GRID1/GluD1 homozygous variants linked to intellectual disability and spastic paraplegia impair mGlu1/5 receptor signaling and excitatory synapses.

The ionotropic glutamate delta receptor GluD1, encoded by the GRID1 gene, is involved in synapse formation, function, and plasticity. GluD1 does not bind glutamate, but instead cerebellin and D-serine, which allow the formation of trans-synaptic bridges, and trigger transmembrane signaling. Despite wide expression in the nervous system, pathogenic GRID1 variants have not been characterized in humans so far. We report homozygous missense GRID1 variants in five individuals from two unrelated consanguineous families presenting with intellectual disability and spastic paraplegia, without (p.Thr752Met) or with (p.Arg161His) diagnosis of glaucoma, a threefold phenotypic association whose genetic bases had not been elucidated previously. Molecular modeling and electrophysiological recordings indicated that Arg161His and Thr752Met mutations alter the hinge between GluD1 cerebellin and D-serine binding domains and the function of this latter domain, respectively. Expression, trafficking, physical interaction with metabotropic glutamate receptor mGlu1, and cerebellin binding of GluD1 mutants were not conspicuously altered. Conversely, upon expression in neurons of dissociated or organotypic slice cultures, we found that both GluD1 mutants hampered metabotropic glutamate receptor mGlu1/5 signaling via Ca2+ and the ERK pathway and impaired dendrite morphology and excitatory synapse density. These results show that the clinical phenotypes are distinct entities segregating in the families as an autosomal recessive trait, and caused by pathophysiological effects of GluD1 mutants involving metabotropic glutamate receptor signaling and neuronal connectivity. Our findings unravel the importance of GluD1 receptor signaling in sensory, cognitive and motor functions of the human nervous system.

Astrocyte Calcium Signaling Shifts the Polarity of Presynaptic Plasticity.

Astrocytes have been increasingly acknowledged to play active roles in regulating synaptic transmission and plasticity. Through a variety of metabotropic and ionotropic receptors expressed on their surface, astrocytes detect extracellular neurotransmitters, and in turn, release gliotransmitters to modify synaptic strength, while they can also alter neuronal membrane excitability by modulating extracellular ionic milieu. Given the seemingly large repertoire of synaptic modulation, when, where and how astrocytes interact with synapses remain to be fully understood. Previously, we have identified a role for astrocyte NMDA receptor and L-VGCCs signaling in heterosynaptic presynaptic plasticity and promoting the heterogeneity of presynaptic strengths at hippocampal synapses. Here, we have sought to further clarify the mode by which astrocytes regulate presynaptic plasticity by exploiting a reduced culture system to globally evoke NMDA receptor-dependent presynaptic plasticity. Recording from a postsynaptic neuron intracellularly loaded with BAPTA, briefly bath applying NMDA and glycine induces a stable decrease in the rate of spontaneous glutamate release, which requires the presence of astrocytes and the activation of A1 adenosine receptors. Upon preventing astrocyte calcium signaling or blocking L-VGCCs, NMDA + glycine application triggers an increase, rather than a decrease, in the rate of spontaneous glutamate release, thereby shifting the presynaptic plasticity to promote an increase in strength. Our findings point to a crucial and surprising role of astrocytes in controlling the polarity of NMDA receptor and adenosine-dependent presynaptic plasticity. Such a pivotal mechanism unveils the power of astrocytes in regulating computations performed by neural circuits and is expected to profoundly impact cognitive processes.

miR-124-dependent tagging of synapses by synaptopodin enables input-specific homeostatic plasticity.

Homeostatic synaptic plasticity is a process by which neurons adjust their synaptic strength to compensate for perturbations in neuronal activity. Whether the highly diverse synapses on a neuron respond uniformly to the same perturbation remains unclear. Moreover, the molecular determinants that underlie synapse-specific homeostatic synaptic plasticity are unknown. Here, we report a synaptic tagging mechanism in which the ability of individual synapses to increase their strength in response to activity deprivation depends on the local expression of the spine-apparatus protein synaptopodin under the regulation of miR-124. Using genetic manipulations to alter synaptopodin expression or regulation by miR-124, we show that synaptopodin behaves as a "postsynaptic tag" whose translation is derepressed in a subpopulation of synapses and allows for nonuniform homeostatic strengthening and synaptic AMPA receptor stabilization. By genetically silencing individual connections in pairs of neurons, we demonstrate that this process operates in an input-specific manner. Overall, our study shifts the current view that homeostatic synaptic plasticity affects all synapses uniformly to a more complex paradigm where the ability of individual synapses to undergo homeostatic changes depends on their own functional and biochemical state.

MDGAs are fast-diffusing molecules that delay excitatory synapse development by altering neuroligin behavior.

MDGA molecules can bind neuroligins and interfere with trans-synaptic interactions to neurexins, thereby impairing synapse development. However, the subcellular localization and dynamics of MDGAs, or their specific action mode in neurons remain unclear. Here, surface immunostaining of endogenous MDGAs and single molecule tracking of recombinant MDGAs in dissociated hippocampal neurons reveal that MDGAs are homogeneously distributed and exhibit fast membrane diffusion, with a small reduction in mobility across neuronal maturation. Knocking-down/out MDGAs using shRNAs and CRISPR/Cas9 strategies increases the density of excitatory synapses, the membrane confinement of neuroligin-1, and the phosphotyrosine level of neuroligins associated with excitatory post-synaptic differentiation. Finally, MDGA silencing reduces the mobility of AMPA receptors, increases the frequency of miniature EPSCs (but not IPSCs), and selectively enhances evoked AMPA-receptor-mediated EPSCs in CA1 pyramidal neurons. Overall, our results support a mechanism by which interactions between MDGAs and neuroligin-1 delays the assembly of functional excitatory synapses containing AMPA receptors.

Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation.

Neuroligins (Nlgns) are adhesion proteins mediating trans-synaptic contacts in neurons. However, conflicting results around their role in synaptic differentiation arise from the various techniques used to manipulate Nlgn expression level. Orthogonally to these approaches, we triggered here the phosphorylation of endogenous Nlgn1 in CA1 mouse hippocampal neurons using a photoactivatable tyrosine kinase receptor (optoFGFR1). Light stimulation for 24 hr selectively increased dendritic spine density and AMPA-receptor-mediated EPSCs in wild-type neurons, but not in Nlgn1 knock-out neurons or when endogenous Nlgn1 was replaced by a non-phosphorylatable mutant (Y782F). Moreover, light stimulation of optoFGFR1 partially occluded LTP in a Nlgn1-dependent manner. Combined with computer simulations, our data support a model by which Nlgn1 tyrosine phosphorylation promotes the assembly of an excitatory post-synaptic scaffold that captures surface AMPA receptors. This optogenetic strategy highlights the impact of Nlgn1 intracellular signaling in synaptic differentiation and potentiation, while enabling an acute control of these mechanisms.

miRNA-Dependent Control of Homeostatic Plasticity in Neurons.

Homeostatic plasticity is a form of plasticity in which neurons compensate for changes in neuronal activity through the control of key physiological parameters such as the number and the strength of their synaptic inputs and intrinsic excitability. Recent studies revealed that miRNAs, which are small non-coding RNAs repressing mRNA translation, participate in this process by controlling the translation of multiple effectors such as glutamate transporters, receptors, signaling molecules and voltage-gated ion channels. In this review, we present and discuss the role of miRNAs in both cell-wide and compartmentalized forms of homeostatic plasticity as well as their implication in pathological processes associated with homeostatic failure.

Differential role of pre- and postsynaptic neurons in the activity-dependent control of synaptic strengths across dendrites.

Neurons receive a large number of active synaptic inputs from their many presynaptic partners across their dendritic tree. However, little is known about how the strengths of individual synapses are controlled in balance with other synapses to effectively encode information while maintaining network homeostasis. This is in part due to the difficulty in assessing the activity of individual synapses with identified afferent and efferent connections for a synapse population in the brain. Here, to gain insights into the basic cellular rules that drive the activity-dependent spatial distribution of pre- and postsynaptic strengths across incoming axons and dendrites, we combine patch-clamp recordings with live-cell imaging of hippocampal pyramidal neurons in dissociated cultures and organotypic slices. Under basal conditions, both pre- and postsynaptic strengths cluster on single dendritic branches according to the identity of the presynaptic neurons, thus highlighting the ability of single dendritic branches to exhibit input specificity. Stimulating a single presynaptic neuron induces input-specific and dendritic branchwise spatial clustering of presynaptic strengths, which accompanies a widespread multiplicative scaling of postsynaptic strengths in dissociated cultures and heterosynaptic plasticity at distant synapses in organotypic slices. Our study provides evidence for a potential homeostatic mechanism by which the rapid changes in global or distant postsynaptic strengths compensate for input-specific presynaptic plasticity.

A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation.

To better understand the molecular mechanisms by which early neuronal connections mature into synapses, we examined the impact of neuroligin-1 (Nlg1) phosphorylation on synapse differentiation, focusing on a unique intracellular tyrosine (Y782), which differentially regulates Nlg1 binding to PSD-95 and gephyrin. By expressing Nlg1 point mutants (Y782A/F) in hippocampal neurons, we show using imaging and electrophysiology that Y782 modulates the recruitment of functional AMPA receptors (AMPARs). Nlg1-Y782F impaired both dendritic spine formation and AMPAR diffusional trapping, but not NMDA receptor recruitment, revealing the assembly of silent synapses. Furthermore, replacing endogenous Nlg1 with either Nlg1-Y782A or -Y782F in CA1 hippocampal neurons impaired long-term potentiation (LTP), demonstrating a critical role of AMPAR synaptic retention. Screening of tyrosine kinases combined with pharmacological inhibitors point to Trk family members as major regulators of endogenous Nlg1 phosphorylation and synaptogenic function. Thus, Nlg1 tyrosine phosphorylation signaling is a critical event in excitatory synapse differentiation and LTP.

Pre-post synaptic alignment through neuroligin-1 tunes synaptic transmission efficiency.

The nanoscale organization of neurotransmitter receptors regarding pre-synaptic release sites is a fundamental determinant of the synaptic transmission amplitude and reliability. How modifications in the pre- and post-synaptic machinery alignments affects synaptic currents, has only been addressed with computer modelling. Using single molecule super-resolution microscopy, we found a strong spatial correlation between AMPA receptor (AMPAR) nanodomains and the post-synaptic adhesion protein neuroligin-1 (NLG1). Expression of a truncated form of NLG1 disrupted this correlation without affecting the intrinsic AMPAR organization, shifting the pre-synaptic release machinery away from AMPAR nanodomains. Electrophysiology in dissociated and organotypic hippocampal rodent cultures shows these treatments significantly decrease AMPAR-mediated miniature and EPSC amplitudes. Computer modelling predicts that ~100 nm lateral shift between AMPAR nanoclusters and glutamate release sites induces a significant reduction in AMPAR-mediated currents. Thus, our results suggest the synapses necessity to release glutamate precisely in front of AMPAR nanodomains, to maintain a high synaptic responses efficiency.

Astrocytes regulate heterogeneity of presynaptic strengths in hippocampal networks.

Dendrites are neuronal structures specialized for receiving and processing information through their many synaptic inputs. How input strengths are modified across dendrites in ways that are crucial for synaptic integration and plasticity remains unclear. We examined in single hippocampal neurons the mechanism of heterosynaptic interactions and the heterogeneity of synaptic strengths of pyramidal cell inputs. Heterosynaptic presynaptic plasticity that counterbalances input strengths requires N-methyl-d-aspartate receptors (NMDARs) and astrocytes. Importantly, this mechanism is shared with the mechanism for maintaining highly heterogeneous basal presynaptic strengths, which requires astrocyte Ca(2+) signaling involving NMDAR activation, astrocyte membrane depolarization, and L-type Ca(2+) channels. Intracellular infusion of NMDARs or Ca(2+)-channel blockers into astrocytes, conditionally ablating the GluN1 NMDAR subunit, or optogenetically hyperpolarizing astrocytes with archaerhodopsin promotes homogenization of convergent presynaptic inputs. Our findings support the presence of an astrocyte-dependent cellular mechanism that enhances the heterogeneity of presynaptic strengths of convergent connections, which may help boost the computational power of dendrites.

Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin.

The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1ß, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures.

miR-92a regulates expression of synaptic GluA1-containing AMPA receptors during homeostatic scaling.

We investigated whether microRNAs could regulate AMPA receptor expression during activity blockade. miR-92a strongly repressed the translation of GluA1 receptors by binding the 3' untranslated region of rat GluA1 (also known as Gria1) mRNA and was downregulated in rat hippocampal neurons after treatment with tetrodotoxin and AP5. Deleting the seed region in GluA1 or overexpressing miR-92a blocked homeostatic scaling, indicating that miR-92a regulates the translation and synaptic incorporation of new GluA1-containing AMPA receptors.

Formation and reformation of climbing fibre synapses in the cerebellum: a similar story?

The assembly of neural circuits involves multiple sequential steps, in particular the formation and maturation of synaptic connections. This often prolonged process involves several stages including the appropriate morphological and physiological maturation of each synaptic partner as well as their mutual interaction in order to ensure correct cellular and subcellular targeting. Understanding the processes involved becomes critical if neural circuits are to be appropriately reassembled following lesion, atrophy or neurodegeneration. We study the climbing fibre to Purkinje cell synapse as an example of a neural circuit which undergoes initial synaptic formation, selective stabilisation and elimination of redundant connections, in order to better understand the relative roles of each synaptic partner in the process of synaptogenesis and post-lesion synapse reformation. In particular, we are interested in the molecules which may underlie these processes. Here, we present data showing that the maturational state of both the target Purkinje cell and the climbing fibre axon influence their capacity for synapse formation. The climbing fibre retains some ability to recapitulate developmental processes irrespective of its maturational state. In contrast, the experience of synaptic formation and selective stabilisation/elimination permanently changes the Purkinje cell so that it cannot be repeated. Thus, if the climbing fibre-Purkinje cell synapse is recreated after the period of normal maturation, the process of synaptic competition, involving the gradual weakening of one climbing fibre synapse and stabilisation of another, no longer takes place. Moreover, we show that these processes of synaptic competition can only proceed during a specific developmental phase. To understand why these changes occur, we have investigated the role of molecules involved in the development of the olivocerebellar path and show that brain-derived neurotrophic factor, through activation of its receptor TrkB, as well as polysialated neural cell adhesion molecule and the transcription factor RORα regulate these processes.

Homeostatic synaptic plasticity: from single synapses to neural circuits.

Homeostatic synaptic plasticity remains an enigmatic form of synaptic plasticity. Increasing interest on the topic has fuelled a surge of recent studies that have identified key molecular players and the signaling pathways involved. However, the new findings also highlight our lack of knowledge concerning some of the basic properties of homeostatic synaptic plasticity. In this review we address how homeostatic mechanisms balance synaptic strengths between the presynaptic and the postsynaptic terminals and across synapses that share the same postsynaptic neuron.

Differential control of presynaptic efficacy by postsynaptic N-cadherin and ß-catenin.

N-cadherin is a homophilic adhesion protein that remains expressed at mature excitatory synapses beyond its developmental role in synapse formation. We investigated the trans-synaptic activity of N-cadherin in regulating synapse function in rodent cultured hippocampal neurons using optical methods and electrophysiology. Interfering with N-cadherin in postsynaptic neurons reduced basal release probability (p(r)) at inputs to the neuron, and this trans-synaptic impairment of release accompanied impaired vesicle endocytosis. Moreover, loss of the GluA2 AMPA-type glutamate receptor subunit, which decreased p(r) by itself, occluded the interference with postsynaptic N-cadherin. The loss of postsynaptic N-cadherin activity, however, did not affect the compensatory upregulation of p(r) induced by chronic activity silencing, whereas postsynaptic ß-catenin deletion blocked this presynaptic homeostatic adaptation. Our findings suggest that postsynaptic N-cadherin helps link basal pre- and postsynaptic strengths to control the p(r) offset, whereas the p(r) gain adjustment requires a distinct trans-synaptic pathway involving ß-catenin.

[Marked for life: synaptic stabilisation leaves an indelible trace on neuronal partners: a question of etiquette for neurons?]. / Faire et refaire une synapse en choisissant un partenaire : une question d'étiquette pour les neurones ?

Recent in vivo and in vitro studies have used the rodent olivocerebellar path to examine developmental synaptogenesis, in particular the mechanisms which permit the simultaneous stabilisation of some synapses and elimination of others. They reveal that while the formation of synapses can take place throughout life, elimination of supernumerary connections is limited to a critical period during development. Synapse elimination involves the strengthening of appropriate synapses and weakening of others, which are subsequently removed. This process is partly dependent on activity and post-synaptic signalling cascades and irreversibly changes each synaptic partner, leaving a trace of previous connectivity. Here we discuss the nature of this << trace >>, which may be a molecule that labels and protects those synapses to be retained, and its functional significance in maintaining the specificity of neuronal circuits needed for coordinate behaviour. double dagger.

Synapse elimination in olivo-cerebellar explants occurs during a critical period and leaves an indelible trace in Purkinje cells.

During developmental synaptogenesis, the pre- and postsynaptic cells undergo specific interactions that lead to the establishment of the mature circuit. We have studied the roles of the pre- and postsynaptic cells in establishing this mature innervation by using an in vitro model of synaptic development. We describe climbing fiber (CF)-Purkinje cell (PC) synaptogenesis in cultured mouse hindbrain explants and show that synaptic competition occurs during early development in vitro. By manipulating the maturation stage of each of the synaptic partners in a coculture experimental paradigm, we found that multi-innervation does not occur when both synaptic partners are mature and have already experienced synapse elimination; in contrast, mature PCs can be multi-innervated when they have never experienced synapse elimination and/or when CFs are immature. However in these cases, the normal process of synapse elimination is impaired. These results show that CF-synapse elimination occurs only during a PC-dependant critical period and triggers indelible signals that prevent synapse competition in the mature system.

Normal adult climbing fiber monoinnervation of cerebellar Purkinje cells in mice lacking MHC class I molecules.

Some immune system proteins have recently been implicated in the development and plasticity of neuronal connections. Notably, proteins of the major histocompatibility complex 1 (MHC class 1) have been shown to be involved in synaptic plasticity in the hippocampus and the development of projection patterns in the visual system. We examined the possible role for the MHC class 1 proteins in one well-characterized example of synaptic exuberance and subsequent refinement, the climbing fiber (CF) to Purkinje cell (PC) synapse. Cerebella from adult mice deficient for two MHC genes, H2-D1 and H2-K1, and for beta2-microglobulin gene were examined for evidence of deficient elimination of supernumerary CF synapses on their PCs. Electrophysiological and morphological evidence showed that, despite the absence of these MHC class 1 molecules, adult PCs in these transgenic mice are monoinnervated as in wild-type animals. These findings indicate that, at the level of restriction of afferent number at this synapse, functional MHC class 1 proteins are not required.