Synapse turnover: a mechanism for acquiring synaptic specificity.

Neurons are generated in chick retina that are able to form synapses with striated muscle cells for only a brief period during embryonic development. The ability to form synapses is lost with a half-life of 21 hr. Retina neuron-myotube synapses form rapidly but soon are terminated. Chick embryo spinal cord neurons also form synapses with muscle cells for only a limited time during development, but these synapses are long lived. These results show that different classes of synapses turn over at different rates and suggest that part of the specificity of synaptic circuits may be acquired during development by a process of selection based on synapse termination rates.     

On the specificity of synapse formation.

Clonal neuroblastome X glioma hybrid cells form synapses with striated muscle cells from different muscles and from different organisms, such as chick, mouse, and rat. Under appropriate conditions 65-80% of the muscle cells exhibited synaptic responses. The results indicate that most striated muscle cells have the same specificity for synapse formation and suggest that synaptogenesis is not dependent on interactions between complementary molecules on neuron and muscle cells that code for different synaptic connections.     

Dissociated ciliary ganglion neurons in vitro: survival and synapse formation.

Normally, about half of the ciliary ganglion neurons in 8-day-old chick embryos die before day 14 in ovo. However, when dissociated ciliary ganglion neurons were prepared from either 8- or 14-day-old embryos and grown in cell culture with skeletal myotubes, essentially all of the neurons survived for at least 3 weeks. Many of the neurons formed functional synapses on myotubes under these conditions; some neuromuscular synapses could be detected as early as 20 hr after addition of the ganglion cells to muscle cultures. In contrast, most neurons from 8-day embryos survived for only a few days when grown alone on either polyornithine- or collagen-coated dishes. These results suggest that neurons destined to die in ovo can be rescued when grown in cell culture with myotubes and that under these conditions the neurons develop and express differentiated properties.     

Emergence of order in visual system development.

Neural connections in the adult central nervous system are highly precise. In the visual system, retinal ganglion cells send their axons to target neurons in the lateral geniculate nucleus (LGN) in such a way that axons originating from the two eyes terminate in adjacent but nonoverlapping eye-specific layers. During development, however, inputs from the two eyes are intermixed, and the adult pattern emerges gradually as axons from the two eyes sort out to form the layers. Experiments indicate that the sorting-out process, even though it occurs in utero in higher mammals and always before vision, requires retinal ganglion cell signaling; blocking retinal ganglion cell action potentials with tetrodotoxin prevents the formation of the layers. These action potentials are endogenously generated by the ganglion cells, which fire spontaneously and synchronously with each other, generating "waves" of activity that travel across the retina. Calcium imaging of the retina shows that the ganglion cells undergo correlated calcium bursting to generate the waves and that amacrine cells also participate in the correlated activity patterns. Physiological recordings from LGN neurons in vitro indicate that the quasiperiodic activity generated by the retinal ganglion cells is transmitted across the synapse between ganglion cells to drive target LGN neurons. These observations suggest that (i) a neural circuit within the immature retina is responsible for generating specific spatiotemporal patterns of neural activity; (ii) spontaneous activity generated in the retina is propagated across central synapses; and (iii) even before the photoreceptors are present, nerve cell function is essential for correct wiring of the visual system during early development. Since spontaneously generated activity is known to be present elsewhere in the developing CNS, this process of activity-dependent wiring could be used throughout the nervous system to help refine early sets of neural connections into their highly precise adult patterns.     

Antibody to a molecular marker of cell position inhibits synapse formation in retina.

A topographic gradient of TOP molecules in retina can be used to identify neuron position. Antibody to TOP from hybridoma cells that were injected into in vivo embryo eyes diffused into the retina and bound in a topographic gradient of [antibody.TOP] ([Ab.TOP]) complexes. Synapse formation in retina was inhibited in the presence of anti-TOP antibody. This suggests that TOP is involved in synapse formation and that recognition of position by neurons is necessary for normal synapse formation.     

Synaptophysin regulates activity-dependent synapse formation in cultured hippocampal neurons.

Synaptophysin is an abundant synaptic vesicle protein without a definite synaptic function. Here, we examined a role for synaptophysin in synapse formation in mixed genotype micro-island cultures of wild-type and synaptophysin-mutant hippocampal neurons. We show that synaptophysin-mutant synapses are poor donors of presynaptic terminals in the presence of competing wild-type inputs. In homogenotypic cultures, however, mutant neurons display no apparent deficits in synapse formation compared with wild-type neurons. The reduced extent of synaptophysin-mutant synapse formation relative to wild-type synapses in mixed genotype cultures is attenuated by blockers of synaptic transmission. Our findings indicate that synaptophysin plays a previously unsuspected role in regulating activity-dependent synapse formation.     

Estrogen increases synaptic connectivity between single presynaptic inputs and multiple postsynaptic CA1 pyramidal cells: a serial electron-microscopic study

Dendritic spines are sites of the vast majority of excitatory synaptic input to hippocampal CA1 pyramidal cells. Estrogen has been shown to increase the density of dendritic spines on CA1 pyramidal cell dendrites in adult female rats. In parallel with increased spine density, estrogen has been shown also to increase the number of spine synapses formed with multiple synapse boutons (MSBs). These findings suggest that estrogen-induced dendritic spines form synaptic contacts with preexisting presynaptic boutons, transforming some previously single synapse boutons (SSBs) into MSBs. The goal of the current study was to determine whether estrogen-induced MSBs form multiple synapses with the same or different postsynaptic cells. To quantify same-cell vs. different-cell MSBs, we filled individual CA1 pyramidal cells with biocytin and serially reconstructed dendrites and dendritic spines of the labeled cells, as well as presynaptic boutons in synaptic contact with labeled and unlabeled (i.e., different-cell) spines. We found that the overwhelming majority of MSBs in estrogen-treated animals form synapses with more than one postsynaptic cell. Thus, in addition to increasing the density of excitatory synaptic input to individual CA1 pyramidal cells, estrogen also increases the divergence of input from individual presynaptic boutons to multiple postsynaptic CA1 pyramidal cells. These findings suggest the formation of new synaptic connections between previously unconnected hippocampal neurons.     

Activity-dependent regulation of synaptic clustering in a hippocampal culture system.

Currently, there is a limited understanding of the factors that influence the localization and density of individual synapses in the central nervous system. Here we have studied the effects of activity on synapse formation between hippocampal dentate granule cells and CA3 pyramidal neurons in culture, taking advantage of FM1-43 as a fluorescent marker of synaptic boutons. We observed an early tendency for synapses to group together, quickly followed by the appearance of synaptic clusters on dendritic processes. These events were strongly influenced by N-methyl-D-aspartic acid receptor- and cyclic AMP-dependent signaling. The microstructure and localization of the synaptic clusters resembled that found in hippocampus, at mossy fiber synapses of stratum lucidum. Activity-dependent clustering of synapses represents a means for synaptic targeting that might contribute to synaptic organization in the brain.     

Estrogen receptor expression in laryngeal muscle in relation to estrogen-dependent increases in synapse strength

In Xenopus laevis, the laryngeal neuromuscular synapse is the final effector for sexually differentiated song production. Females have stronger laryngeal synapses than males, and synapse strength is estrogen dependent. Estrogen-induced increases in synaptic strength require at least 3 weeks of exposure, suggesting that the hormone acts via a classical genomic mechanism involving the estrogen receptor (ER). The locus of the sex difference in synapse strength, determined using quantal analysis, is presynaptic, leading to the prediction that estrogen acts directly on vocal motor neurons. However, laryngeal motor neurons do not accumulate estrogen. Estrogen might instead affect motor neuron transmitter release via a retrograde signal from its target muscle. To test this hypothesis, we determined whether laryngeal muscle expresses ER. With RT-PCR using primers that recognize highly conserved domains of the ERalpha, mRNA products of the predicted size were amplified from laryngeal muscle as well as from other classical target tissues (forebrain and oviduct). Northern blots using a portion of the PCR product as primer revealed the same-sized band in oviduct and laryngeal muscle. Immunocytochemistry and Western blots confirmed the presence of ER protein in laryngeal muscle fibers and revealed several proteins in laryngeal muscle, brain and liver; among these was an approximately 66-kD protein - presumed to be full-length ER - that was the only one found in oviduct. Estrogen treatment of juveniles resulted in an upregulation of the 66-kD ER protein concomitant with an increase in quantal content. Taken together, these experiments strongly suggest that the ER is expressed by laryngeal muscle; this receptor could mediate estrogen-dependent changes in synaptic strength via retrograde signaling.     

gamma-Aminobutyric acid exerts a local inhibitory action on the axon terminal of bipolar cells: evidence for negative feedback from amacrine cells.

It is well-established morphologically that bipolar cells, the second-order neurons in the vertebrate retina, make reciprocal synapses with amacrine cells in the inner plexiform layer. However, neither the property nor the physiological function of the feedback synapse is understood. Autoradiographic and immunohistochemical studies suggest the presence of gamma-aminobutyric acid (GABA)-ergic amacrine cells, and therefore the bipolar cells are thought to receive GABAergic inputs from amacrine cells. This possibility was investigated in the present study, in which we used solitary bipolar cells dissociated from the goldfish retina enzymatically. Dissociated solitary bipolar cells showed a large variety in morphology. In the present study, we selected the bipolar cells with a huge bulbous axon terminal. Bipolar cells of this subtype were identical in morphology to the on-center cells with rod-dominant inputs as revealed in earlier studies by intracellular staining. Membrane currents were measured under voltage clamp with a patch pipette in the whole cell configuration. In some experiments, GABA-sensitive membrane was excised as an outside-out patch from the axon terminal bulb of solitary bipolar cells. All cells of this type responded to GABA. The highest sensitivity was located at the axon terminal. The minimal effective dose was on the order of 10(-7) M. GABA increased the chloride conductance and evoked a membrane hyperpolarization. Partial desensitization was observed during the application of GABA. The bipolar cells had GABA type A receptors. These results are consistent with the idea that the rod-dominant on-center bipolar cells receive negative feedback inputs from GABAergic amacrine cells.     

Trans-synaptic EphB2–ephrin–B3 interaction regulates excitatory synapse density by inhibition of postsynaptic MAPK signaling

Nervous system function requires tight control over the number of synapses individual neurons receive, but the underlying cellular and molecular mechanisms that regulate synapse number remain obscure. Here we present evidence that a trans-synaptic interaction between EphB2 in the presynaptic compartment and ephrin-B3 in the postsynaptic compartment regulates synapse density and the formation of dendritic spines. Observations in cultured cortical neurons demonstrate that synapse density scales with ephrin-B3 expression level and is controlled by ephrin-B3–dependent competitive cell–cell interactions. RNA interference and biochemical experiments support the model that ephrin-B3 regulates synapse density by directly binding to Erk1/2 to inhibit postsynaptic Ras/mitogen-activated protein kinase signaling. Together these findings define a mechanism that contributes to synapse maturation and controls the number of excitatory synaptic inputs received by individual neurons.     

Multiple signaling pathways are essential for synapse formation induced by synaptic adhesion molecules

Little is known about the cellular signals that organize synapse formation. To explore what signaling pathways may be involved, we employed heterologous synapse formation assays in which a synaptic adhesion molecule expressed in a nonneuronal cell induces pre- or postsynaptic specializations in cocultured neurons. We found that interfering pharmacologically with microtubules or actin filaments impaired heterologous synapse formation, whereas blocking protein synthesis had no effect. Unexpectedly, pharmacological inhibition of c-jun N-terminal kinases (JNKs), protein kinase-A (PKA), or AKT kinases also suppressed heterologous synapse formation, while inhibition of other tested signaling pathways—such as MAP kinases or protein kinase C—did not alter heterologous synapse formation. JNK and PKA inhibitors suppressed formation of both pre- and postsynaptic specializations, whereas AKT inhibitors impaired formation of post- but not presynaptic specializations. To independently test whether heterologous synapse formation depends on AKT signaling, we targeted PTEN, an enzyme that hydrolyzes phosphatidylinositol 3-phosphate and thereby prevents AKT kinase activation, to postsynaptic sites by fusing PTEN to Homer1. Targeting PTEN to postsynaptic specializations impaired heterologous postsynaptic synapse formation induced by presynaptic adhesion molecules, such as neurexins and additionally decreased excitatory synapse function in cultured neurons. Taken together, our results suggest that heterologous synapse formation is driven via a multifaceted and multistage kinase network, with diverse signals organizing pre- and postsynaptic specializations.

Synapse formation is the universal process that underlies construction of all of the brain’s circuits, but little is known about its mechanisms. Unknown signaling pathways presumably organize synapses, but what pathways are involved remains unclear. Synapse formation likely requires interactions between pre- and postsynaptic neurons via adhesion molecules that transmit bidirectional signals to pre- and postsynaptic neurons and organize pre- and postsynaptic specializations (reviewed in refs. 1–3). Synapses exhibit canonical features that include a presynaptic side that releases neurotransmitters rapidly and transiently and a postsynaptic side that recognizes these neurotransmitters. Interestingly, only the presynaptic side of a synapse harbors canonical features that are shared by all synapses, such as synaptic vesicles and active zones with the same components in excitatory and inhibitory synapses. In contrast, the postsynaptic sides differ dramatically between excitatory and inhibitory synapses. Even excitatory and inhibitory neurotransmitter receptors exhibit no homology, and few if any molecular components are shared among excitatory and inhibitory postsynaptic specializations.At present, it is unknown what intracellular signaling pathways are involved in the assembly of pre- and postsynaptic specializations, whether different types of signaling pathways exist for pre- vs. postsynaptic specializations, and how excitatory vs. inhibitory synapses are organized. In the present study, we chose the heterologous synapse formation assay as an approach in order to begin to address these fundamental questions (4). In the heterologous synapse formation assay, nonneuronal cells, such as HEK293T cells, express a synaptic adhesion molecule that then induces pre- or postsynaptic specializations when these nonneuronal cells are cocultured with neurons (5–9). For example, if a postsynaptic adhesion molecule, such as neuroligin-1 (Nlgn1) or latrophilin-3, is expressed in HEK293T cells, and the HEK293T cells are cocultured with neurons, these neurons form presynaptic specializations on the HEK293T cells (5, 10). If, conversely, a presynaptic adhesion molecule, such as a neurexin or teneurin, is expressed in HEK293T cells, postsynaptic specializations are induced in cocultured neurons (8, 9, 11).Many adhesion molecules have been shown to induce heterologous synapse formation, including neurexins, neuroligins, latrophilins, teneurins, SynCAMs, neuronal pentraxin receptors, SALMs, LAR-type PTPRs, and others (5, 6, 8–15), suggesting that there are common “synapse signaling” pathways and that the heterologous synapse formation assay nonspecifically transduces different adhesion molecules signals into a response that organizes pre- or postsynaptic specializations. Even engagement of neuronal AMPA-type glutamate receptors by the neuronal pentraxin receptor, when expressed in HEK293T cells, causes organization of postsynaptic specializations in the heterologous synapse formation assay, testifying to the broad nature of the signals that mediate heterologous synapse formation (12). Strikingly, any given adhesion molecule triggers only either pre- or postsynaptic specializations, but not both, indicating signaling specificity. Most adhesion molecules—with the exception of teneurin splice variants (11)—induce both excitatory and inhibitory synaptic specializations at the same time. Heterologous synapses resemble real synapses and are functional (6, 7). Overall, these observations suggest that specific signaling pathways regulate synapse formation and that the heterologous synapse formation assay provides a plausible and practical paradigm to dissect such signaling pathways, even though it represents an artificial system that lacks much of the specificity of physiological synapse formation.In the present study, we have employed pharmacological inhibitors and molecular interventions to probe the nature of the signals mediating heterologous synapse formation. Our data reveal that multiple parallel protein kinase signaling pathways are required for heterologous synapse formation. We identified a role for both JNK and PKA signaling in the formation of pre- and postsynaptic specializations and found that the PI3 kinase pathway is specifically required for the formation of post- but not presynaptic specializations. Thus, our data provide initial insight into the signaling mechanisms underlying heterologous synapse formation that may be relevant for synapse formation in general.     

Cooperative synapse formation in the neocortex

Neuron morphology plays an important role in defining synaptic connectivity. Clearly, only pairs of neurons with closely positioned axonal and dendritic branches can be synaptically coupled. For excitatory neurons in the cerebral cortex, such axo-dendritic oppositions, termed potential synapses, must be bridged by dendritic spines to form synaptic connections. To explore the rules by which synaptic connections are formed within the constraints imposed by neuron morphology, we compared the distributions of the numbers of actual and potential synapses between pre- and postsynaptic neurons forming different laminar projections in rat barrel cortex. Quantitative comparison explicitly ruled out the hypothesis that individual synapses between neurons are formed independently of each other. Instead, the data are consistent with a cooperative scheme of synapse formation where multiple-synaptic connections between neurons are stabilized while neurons that do not establish a critical number of synapses are not likely to remain synaptically coupled.     

Activity-dependent neurotransmitter-receptor matching at the neuromuscular junction

Signaling in the nervous system requires matching of neurotransmitter receptors with cognate neurotransmitters at synapses. The vertebrate neuromuscular junction is the best studied cholinergic synapse, but the mechanisms by which acetylcholine is matched with acetylcholine receptors are not fully understood. Because alterations in neuronal calcium spike activity alter transmitter specification in embryonic spinal neurons, we hypothesized that receptor expression in postsynaptic cells follows changes in transmitter expression to achieve this specific match. We find that embryonic vertebrate striated muscle cells normally express receptors for glutamate, GABA, and glycine as well as for acetylcholine. As maturation progresses, acetylcholine receptor expression prevails. Receptor selection is altered when early neuronal calcium-dependent activity is perturbed, and remaining receptor populations parallel changes in transmitter phenotype. In these cases, glutamatergic, GABAergic, and glycinergic synaptic currents are recorded from muscle cells, demonstrating that activity regulates matching of transmitters and their receptors in the assembly of functional synapses.     

The immunoglobulin family member dendrite arborization and synapse maturation 1 (Dasm1) controls excitatory synapse maturation

In the developing mammalian brain, a large fraction of excitatory synapses initially contain only N-methyl-D-aspartate receptor and thus are "silent" at the resting membrane potential. As development progresses, synapses acquire alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs). Although this maturation of excitatory synapses has been well characterized, the molecular basis for this developmental change is not known. Here, we report that dendrite arborization and synapse maturation 1 (Dasm1), an Ig superfamily member, controls excitatory synapse maturation. Dasm1 is localized at the excitatory synapses. Suppression of Dasm1 expression by using RNA interference or expression of dominant negative deletion mutants of Dasm1 in hippocampal neurons at late developmental stage specifically impairs AMPA-R-mediated, but not N-methyl-D-aspartate receptor-mediated, synaptic transmission. The ability of Dasm1 to regulate synaptic AMPA-Rs requires its intracellular C-terminal PDZ domain-binding motif, which interacts with two synaptic PDZ domain-containing proteins involved in spine/synapse maturation, Shank and S-SCAM. Moreover, expression of dominant negative deletion mutants of Dasm1 leads to more immature silent synapses. These results suggest that Dasm1, as a transmembrane molecule, likely provides a link to bridge extracellular signals and intracellular signaling complexes in controlling excitatory synapse maturation.     

Expression of a conserved cell-type-specific protein in nerve terminals coincides with synaptogenesis.

Contact of axons with target territories results in the formation of synapses, specific junctional complexes that may represent a final stage of neuronal maturation. Synaptosomal-associated protein 25 (SNAP-25) is a component of particular nerve terminals recently identified in rodent brain. To evaluate the structure and regulation of molecular components of the synapse, we investigated the expression of SNAP-25 in the developing chicken nervous system. Analysis of SNAP-25 cDNA clones demonstrated that the chicken homologue is identical in amino acid sequence to the mouse protein. In chicken retina and neural tube, the onset of SNAP-25 mRNA and protein expression was found to correspond to the time of synaptogenesis. These results suggest that SNAP-25 plays a role in the physiology of mature nerve terminals and that its expression may be regulated by specific cell-cell interactions occurring during synapse formation.     

Differentiation of neuronal growth cones: specialization of filopodial tips for adhesive interactions.

Adhesive contacts made by filopodia of developing neurons are important in neurite growth and in the formation of synaptic junctions. In the present work, filopodial interactions of cultured chicken retina neurons were studied by using video-enhanced contrast, differential interference contrast (VEC-DIC) microscopy and the high-voltage electron microscope (HVEM). Use of the HVEM to examine whole mounts of fixed cells showed that filopodia in older cultures developed an appearance that might be expected of nascent synapses, becoming enlarged at their endings and accumulating organelles resembling synaptic vesicles. VEC-DIC microscopy, used to observe the motility and adhesive properties of filopodia in living cells, showed there was a particularly high affinity between filopodia tips. Contacting filopodia typically repositioned themselves so they could attach at a tip-to-tip position, occasionally bending as much as 90 degrees to achieve this preferred orientation. Interacting filopodia frequently remained together as they pushed or pulled on each other, moved laterally together, or stretched tightly and underwent intense vibratory movements. Such linked motility occurred even when apparent gaps existed between the filopodia. Examination of these gaps with the HVEM revealed filamentous structures linking the apposed membranes. The filamentous links were 10-13 nm in diameter and 30-100 nm long. Although it has not yet been established that the filaments reflect the native configuration of the interconnecting materials, the structures seem likely to be associated with the strongly adhesive behavior of the filopodial tips. The possible significance of these structural and functional properties of filopodia tips to axon growth and synapse formation is discussed.     

Synapse formation and clustering of neuroligin-2 in the absence of GABAA receptors

GABAergic synapses are crucial for brain function, but the mechanisms underlying inhibitory synaptogenesis are unclear. Here, we show that postnatal Purkinje cells (PCs) of GABA_Aα1 knockout (KO) mice express transiently the α3 subunit, leading to the assembly of functional GABA_A receptors and initial normal formation of inhibitory synapses, that are retained until adulthood. Subsequently, down-regulation of the α3 subunit causes a complete loss of GABAergic postsynaptic currents, resulting in a decreased rate of inhibitory synaptogenesis and formation of mismatched synapses between GABAergic axons and PC spines. Notably, the postsynaptic adhesion molecule neuroligin-2 (NL2) is correctly targeted to inhibitory synapses lacking GABA_A receptors and the scaffold molecule gephyrin, but is absent from mismatched synapses, despite innervation by GABAergic axons. Our data indicate that GABA_A receptors are dispensable for synapse formation and maintenance and for targeting NL2 to inhibitory synapses. However, GABAergic signaling appears to be crucial for activity-dependent regulation of synapse density during neuronal maturation.     

Neural regulation of acetylcholine sensitivity in embryonic sympathetic neurons.

The development of transmitter sensitivity is an important component of synaptic differentiation. Despite a wealth of information about the appearance of acetylcholine (AcCho) sensitivity at the neuromuscular junction, the onset and regulation of this critical aspect of synaptogenesis has not previously been examined for synapse formation between neurons. To determine whether there is a role of presynaptic input in the induction of AcCho sensitivity at interneuronal synapses, AcCho-induced currents were measured in embryonic sympathetic neurons before and after synapse formation in vitro. The total AcCho sensitivity of postsynaptic neurons was increased nearly 10-fold after innervation. The effects of innervation are mimicked by medium conditioned by preganglionic neurons, suggesting that presynaptic neurons regulate postsynaptic AcCho sensitivity by release of a soluble factor. These observations provide evidence that presynaptic input regulates neuronal sensitivity to an identified synaptic transmitter.     

The neurexin ligands, neuroligins and leucine-rich repeat transmembrane proteins, perform convergent and divergent synaptic functions in vivo

Synaptic cell adhesion molecules, including the neurexin ligands, neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs), are thought to organize synapse assembly and specify synapse function. To test the synaptic role of these molecules in vivo, we performed lentivirally mediated knockdown of NL3, LRRTM1, and LRRTM2 in CA1 pyramidal cells of WT and NL1 KO mice at postnatal day (P)0 (when synapses are forming) and P21 (when synapses are largely mature). P0 knockdown of NL3 in WT or NL1 KO neurons did not affect excitatory synaptic transmission, whereas P0 knockdown of LRRTM1 and LRRTM2 selectively reduced AMPA receptor-mediated synaptic currents. P0 triple knockdown of NL3 and both LRRTMs in NL1 KO mice yielded greater reductions in AMPA and NMDA receptor-mediated currents, suggesting functional redundancy between NLs and LRRTMs during early synapse development. In contrast, P21 knockdown of LRRTMs did not alter excitatory transmission, whereas NL manipulations supported a role for NL1 in maintaining NMDA receptor-mediated transmission. These results show that neurexin ligands in vivo form a dynamic synaptic cell adhesion network, with compensation between NLs and LRRTMs during early synapse development and functional divergence upon synapse maturation.