Visual information is relayed from the eye to the brain via retinal ganglion cell (RGC) axons. Mice lacking NRP1 or NRP1-binding VEGF-A isoforms have defective RGC axon organisation alongside brain vascular defects. It is not known whether axonal defects are caused exclusively by defective VEGF-A signalling in RGCs or are exacerbated by abnormal vascular morphology. Targeted NRP1 ablation in RGCs with a Brn3bCre knock-in allele reduced axonal midline crossing at the optic chiasm and optic tract fasciculation. In contrast, Tie2-Cre-mediated endothelial NRP1 ablation induced axon exclusion zones in the optic tracts without impairing axon crossing. Similar defects were observed in Vegfa120/120 and Vegfa188/188 mice, which have vascular defects as a result of their expression of single VEGF-A isoforms. Ectopic midline vascularisation in endothelial Nrp1 and Vegfa188/188 mutants caused additional axonal exclusion zones within the chiasm. As in vitro and in vivo assays demonstrated that vessels do not repel axons, abnormally large or ectopically positioned vessels are likely to present physical obstacles to axon growth. We conclude that proper axonal wiring during brain development depends on the precise molecular control of neurovascular co-patterning.

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the best-described monogenic cause of autism. CGG-repeat expansion in the FMR1 gene leads to FMR1 silencing, loss-of-expression of the Fragile X Mental Retardation Protein (FMRP), and is a common cause of FXS. Missense mutations in the FMR1 gene were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS caused by this mutation, we generated a knock-in mouse model (Fmr1R138Q) expressing the FMRP-R138Q protein. We demonstrate that, in the hippocampus of the Fmr1R138Q mice, neurons show an increased spine density associated with synaptic ultrastructural defects and increased AMPA receptor-surface expression. Combining biochemical assays, high-resolution imaging, electrophysiological recordings, and behavioural testing, we also show that the R138Q mutation results in impaired hippocampal long-term potentiation and socio-cognitive deficits in mice. These findings reveal the functional impact of the FMRP-R138Q mutation in a mouse model of FXS.

Regulation of synaptic neurotransmitter receptor content is a fundamental mechanism for tuning synaptic efficacy during experience-dependent plasticity and behavioral adaptation. However, experimental approaches to track and modify receptor movements in integrated experimental systems are limited. Exploiting AMPA-type glutamate receptors (AMPARs) as a model, we generated a knock-in mouse expressing the biotin acceptor peptide (AP) tag on the GluA2 extracellular N-terminal. Cell-specific introduction of biotin ligase allows the use of monovalent or tetravalent avidin variants to respectively monitor or manipulate the surface mobility of endogenous AMPAR containing biotinylated AP–GluA2 in neuronal subsets. AMPAR immobilization precluded the expression of long-term potentiation and formation of contextual fear memory, allowing target-specific control of the expression of synaptic plasticity and animal behavior. The AP tag knock-in model offers unprecedented access to resolve and control the spatiotemporal dynamics of endogenous receptors, and opens new avenues to study the molecular mechanisms of synaptic plasticity and learning.

Glutamate secretion at excitatory synapses is tightly regulated to allow for the precise tuning of synaptic strength. Vesicular Glutamate Transporters (VGLUT) accumulate glutamate into synaptic vesicles (SV) and thereby regulate quantal size. Further, the number of release sites and the release probability of SVs maybe regulated by the organization of active-zone proteins and SV clusters. In the present work, we uncover a mechanism mediating an increased SV clustering through the interaction of VGLUT1 second proline-rich domain, endophilinA1 and intersectin1. This strengthening of SV clusters results in a combined reduction of axonal SV super-pool size and miniature excitatory events frequency. Our findings support a model in which clustered vesicles are held together through multiple weak interactions between Src homology three and proline-rich domains of synaptic proteins. In mammals, VGLUT1 gained a proline-rich sequence that recruits endophilinA1 and turns the transporter into a regulator of SV organization and spontaneous release.

Consolidation of recent memory depends on hippocampal activities during resting periods that immediately follows the memory encoding. There, Slow Save Sleep phases appear as privileged periods for memory consolidation as hosting the ripple activities, which are fast oscillations generated within the hippocampus whose inactivation leads to memory impairment. If a strong correlation exists between these replays of recent experience and the persistence of behavioural adaptations, the mobilisation, the localization and the importance of synaptic plasticity events in this process is largely unknown. To question this issue, we used cell-surface AMPAR immobilisation to block post-synaptic LTP within the hippocampal region at various steps of the memory process. 1- Our results show that hippocampal synaptic plasticity is engaged during the consolidation but is dispensable during the encoding or recall of a working memory based spatial memory task. 2- Blockade of plasticity during sleep leads to apparent forgetting of the encoded rule. 3- In vivo recordings of ripple activities during resting periods show a strong impact of AMPAR immobilization solely, prominent when a rule has been recently encoded. 4- In situ examination of the interplay between AMPAR mobility, hippocampal plasticity and spontaneous ripple activities pointed that post-synaptic plasticity at CA3-CA3 recurrent synapses support ripple generation. As crucial results were reproduced using another AMPARM blockade strategy, we propose that after rule encoding, post-synaptic AMPAR mobility at CA3 recurrent synapses support the generation of ripples necessary for rule consolidation.

Keywords: synaptic vesicle; synaptic vesicle super-pool; vesicular glutamate transport; liquid phase separation; EndophilinA1; quantal neurotransmitter release; fluorescence live cell microscopy; synaptic vesicle cycle. Zhang X.M. et al., 2018 2 ABSTRACTGlutamate secretion at excitatory synapses is tightly regulated to allow for the precise tuning of synaptic strength. Vesicular Glutamate Transporters (VGLUT) accumulate glutamate into synaptic vesicles (SV) and thereby regulate quantal size. Further, the number of release sites and the release probability of SVs maybe regulated by the organization of active zone proteins and SV clusters. In the present work, we uncover a mechanism mediating an increased SV clustering through a tripartite interaction of VGLUT1, endophilinA1 and intersectin1. This strengthening of SV clusters results in a combined reduction of axonal SV super-pool size and miniature excitatory events frequency. Our findings support a model in which clustered vesicles are held together through multiple weak interactions between SH3 domains and proline rich sequences of synaptic proteins. In mammals, VGLUT1 gained a poly-proline sequence that recruits endophilinA1 and turns the transporter into a dual regulator of quantal release parameters at excitatory synapses.

Regulation of synaptic neurotransmitter receptor content is a fundamental mechanism for tuning synaptic efficacy during experience-dependent plasticity and behavioral adaptation. However, experimental approaches to track and modify receptor movements in integrated experimental systems are limited. Exploiting AMPA-type glutamate receptors (AMPAR) as a model, we generated a knock-in mouse expressing the biotin acceptor peptide (AP) tag on the GluA2 extracellular N-terminus. Cell-specific introduction of biotin ligase allows the use of monovalent or tetravalent avidin variants to respectively monitor or manipulate the surface mobility of endogenous AMPAR containing biotinylated AP-GluA2 in neuronal subsets. AMPAR immobilization precluded the expression of long-term potentiation and formation of contextual fear memory, allowing for target-specific control of the expression of synaptic plasticity and animal behavior. The AP tag knock-in model offers unprecedented access to resolve and control the spatiotemporal dynamics of endogenous receptors, and opens new avenues to study the molecular mechanisms of synaptic plasticity and learning.

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the bestdescribed monogenic cause of autism. FXS is usually caused by a CGG-repeat expansion in the FMR1 gene leading to its silencing and the loss-of-expression of the Fragile X Mental Retardation Protein (FMRP). Missense mutations were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS in these patients, we generated a knock-in mouse model (Fmr1 R138Q ) expressing the FMRP-R138Q protein. We demonstrate that the Fmr1 R138Q hippocampus has an increased spine density associated with postsynaptic ultrastructural defects and increased AMPA receptor surface expression. Combining biochemical assays, highresolution imaging and electrophysiological recordings, we also show that the mutation impairs the hippocampal long-term potentiation (LTP) and leads to socio-cognitive deficits in Fmr1 R138Q mice.These findings reveal that the R138Q mutation impacts the postsynaptic function of FMRP and highlight potential mechanisms causing FXS in FMRP-R138Q patients. IntroductionThe formation of functional synapses in the developing brain is fundamental to establishing efficient neuronal communication and plasticity, which underlie cognitive processes. In the past years, synaptic dysfunction has clearly emerged as a critical factor in the aetiology of neurodevelopmental disorders including Autism Spectrum disorder (ASD) and Intellectual Disability (ID). X-linked ID (XLID) accounts for 5-10% of ID patients and is caused by mutations in genes located on the X chromosome.The Fragile X Syndrome (FXS) is the most frequent form of inherited XLID and the first monogenic cause of ASD with a prevalence of 1:4000 males and 1:7000 females 1 . The majority of FXS patients exhibit mild-to-severe ID associated with significant learning and memory impairments, Attention Deficit Hyperactivity Disorder (ADHD) and autistic-like features 2-4 . To date, no effective therapeutic strategies are available.FXS generally results from a massive expansion of the trinucleotide CGG (>200 repeats) in the 5'-UTR region of the FMR1 gene leading to its transcriptional silencing and consequently, the lack of expression of the encoded Fragile X Mental Retardation Protein (FMRP) 2,5 . FMRP is an RNA-binding protein that binds a large subset of mRNAs in the mammalian brain and is a key component of RNA granules. These granules transport translationally-repressed mRNAs essential for the synaptic function along axons and dendrites 1,3 . Neuronal activation triggers the local translation of these critical mRNAs at synapses allowing spine maturation and elimination, which are essential processes to shape a functional neuronal network in the developing brain. Accordingly, the lack of FMRP expression in FXS patients and Fmr1 knockout (Fmr1-KO) animal models leads to a pathological increase in immature dendritic protrusions due to a failure in the synapse maturation and/or elimination processes 6 . These defects co...