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Electrically non-excitable astroglia take up neurotransmitters, buffer extracellular K + and generate Ca 2+ signals that release molecular regulators of neural circuitry. The underlying machinery remains enigmatic, mainly because the sponge-like astrocyte morphology has been difficult to access experimentally or explore theoretically. Here, we systematically incorporate multi-scale, tri-dimensional astroglial architecture into a realistic multi-compartmental cell model, which we constrain by empirical tests and integrate into the NEURON computational biophysical environment. This approach is implemented as a flexible astrocyte-model builder ASTRO. As a proof-of-concept, we explore an in silico astrocyte to evaluate basic cell physiology features inaccessible experimentally. Our simulations suggest that currents generated by glutamate transporters or K + channels have negligible distant effects on membrane voltage and that individual astrocytes can successfully handle extracellular K + hotspots. We show how intracellular Ca 2+ buffers affect Ca 2+ waves and why the classical Ca 2+ sparks-and-puffs mechanism is theoretically compatible with common readouts of astroglial Ca 2+ imaging. Astrocytes have gained increasing attention for their roles in regulating neural circuits via neurotransmitter uptake, K + buffering, and ability to signal via Ca 2 +  transients. Here, the authors develop a computational modelling environment for astrocytes, akin to the NEURON environment, called ASTRO.

Biomimetic divalent ligands based on the PDZ domain–binding motifs from the AMPA receptor auxiliary subunit Stargazin disrupt the receptor's interaction with the scaffold protein PSD-95 and show that AMPARs are stabilized at synapses by engaging in multivalent interactions with PDZ domain-containing proteins. The interactions of the AMPA receptor (AMPAR) auxiliary subunit Stargazin with PDZ domain–containing scaffold proteins such as PSD-95 are critical for the synaptic stabilization of AMPARs. To investigate these interactions, we have developed biomimetic competing ligands that are assembled from two Stargazin-derived PSD-95/DLG/ZO-1 (PDZ) domain–binding motifs using 'click' chemistry. Characterization of the ligands in vitro and in a cellular FRET-based model revealed an enhanced affinity for the multiple PDZ domains of PSD-95 compared to monovalent peptides. In cultured neurons, the divalent ligands competed with transmembrane AMPAR regulatory protein (TARP) for the intracellular membrane-associated guanylate kinase resulting in increased lateral diffusion and endocytosis of surface AMPARs, while showing strong inhibition of synaptic AMPAR currents. This provides evidence for a model in which the TARP-containing AMPARs are stabilized at the synapse by engaging in multivalent interactions. In light of the prevalence of PDZ domain clusters, these new biomimetic chemical tools could find broad application for acutely perturbing multivalent complexes.

The relative content of NR2 subunits in the NMDA receptor confers specific signaling properties and plasticity to synapses. However, the mechanisms that dynamically govern the retention of synaptic NMDARs, in particular 2A-NMDARs, remain poorly understood. Here, we investigate the dynamic interaction between NR2 C termini and proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) scaffold proteins at the single molecule level by using high-resolution imaging. We report that a biomimetic divalent competing ligand, mimicking the last 15 amino acids of NR2A C terminus, specifically and efficiently disrupts the interaction between 2A-NMDARs, but not 2B-NMDARs, and PDZ proteins on the time scale of minutes. Furthermore, displacing 2A-NMDARs out of synapses lead to a compensatory increase in synaptic NR2B-NMDARs, providing functional evidence that the anchoring mechanism of 2A- or 2B-NMDARs is different. These data reveal an unexpected role of the NR2 subunit divalent arrangement in providing specific anchoring within synapses, highlighting the need to study such dynamic interactions in native conditions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neuronal N-methyl- d -aspartate receptors (NMDARs) play a critical role in synaptic plasticity. Their activation requires not only binding of their ligand glutamate and membrane depolarization but also the presence of a co-agonist, glycine or d -serine. An increasing body of experimental evidence suggests that different populations of NMDARs could be gated by different co-agonists. Here we discuss how the spatial distribution of co-agonist sources and uptake mechanisms, together with diffusional properties of the synaptic environment, could shape NMDAR co-agonist supply and therefore NMDAR-dependent plasticity.

Activity-dependent remodeling of excitatory connections underpins memory formation in the brain. Serotonin receptors are known to contribute to such remodeling, yet the underlying molecular machinery remains poorly understood. Here, we employ high-resolution time-lapse FRET imaging in neuroblastoma cells and neuronal dendrites to establish that activation of serotonin receptor 5-HT 4 (5-HT 4 R) rapidly triggers spatially-restricted RhoA activity and G13-mediated phosphorylation of cofilin, thus locally boosting the filamentous actin fraction. In neuroblastoma cells, this leads to cell rounding and neurite retraction. In hippocampal neurons in situ, 5-HT 4 R-mediated RhoA activation triggers maturation of dendritic spines. This is paralleled by RhoA-dependent, transient alterations in cell excitability, as reflected by increased spontaneous synaptic activity, apparent shunting of evoked synaptic responses, and enhanced long-term potentiation of excitatory transmission. The 5-HT 4 R/G13/RhoA signaling thus emerges as a previously unrecognized molecular pathway underpinning use-dependent functional remodeling of excitatory synaptic connections. Schill, Bijata, Kopach et al. show that 5-HT 4 R activation leads to maturation of dendritic spines in hippocampal neurons via G13/RhoA signaling. The dendritic changes are complemented with corresponding changes in excitatory transmission, suggesting a role for 5-HT 4 R in dendritic spine remodeling.

Activity-dependent remodeling of excitatory connections underpins memory formation in the brain. Serotonin receptors are known to contribute to such remodeling, yet the underlying molecular machinery remains poorly understood. Here, we employ high-resolution time-lapse FRET imaging in neuroblastoma cells and neuronal dendrites to establish that activation of serotonin receptor 5-HT (5-HT R) rapidly triggers spatially-restricted RhoA activity and G13-mediated phosphorylation of cofilin, thus locally boosting the filamentous actin fraction. In neuroblastoma cells, this leads to cell rounding and neurite retraction. In hippocampal neurons in situ, 5-HT R-mediated RhoA activation triggers maturation of dendritic spines. This is paralleled by RhoA-dependent, transient alterations in cell excitability, as reflected by increased spontaneous synaptic activity, apparent shunting of evoked synaptic responses, and enhanced long-term potentiation of excitatory transmission. The 5-HT R/G13/RhoA signaling thus emerges as a previously unrecognized molecular pathway underpinning use-dependent functional remodeling of excitatory synaptic connections.

The relative content of NR2 subunits in the NMDA receptor confers specific signaling properties and plasticity to synapses. However, the mechanisms that dynamically govern the retention of synaptic NMDARs, in particular 2A-NMDARs, remain poorly understood. Here, we investigate the dynamic interaction between NR2 C termini and proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) scaffold proteins at the single molecule level by using high-resolution imaging. We report that a biomimetic divalent competing ligand, mimicking the last 15 amino acids of NR2A C terminus, specifically and efficiently disrupts the interaction between 2A-NMDARs, but not 2B-NMDARs, and PDZ proteins on the time scale of minutes. Furthermore, displacing 2A-NMDARs out of synapses lead to a compensatory increase in synaptic NR2B-NMDARs, providing functional evidence that the anchoring mechanism of 2A- or 2B-NMDARs is different. These data reveal an unexpected role of the NR2 subunit divalent arrangement in providing specific anchoring within synapses, highlighting the need to study such dynamic interactions in native conditions.

This paper describes applications of two optical microscopy techniques, namely laser tweezers and fluorescence recovery after photo-bleaching (FRAP), to the measurement of ligand/receptor/cytoskeleton interactions. These methods are used in combination with ligand-coated microspheres, binding to specific membrane receptors at the dorsal cell surface. A first application exploits the possibility to impose piconewton forces on microspheres. At low ligand density, one can identify the rupture of individual bonds between ligand/receptor complexes and the motile actin cytoskeleton. The second application uses control of the initial contact time by optical tweezers, together with time lapse imaging of GFP-tagged receptor accumulation around the microspheres. Quantification of fluorescence enrichment together with a simple chemical reaction model allows characterization of global ligand/receptor on-rates. These contacts eventually reach steady-state corresponding to continuous formation and dissociation of ligand/receptor bonds. FRAP is then used to probe the equilibrium dynamics of this system, by photo-bleaching GFP-tagged receptors recruited at ligand-coated beads. The recovery curves are fitted by a diffusion/reaction model, to yield turnover rates between adhesion proteins. To illustrate the potential of these techniques, we take examples from our previously published data on neuronal adhesion proteins involved in growth cone locomotion, in particular N-cadherin and immunoglobulin cell adhesion molecules.

Neutrophils are white blood cells that are critical to the acute inflammatory and adaptive immune responses. Their swarming-pattern behaviour is controlled by multiple cellular cascades involving calcium-dependent release of various signalling molecules. Previous studies have reported that neutrophils express glutamate receptors and can release glutamate but evidence of direct neutrophil-neutrophil communication has been elusive. Here, we hold semi-suspended cultured human neutrophils in patch-clamp whole-cell mode to find that calcium mobilisation induced by stimulating one neutrophil can trigger an NMDA receptor-driven membrane current and calcium signal in neighbouring neutrophils. We employ an enzymatic-based imaging assay to image, in real time, glutamate release from neutrophils induced by glutamate released from their neighbours. These observations provide direct evidence for a positive-feedback inter-neutrophil communication that could contribute to mechanisms regulating communal neutrophil behaviour.Competing Interest StatementThe authors have declared no competing interest.Footnotes* This version has updated data selection and analyses, focusing on a shorter time span after cell isolation and suspension.