaptic plasticity. Rather, elimination is a consequence of postsynaptic activation and a function of location of the muscle cell relative to the neuron. An interaction between spatial and activity-dependent effects on synapse elimination could help produce optimal refinement of synaptic connections during postnatal development. © 1993 John Wiley & Sons, Inc.     

Remodeling of synaptic actin induced by photoconductive stimulation.

Use-dependent synapse remodeling is thought to provide a cellular mechanism for encoding durable memories, yet whether activity triggers an actual structural change has remained controversial. We use photoconductive stimulation to demonstrate activity-dependent morphological synaptic plasticity by video imaging of GFP-actin at individual synapses. A single tetanus transiently moves presynaptic actin toward and postsynaptic actin away from the synaptic junction. Repetitive spaced tetani induce glutamate receptor-dependent stable restructuring of synapses. Presynaptic actin redistributes and forms new puncta that label for an active synapse marker FM5-95 within 2 hr. Postsynaptic actin sprouts projections toward the new presynaptic actin puncta, resembling the axon-dendrite interaction during synaptogenesis. Our results indicate that activity-dependent presynaptic structural plasticity facilitates the formation of new active presynaptic terminals.