Neuroligin-1 Overexpression in Newborn Granule Cells In Vivo

Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons.     

SALM synaptic cell adhesion-like molecules regulate the differentiation of excitatory synapses

Synaptic cell adhesion molecules (CAMs) are known to play key roles in various aspects of synaptic structures and functions, including early differentiation, maintenance, and plasticity. We herein report the identification of a family of cell adhesion-like molecules termed SALM that interacts with the abundant postsynaptic density (PSD) protein PSD-95. SALM2, a SALM isoform, distributes to excitatory, but not inhibitory, synaptic sites. Overexpression of SALM2 increases the number of excitatory synapses and dendritic spines. Mislocalized expression of SALM2 disrupts excitatory synapses and dendritic spines. Bead-induced direct aggregation of SALM2 results in coclustering of PSD-95 and other postsynaptic proteins, including GKAP and AMPA receptors. Knockdown of SALM2 by RNA interference reduces the number of excitatory synapses and dendritic spines and the frequency, but not amplitude, of miniature excitatory postsynaptic currents. These results suggest that SALM2 is an important regulator of the differentiation of excitatory synapses.     

A Combined Transgenic Proteomic Analysis and Regulated Trafficking of Neuroligin-2

Synapses, the basic units of communication in the brain, require complex molecular machinery for neurotransmitter release and reception. Whereas numerous components of excitatory postsynaptic sites have been identified, relatively few proteins are known that function at inhibitory postsynaptic sites. One such component is neuroligin-2 (NL2), an inhibitory synapse-specific cell surface protein that functions in cell adhesion and synaptic organization via binding to neurexins. In this study, we used a transgenic tandem affinity purification and mass spectrometry strategy to isolate and characterize NL2-associated complexes. Complexes purified from brains of transgenic His₆-FLAG-YFP-NL2 mice showed enrichment in the Gene Ontology terms cell-cell signaling and synaptic transmission relative to complexes purified from wild type mice as a negative control. In addition to expected components including GABA receptor subunits and gephyrin, several novel proteins were isolated in association with NL2. Based on the presence of multiple components involved in trafficking and endocytosis, we showed that NL2 undergoes dynamin-dependent endocytosis in response to soluble ligand and colocalizes with VPS35 retromer in endosomes. Inhibitory synapses in brain also present a particular challenge for imaging. Whereas excitatory synapses on spines can be imaged with a fluorescent cell fill, inhibitory synapses require a molecular tag. We find the His₆-FLAG-YFP-NL2 to be a suitable tag, with the unamplified YFP signal localizing appropriately to inhibitory synapses in multiple brain regions including cortex, hippocampus, thalamus, and basal ganglia. Altogether, we characterize NL2-associated complexes, demonstrate regulated trafficking of NL2, and provide tools for further proteomic and imaging studies of inhibitory synapses.     

Long-term depression-inducing stimuli promote cleavage of the synaptic adhesion molecule NGL-3 through NMDA receptors,matrix metalloproteinases and presenilin/γ-secretase

Long-term depression (LTD) reduces the functional strength of excitatory synapses through mechanisms that include the removal of AMPA glutamate receptors from the postsynaptic membrane. LTD induction is also known to result in structural changes at excitatory synapses, including the shrinkage of dendritic spines. Synaptic adhesion molecules are thought to contribute to the development, function and plasticity of neuronal synapses largely through their trans-synaptic adhesions. However, little is known about how synaptic adhesion molecules are altered during LTD. We report here that NGL-3 (netrin-G ligand-3), a postsynaptic adhesion molecule that trans-synaptically interacts with the LAR family of receptor tyrosine phosphatases and intracellularly with the postsynaptic scaffolding protein PSD-95, undergoes a proteolytic cleavage process. NGL-3 cleavage is induced by NMDA treatment in cultured neurons and low-frequency stimulation in brain slices and requires the activities of NMDA glutamate receptors, matrix metalloproteinases (MMPs) and presenilin/γ-secretase. These results suggest that NGL-3 is a novel substrate of MMPs and γ-secretase and that NGL-3 cleavage may regulate synaptic adhesion during LTD.     

Multiple association states between glycine receptors and gephyrin identified by SPT analysis

The scaffolding protein gephyrin is known to anchor glycine receptors (GlyR) at synapses and to participate in the dynamic equilibrium between synaptic and extrasynaptic GlyR in the neuronal membrane. Here we investigated the properties of this interaction in cells cotransfected with YFP-tagged gephyrin and GlyR subunits possessing an extracellular myc-tag. In HeLa cells and young neurons, single particle tracking was used to follow in real time individual GlyR, labeled with quantum dots, traveling into and out of gephyrin clusters. Analysis of the diffusion properties of two GlyR subunit types--able or unable to bind gephyrin--gave access to the association states of GlyR with its scaffolding protein. Our results indicated that an important portion of GlyR could be linked to a few molecules of gephyrin outside gephyrin clusters. This emphasizes the role of scaffolding proteins in the extrasynaptic membrane and supports the implication of gephyrin-gephyrin interactions in the stabilization of GlyR at synapses. The kinetic parameters controlling the equilibrium between GlyR inside and outside clusters were also characterized. Within clusters, we identified two subpopulations of GlyR with distinct degrees of stabilization between receptors and scaffolding proteins.     

Diffusion barriers constrain receptors at synapses

The flux of neurotransmitter receptors in and out of synapses depends on receptor interaction with scaffolding molecules. However, the crowd of transmembrane proteins and the rich cytoskeletal environment may constitute obstacles to the diffusion of receptors within the synapse. To address this question, we studied the membrane diffusion of the γ-aminobutyric acid type A receptor (GABA(A)R) subunits clustered (γ2) or not (α5) at inhibitory synapses in rat hippocampal dissociated neurons. Relative to the extrasynaptic region, γ2 and α5 showed reduced diffusion and increased confinement at both inhibitory and excitatory synapses but they dwelled for a short time at excitatory synapses. In contrast, γ2 was ～3-fold more confined and dwelled ～3-fold longer in inhibitory synapses than α5, indicating faster synaptic escape of α5. Furthermore, using a gephyrin dominant-negative approach, we showed that the increased residency time of γ2 at inhibitory synapses was due to receptor-scaffold interactions. As shown for GABA(A)R, the excitatory glutamate receptor 2 subunit (GluA2) of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) had lower mobility in both excitatory and inhibitory synapses but a higher residency time at excitatory synapses. Therefore barriers impose significant diffusion constraints onto receptors at synapses where they accumulate or not. Our data further reveal that the confinement and the dwell time but not the diffusion coefficient report on the synapse specific sorting, trapping and accumulation of receptors.     

Casting a net on dendritic spines: the extracellular matrix and its receptors

Dendritic spines are dynamic structures that accommodate the majority of excitatory synapses in the brain and are influenced by extracellular signals from presynaptic neurons, glial cells, and the extracellular matrix (ECM). The ECM surrounds dendritic spines and extends into the synaptic cleft, maintaining synapse integrity as well as mediating trans-synaptic communications between neurons. Several scaffolding proteins and glycans that compose the ECM form a lattice-like network, which serves as an attractive ground for various secreted glycoproteins, lectins, growth factors, and enzymes. ECM components can control dendritic spines through the interactions with their specific receptors or by influencing the functions of other synaptic proteins. In this review, we focus on ECM components and their receptors that regulate dendritic spine development and plasticity in the normal and diseased brain.     

Gephyrin regulates GABAergic and glutamatergic synaptic transmission in hippocampal cell cultures

Gephyrin is a scaffold protein essential for stabilizing glycine and GABA(A) receptors at inhibitory synapses. Here, recombinant intrabodies against gephyrin (scFv-gephyrin) were used to assess whether this protein exerts a transynaptic action on GABA and glutamate release. Pair recordings from interconnected hippocampal cells in culture revealed a reduced probability of GABA release in scFv-gephyrin-transfected neurons compared with controls. This effect was associated with a significant decrease in VGAT, the vesicular GABA transporter, and in neuroligin 2 (NLG2), a protein that, interacting with neurexins, ensures the cross-talk between the post- and presynaptic sites. Interestingly, hampering gephyrin function also produced a significant reduction in VGLUT, the vesicular glutamate transporter, an effect accompanied by a significant decrease in frequency of miniature excitatory postsynaptic currents. Overexpressing NLG2 in gephyrin-deprived neurons rescued GABAergic but not glutamatergic innervation, suggesting that the observed changes in the latter were not due to a homeostatic compensatory mechanism. Pulldown experiments demonstrated that gephyrin interacts not only with NLG2 but also with NLG1, the isoform enriched at excitatory synapses. These results suggest a key role of gephyrin in regulating transynaptic signaling at both inhibitory and excitatory synapses.     

Gephyrin-independent GABA(A)R mobility and clustering during plasticity

The activity-dependent modulation of GABA-A receptor (GABA(A)R) clustering at synapses controls inhibitory synaptic transmission. Several lines of evidence suggest that gephyrin, an inhibitory synaptic scaffold protein, is a critical factor in the regulation of GABA(A)R clustering during inhibitory synaptic plasticity induced by neuronal excitation. In this study, we tested this hypothesis by studying relative gephyrin dynamics and GABA(A)R declustering during excitatory activity. Surprisingly, we found that gephyrin dispersal is not essential for GABA(A)R declustering during excitatory activity. In cultured hippocampal neurons, quantitative immunocytochemistry showed that the dispersal of synaptic GABA(A)Rs accompanied with neuronal excitation evoked by 4-aminopyridine (4AP) or N-methyl-D-aspartic acid (NMDA) precedes that of gephyrin. Single-particle tracking of quantum dot labeled-GABA(A)Rs revealed that excitation-induced enhancement of GABA(A)R lateral mobility also occurred before the shrinkage of gephyrin clusters. Physical inhibition of GABA(A)R lateral diffusion on the cell surface and inhibition of a Ca(2+) dependent phosphatase, calcineurin, completely eliminated the 4AP-induced decrease in gephyrin cluster size, but not the NMDA-induced decrease in cluster size, suggesting the existence of two different mechanisms of gephyrin declustering during activity-dependent plasticity, a GABA(A)R-dependent regulatory mechanism and a GABA(A)R-independent one. Our results also indicate that GABA(A)R mobility and clustering after sustained excitatory activity is independent of gephyrin.     

Posttranslational Modifications Regulate the Postsynaptic Localization of PSD-95

The postsynaptic density (PSD) consists of a lattice-like array of interacting proteins that organizes and stabilizes synaptic receptors, ion channels, structural proteins, and signaling molecules required for normal synaptic transmission and synaptic function. The scaffolding and hub protein postsynaptic density protein-95 (PSD-95) is a major element of central chemical synapses and interacts with glutamate receptors, cell adhesion molecules, and cytoskeletal elements. In fact, PSD-95 can regulate basal synaptic stability as well as the activity-dependent structural plasticity of the PSD and, therefore, of the excitatory chemical synapse. Several studies have shown that PSD-95 is highly enriched at excitatory synapses and have identified multiple protein structural domains and protein-protein interactions that mediate PSD-95 function and trafficking to the postsynaptic region. PSD-95 is also a target of several signaling pathways that induce posttranslational modifications, including palmitoylation, phosphorylation, ubiquitination, nitrosylation, and neddylation; these modifications determine the synaptic stability and function of PSD-95 and thus regulate the fates of individual dendritic spines in the nervous system. In the present work, we review the posttranslational modifications that regulate the synaptic localization of PSD-95 and describe their functional consequences. We also explore the signaling pathways that induce such changes.     

外周输入依赖的嗅球颗粒细胞的突触结构可塑性

活动依赖的突触结构可塑性是学习和记忆的基础.哺乳动物,尤其是啮齿类动物,具有高度发达的嗅觉系统和惊人的气味学习和记忆能力.本研究以CNGA2敲除而导致外周输入缺失的小鼠为模型,研究嗅球内活动依赖的突触结构可塑性.利用特异性的突触前和突触后标记物,发现外周输入缺失减少了突触标记蛋白突触素(synaptophysin)和抑制性突触标记蛋白桥蛋白(gephyrin)在嗅球外网状层和颗粒细胞层中的表达;兴奋性突触标记蛋白囊泡谷氨酸转运蛋白1(VGluT1)的表达水平只在外网状层中有显著下降,而在颗粒细胞层中没有明显变化.进一步通过活体质粒电转标记嗅球颗粒细胞后发现,CNGA2敲除小鼠颗粒细胞上位于外网状层中的远端树突棘密度显著减小,而位于颗粒细胞层中的近端树突棘密度没有明显变化.这些结果表明颗粒细胞上的树-树突触具有对外周活动依赖的结构可塑性,而轴-树突触则无.     

The adhesion protein IgSF9b is coupled to neuroligin 2 via S-SCAM to promote inhibitory synapse development

Synaptic adhesion molecules regulate diverse aspects of synapse formation and maintenance. Many known synaptic adhesion molecules localize at excitatory synapses, whereas relatively little is known about inhibitory synaptic adhesion molecules. Here we report that IgSF9b is a novel, brain-specific, homophilic adhesion molecule that is strongly expressed in GABAergic interneurons. IgSF9b was preferentially localized at inhibitory synapses in cultured rat hippocampal and cortical interneurons and was required for the development of inhibitory synapses onto interneurons. IgSF9b formed a subsynaptic domain distinct from the GABA_A receptor– and gephyrin-containing domain, as indicated by super-resolution imaging. IgSF9b was linked to neuroligin 2, an inhibitory synaptic adhesion molecule coupled to gephyrin, via the multi-PDZ protein S-SCAM. IgSF9b and neuroligin 2 could reciprocally cluster each other. These results suggest a novel mode of inhibitory synaptic organization in which two subsynaptic domains, one containing IgSF9b for synaptic adhesion and the other containing gephyrin and GABA_A receptors for synaptic transmission, are interconnected through S-SCAM and neuroligin 2.     

Loss of sensory input causes rapid structural changes of inhibitory neurons in adult mouse visual cortex

A fundamental property of neuronal circuits is the ability to adapt to altered sensory inputs. It is well established that the functional synaptic changes underlying this adaptation are reflected by structural modifications in excitatory neurons. In contrast, the degree to which structural plasticity in inhibitory neurons accompanies functional changes is less clear. Here, we use two-photon imaging to monitor the fine structure of inhibitory neurons in mouse visual cortex after deprivation induced by retinal lesions. We find that a subset of inhibitory neurons carry dendritic spines, which form glutamatergic synapses. Removal of visual input correlates with a rapid and lasting reduction in the number of inhibitory cell spines. Similar to the effects seen for dendritic spines, the number of inhibitory neuron boutons dropped sharply after retinal lesions. Together, these data suggest that structural changes in inhibitory neurons may precede structural changes in excitatory circuitry, which ultimately result in functional adaptation following sensory deprivation.     

Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2

Neuroligins enhance synapse formation in vitro, but surprisingly are not required for the generation of synapses in vivo. We now show that in cultured neurons, neuroligin-1 overexpression increases excitatory, but not inhibitory, synaptic responses, and potentiates synaptic NMDAR/AMPAR ratios. In contrast, neuroligin-2 overexpression increases inhibitory, but not excitatory, synaptic responses. Accordingly, deletion of neuroligin-1 in knockout mice selectively decreases the NMDAR/AMPAR ratio, whereas deletion of neuroligin-2 selectively decreases inhibitory synaptic responses. Strikingly, chronic inhibition of NMDARs or CaM-Kinase II, which signals downstream of NMDARs, suppresses the synapse-boosting activity of neuroligin-1, whereas chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin-2. Taken together, these data indicate that neuroligins do not establish, but specify and validate, synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses. This hypothesis reconciles the overexpression and knockout phenotypes and suggests that neuroligins contribute to the use-dependent formation of neural circuits.     

The synaptic scaffold protein MPP2 interacts with GABAA receptors at the periphery of the postsynaptic density of glutamatergic synapses

Recent advances in imaging technology have highlighted that scaffold proteins and receptors are arranged in subsynaptic nanodomains. The synaptic membrane-associated guanylate kinase (MAGUK) scaffold protein membrane protein palmitoylated 2 (MPP2) is a component of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor–associated protein complexes and also binds to the synaptic cell adhesion molecule SynCAM 1. Using superresolution imaging, we show that—like SynCAM 1—MPP2 is situated at the periphery of the postsynaptic density (PSD). In order to explore MPP2-associated protein complexes, we used a quantitative comparative proteomics approach and identified multiple γ-aminobutyric acid (GABA)_A receptor subunits among novel synaptic MPP2 interactors. In line with a scaffold function for MPP2 in the assembly and/or modulation of intact GABA_A receptors, manipulating MPP2 expression had effects on inhibitory synaptic transmission. We further show that GABA_A receptors are found together with MPP2 in a subset of dendritic spines and thus highlight MPP2 as a scaffold that serves as an adaptor molecule, linking peripheral synaptic elements critical for inhibitory regulation to central structures at the PSD of glutamatergic synapses.

This study shows that the MAGUK scaffold protein MPP2 is located at the periphery of postsynaptic densities in excitatory neurons, where it interacts with GABA-A receptors, thereby serving as a functional adaptor that links excitatory and inhibitory components of synaptic transmission at glutamatergic synapses.     

The enigma of transmitter-selective receptor accumulation at developing inhibitory synapses

The control of synaptic inhibition is crucial for normal brain function. More than 20 years ago, glycine and gamma-aminobutyric acid (GABA) were shown to be the two major inhibitory neurotransmitters. They can be released independently from different terminals or co-released from the same terminal to activate postsynaptic glycine and GABA(A) receptors. The anchoring protein gephyrin is involved in the postsynaptic accumulation of both glycine and GABA(A) receptors. In lower brain regions, both receptors can be concentrated in synapses, whereas in higher brain regions, glycine receptors are mostly excluded from postsynaptic sites. The activation of glycine and/or GABA(A) receptors determines the strength and precise timing of inhibition. Therefore, tight regulation of postsynaptic glycine versus GABA(A) receptor localization is crucial for optimizing synaptic inhibition in neurons. This review focuses on recent findings and discusses questions concerning the specificity of postsynaptic inhibitory neurotransmitter receptor accumulation during inhibitory synapse formation and development.     

Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin

The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABA(A) receptor (GABA(A)R) subtypes. We report a direct interaction between the GABA(A)R α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA(A)R α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABA(A)R α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA(A)Rs in recombinant systems. Interestingly, motifs responsible for interactions with GABA(A)R α2, GABA(A)R α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABA(A)R α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABA(A)R α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABA(A)R α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.     

Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice

Collybistin (Cb) is a brain-specific guanine nucleotide exchange factor that has been implicated in plasma membrane targeting of the postsynaptic scaffolding protein gephyrin found at glycinergic and GABAergic synapses. Here we show that Cb-deficient mice display a region-specific loss of postsynaptic gephyrin and GABA(A) receptor clusters in the hippocampus and the basolateral amygdala. Cb deficiency is accompanied by significant changes in hippocampal synaptic plasticity, due to reduced dendritic GABAergic inhibition. Long-term potentiation is enhanced, and long-term depression reduced, in Cb-deficient hippocampal slices. Consistent with the anatomical and electrophysiological findings, the animals show increased levels of anxiety and impaired spatial learning. Together, our data indicate that Cb is essential for gephyrin-dependent clustering of a specific set of GABA(A) receptors, but not required for glycine receptor postsynaptic localization.     

Neuronal Synapse Formation Induced by Microglia and Interleukin 10

Recently, it was found that microglia regulated synaptic remodeling of the developing brain, but their mechanisms have not been well understood. In this study, the action of microglia on neuronal synapse formation was investigated, and the primary target of microglial processes was discovered. When the developing microglia were applied to cultured hippocampal neurons without direct contact, the numbers of dendritic spines and excitatory and inhibitory synapses significantly increased. In order to find out the main factor for synaptic formation, the effects of cytokines released from microglia were examined. When recombinant proteins of cytokines were applied to neuronal culture media, interleukin 10 increased the numbers of dendritic spines in addition to excitatory and inhibitory synapses. Interestingly, without external stimuli, the amount of interleukin 10 released from the intact microglia appeared to be sufficient for the induction of synaptic formation. The neutralizing antibodies of interleukin 10 receptors attenuated the induction of the synaptic formation by microglia. The expression of interleukin 10 receptor was newly found in the hippocampal neurons of early developmental stage. When interleukin 10 receptors on the hippocampal neurons were knocked down with specific shRNA, the induction of synaptic formation by microglia and interleukin 10 disappeared. Pretreatment with lipopolysaccharide inhibited microglia from inducing synaptic formation, and interleukin 1β antagonized the induction of synaptic formation by interleukin 10. In conclusion, the developing microglia regulated synaptic functions and neuronal development through the interactions of the interleukin 10 released from the microglia with interleukin 10 receptors expressed on the hippocampal neurons.