SUMMARY Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA2 receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA2 receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA2 receptor signaling.

Neurotransmission depends on the exocytic fusion of synaptic vesicles (SVs) and their subsequent reformation either by clathrinmediated endocytosis or budding from bulk endosomes. How synapses are able to rapidly recycle SVs to maintain SV pool size, yet preserve their compositional identity, is poorly understood. We demonstrate that deletion of the endocytic adaptor stonin 2 (Stn2) in mice compromises the fidelity of SV protein sorting, whereas the apparent speed of SV retrieval is increased. Loss of Stn2 leads to selective missorting of synaptotagmin 1 to the neuronal surface, an elevated SV pool size, and accelerated SV protein endocytosis. The latter phenotype is mimicked by overexpression of endocytosisdefective variants of synaptotagmin 1. Increased speed of SV protein retrieval in the absence of Stn2 correlates with an upregulation of SV reformation from bulk endosomes. Our results are consistent with a model whereby Stn2 is required to preserve SV protein composition but is dispensable for maintaining the speed of SV recycling.pHluorin | hippocampus | mossy fibers N eurotransmission involves the calcium-regulated fusion of synaptic vesicles (SVs), a process that requires the SV calcium sensor synaptotagmin (Syt) (1) and components of the active zone (AZ) that define sites of neurotransmitter release (2). Postexocytic fusion SV membranes are retrieved by endocytosis from the plasma membrane (2-5) to regenerate SVs of the correct size and composition (6). Alternatively, SVs can also be reformed from large plasma membrane infoldings and from endosomes (7) via a brefeldin A-sensitive pathway (8) that may become particularly important under conditions of sustained high-level activity and involves endosomal adaptor complexes such as adaptor protein complex 1 (AP-1) (9, 10). Maintenance of the SV pool requires that the number of recycled SVs closely matches those having undergone exocytosis. As SVs are characterized by a precise protein composition (11) that at least for some SV proteins including Syt1, VGLUT1, and SV2A displays little intervesicle variation (12), molecular mechanisms must exist to control the fidelity of SV protein sorting while maintaining the speed of exo-endocytosis.The mechanisms by which exo-endocytic balance and the fidelity of SV protein sorting are maintained are unknown. One possibility is that retrieval of SV proteins involves clustering (13,14), which would alleviate a need for specific sorting, even if multiple pathways of SV reformation are used (9, 10). However, data based on imaging of SV proteins tagged with the GFPderived pH sensor pHluorin indicate that exocytosed and newly endocytosed SV proteins are not identical, suggesting that intermixing between exocytosed and preexisting surface pools of vesicle proteins occurs (15,16). If SVs lose their identity over multiple rounds of exo-endocytosis, specific mechanisms should exist for the cargo-specific recognition and sorting of SV proteins, e.g., by adaptors (4, 15).Several components of the endocytic machinery may function as a...

The amygdala is a brain area critical for the formation of fear memories. However, the nature of the teaching signal(s) that drive plasticity in the amygdala are still under debate. Here, we use optogenetic methods to investigate the contribution of ventral tegmental area (VTA) dopamine neurons to auditory-cued fear learning in male mice. Using anterograde and retrograde labeling, we found that a sparse and relatively evenly distributed population of VTA neurons projects to the basal amygdala (BA). In vivo optrode recordings in behaving mice showed that many VTA neurons, among them putative dopamine neurons, are excited by footshocks, and acquire a response to auditory stimuli during fear learning. Combined cfos imaging and retrograde labeling in dopamine transporter (DAT) Cre mice revealed that a large majority of BA projectors (.95%) are dopamine neurons, and that BA projectors become activated by the tone-footshock pairing of fear learning protocols. Finally, silencing VTA dopamine neurons, or their axon terminals in the BA during the footshock, reduced the strength of fear memory as tested 1 d later, whereas silencing the VTA-central amygdala (CeA) projection had no effect. Thus, VTA dopamine neurons projecting to the BA contribute to fear memory formation, by coding for the saliency of the footshock event and by signaling such events to the basal amygdala.

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IRSp53 is an essential intermediate between the activation of Rac and Cdc42GTPases and the formation of cellular protrusions; it affects cell shape by coupling membrane-deforming activity with the actin cytoskeleton. IRSp53 is highly expressed in neurons where it is also an abundant component of the postsynaptic density (PSD). Here we analyze the physiological function of this protein in the mouse brain by generating IRSp53-deficient mice. Neurons in the hippocampus of young and adult knock-out (KO) mice do not exhibit morphological abnormalities in vivo. Conversely, primary cultured neurons derived from IRSp53 KO mice display retarded dendritic development in vitro. On a molecular level, Eps8 cooperates with IRSp53 to enhance actin bundling and interacts with IRSp53 in developing neurons. However, postsynaptic Shank proteins which are expressed at high levels in mature neurons compete with Eps8 to block actin bundling. In electrophysiological experiments the removal of IRSp53 increases synaptic plasticity as measured by augmented long term potentiation and pairedpulse facilitation. A primarily postsynaptic role of IRSp53 is underscored by the decreased size of the PSDs, which display increased levels of N-methyl-D-aspartate receptor subunits in IRSp53 KO animals. Our data suggest that the incorporation of IRSp53 into the PSD enables the protein to limit the number of postsynaptic glutamate receptors and thereby affect synaptic plasticity rather than dendritic morphology. Consistent with altered synaptic plasticity, IRSp53-deficient mice exhibit cognitive deficits in the contextual fear-conditioning paradigm.Rho GTPases such as Cdc42, Rac, and Rho control key events in neuronal cell biology, including the generation of neuronal polarity and morphology, establishment of dendritic spines, the generation of postsynaptic specializations and synaptic plasticity (1, 2). Specificity in these processes is thought to arise through control of different downstream targets which are recognized and activated by the active, GTP-bound forms of Rho family members. The insulin receptor substrate of 53 kDa (IRSp53) 3 is an essential mediator between activated Rac or Cdc42 and the formation of lamellipodia or filopodia, respectively. GTPase binding to IRSp53 enables interactions of its SH3 domain with downstream effectors WAVE2, Mena, Eps8, or N-WASP, all of which are known regulators of actin dynamics (3-6). In addition, the N-terminal IRSp53/missing in metastasis homology domain of IRSp53 assists in generating cellular protrusions by bundling actin filaments (5, 7, 8) and promoting membrane curvature (9, 10). Expression of IRSp53 is particularly high in the brain, and consequently IRSp53 contributes to the formation of dendritic spines in the cultured hippocampal neuron model (11).Via the SH3 domain and a C-terminal PDZ binding motif, IRSp53 also bridges postsynaptic shank and PSD-95 family members (11)(12)(13)(14). A significant enrichment in the postsynaptic density (PSD) of excitatory synapses suggests that Rac/Cdc42 signalin...