ABSTRACT
Selective synapse development determines how complex neuronal networks in the brain are formed. Complexes of postsynaptic neuroligins and LRRTMs with presynaptic neurexins contribute widely to excitatory synapse development, and mutations in these gene families increase the risk of developing psychiatric disorders. We find that LRRTM4 has distinct presynaptic binding partners, heparan sulfate proteoglycans (HSPGs). HSPGs are required to mediate the synaptogenic activity of LRRTM4. LRRTM4 shows highly selective expression in the brain. Within the hippocampus, we detected LRRTM4 specifically at excitatory postsynaptic sites on dentate gyrus granule cells. LRRTM4(-/-) dentate gyrus granule cells, but not CA1 pyramidal cells, exhibit reductions in excitatory synapse density and function. Furthermore, LRRTM4(-/-) dentate gyrus granule cells show impaired activity-regulated AMPA receptor trafficking. These results identifying cell-type-specific functions and multiple presynaptic binding partners for different LRRTM family members reveal an unexpected complexity in the design and function of synapse-organizing proteins.
Subject(s)
Dentate Gyrus/cytology , Excitatory Postsynaptic Potentials/physiology , Heparan Sulfate Proteoglycans/metabolism , Nerve Tissue Proteins/metabolism , Neurons/physiology , Synapses/metabolism , Amino Acids/metabolism , Animals , Animals, Newborn , Cells, Cultured , Chlorocebus aethiops , Disks Large Homolog 4 Protein , Embryo, Mammalian , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/genetics , Guanylate Kinases , Heparan Sulfate Proteoglycans/genetics , Humans , In Vitro Techniques , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Mutation/genetics , Nerve Tissue Proteins/genetics , Neurons/cytology , Neurons/ultrastructure , Protein Transport/genetics , Rats , Receptors, AMPA/metabolism , Synapses/ultrastructure , Synapsins/metabolismABSTRACT
Although the contribution of postsynaptic mechanisms to long-term synaptic plasticity has been studied extensively, understanding the contribution of presynaptic modifications to this process lags behind, primarily because of a lack of techniques with which to directly and quantifiably measure neurotransmitter release from synaptic terminals. Here, we developed a method to measure presynaptic activity through the biotinylation of vesicular transporters in vesicles fused with presynaptic membranes during neurotransmitter release. This method allowed us for the first time to selectively quantify the spontaneous or evoked release of glutamate or GABA at their respective synapses. Using this method to investigate presynaptic changes during the expression of group I metabotropic glutamate receptor (mGluR1/5)-mediated long-term depression (LTD) in cultured rat hippocampal neurons, we discovered that this form of LTD was associated with increased presynaptic release of glutamate, despite reduced miniature EPSCs measured with whole-cell recording. Moreover, we found that specific blockade of AMPA receptor (AMPAR) endocytosis with a membrane-permeable GluR2-derived peptide not only prevented the expression of LTD but also eliminated LTD-associated increase in presynaptic release. Thus, our work not only demonstrates that mGluR1/5-mediated LTD is associated with increased endocytosis of postsynaptic AMPARs but also reveals an unexpected homeostatic/compensatory increase in presynaptic release. In addition, this study indicates that biotinylation of vesicular transporters in live cultured neurons is a valuable tool for studying presynaptic function.
Subject(s)
Long-Term Synaptic Depression/physiology , Neurons/cytology , Neurotransmitter Agents/metabolism , Presynaptic Terminals/metabolism , Receptors, Metabotropic Glutamate/metabolism , Analysis of Variance , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Biophysics , Biotin/analogs & derivatives , Biotin/pharmacology , Biotinylation , Electric Stimulation , Endocytosis/drug effects , Endocytosis/physiology , Excitatory Amino Acid Antagonists/pharmacology , GABA-A Receptor Antagonists/pharmacology , Hippocampus/cytology , Long-Term Synaptic Depression/drug effects , Methoxyhydroxyphenylglycol/analogs & derivatives , Methoxyhydroxyphenylglycol/pharmacology , Microtubule-Associated Proteins/metabolism , Neurons/physiology , Organ Culture Techniques , Patch-Clamp Techniques , Peptides/pharmacology , Potassium/pharmacology , Protein Transport/drug effects , Protein Transport/physiology , Quinoxalines/pharmacology , Rats , Receptors, AMPA/chemistry , Receptors, Transferrin/metabolism , Sodium Channel Blockers/pharmacology , Succinimides/pharmacology , Synaptotagmin I/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Tetrodotoxin/pharmacology , Vesicular Glutamate Transport Protein 1/metabolism , Vesicular Inhibitory Amino Acid Transport Proteins/metabolismABSTRACT
Dynamics of GABAergic synaptic components have been studied previously over milliseconds to minutes, revealing mobility of postsynaptic scaffolds and receptors. Here we image inhibitory synapses containing fluorescently tagged postsynaptic scaffold Gephyrin, together with presynaptic vesicular GABA transporter (VGAT) or postsynaptic GABA(A) receptor γ2 subunit (GABA(A)Rγ2), over seconds to days in cultured rat hippocampal neurons, revealing modes of inhibitory synapse formation and remodeling. Entire synapses were mobile, translocating rapidly within a confined region and exhibiting greater nonstochastic motion over multihour periods. Presynaptic and postsynaptic components moved in unison, maintaining close apposition while translocating distances of several micrometers. An observed flux in the density of synaptic puncta partially resulted from the apparent merging and splitting of preexisting clusters. De novo formation of inhibitory synapses was observed, marked by the appearance of stably apposed Gephyrin and VGAT clusters at sites previously lacking either component. Coclustering of GABA(A)Rγ2 supports the identification of such new clusters as synapses. Nascent synapse formation occurred by gradual accumulation of components over several hours, with VGAT clustering preceding that of Gephyrin and GABA(A)Rγ2. Comparing VGAT labeling by active uptake of a luminal domain antibody with post hoc immunocytochemistry indicated that recycling vesicles from preexisting boutons significantly contribute to vesicle pools at the majority of new inhibitory synapses. Although new synapses formed primarily on dendrite shafts, some also formed on dendritic protrusions, without apparent interconversion. Altogether, the long-term imaging of GABAergic presynaptic and postsynaptic components reveals complex dynamics and perpetual remodeling with implications for mechanisms of assembly and synaptic integration.
Subject(s)
Dendrites/physiology , Neurons/cytology , Neurons/physiology , Presynaptic Terminals/physiology , Synapses/physiology , Synaptic Vesicles/physiology , Animals , Carrier Proteins/genetics , Cells, Cultured , Dendrites/ultrastructure , Embryo, Mammalian , Female , Hippocampus/cytology , Humans , Luminescent Proteins/genetics , Male , Membrane Proteins/genetics , Microscopy, Confocal/methods , Microtubule-Associated Proteins/metabolism , Mutagenesis, Site-Directed/methods , Neural Inhibition/physiology , Neurons/metabolism , Presynaptic Terminals/metabolism , Protein Transport/physiology , Rats , Receptors, GABA-A/genetics , Synapses/ultrastructure , Synaptic Vesicles/metabolism , Time Factors , Transfection/methods , Vesicular Inhibitory Amino Acid Transport Proteins/metabolismABSTRACT
Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.
