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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.

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.

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.

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-1β, 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. The advent of fluorescence-based super-resolution microscopy has created a need for labeling strategies relying on small probes that minimally perturb protein function. Here the authors describe a labeling method that reduces protein tag and label sizes, allowing for accurate protein targeting and measurements of protein dynamics in tight cellular spaces.

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.

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.

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 Ca2+ signaling involving NMDAR activation, astrocyte membrane depolarization, and L-type Ca2+ channels. Intracellular infusion of NMDARs or Ca2+-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.

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. Neuroligins are postsynaptic cell adhesion molecules thought to play roles in synaptic development and function. Here, authors show that phosphorylation of Y782 in neuroligin-1 modulates its role in differentiation and ability to recruit AMPARs including during long-term potentiation.

[...]as it is often the case for epilepsy, ASD or addiction, homeostatic plasticity can become maladaptive. Homeostatic compensations are often presented as relatively slow processes developing in response to prolonged perturbations of neuronal activity and relying on the synthesis of new proteins that regulate key physiological parameters, such as synaptic efficacy, synapse number and membrane excitability. [...]external genetic interventions (e.g., by enhancing the expression of the potassium channel Kv1.1) may push back neural networks within their physiological boundaries and allow them to take back control of their own homeostasis. In their review, the authors highlight the possible role of weight-dependent heterosynaptic plasticity in normalizing the excitatory drive to hippocampal inhibitory neurons. Besides synapse-specific mechanisms, mounting evidence point to both permissive and instructive roles of the extracellular matrix (ECM) and glial cells in homeostatic plasticity.

Synapses are organized into nanocolumns that control synaptic transmission efficacy through precise alignment of postsynaptic neurotransmitter receptors and presynaptic release sites. Recent evidence show that Leucine-Rich Repeat Transmembrane protein LRRTM2, highly enriched and confined at synapses, interacts with Neurexins through its C-terminal cap, but the role of this binding interface has not been explored in synapse formation and function. Here, we develop a conditional knock-out mouse model (cKO) to address the molecular mechanisms of LRRTM2 regulation, and its role in synapse organization and function. We show that LRRTM2 cKO specifically impairs excitatory synapse formation and function in mice. Surface expression, synaptic clustering, and membrane dynamics of LRRTM2 are tightly controlled by selective motifs in the C-terminal domain. Conversely, the N-terminal domain controls presynapse nano-organization and postsynapse AMPAR sub-positioning and stabilization through the recently identified Neurexin-binding interface. Thus, we identify LRRTM2 as a central organizer of pre- and post- excitatory synapse nanostructure through interaction with presynaptic Neurexins. Synapse nanocolumns control neural transmission through precise alignment of neurotransmitter receptors and release sites. Here, the authors identify the Neurexin-LRRTM2 complex as a central organiser of excitatory synapse nanostructure and function.