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 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.

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 astrocytedependent cellular mechanism that enhances the heterogeneity of presynaptic strengths of convergent connections, which may help boost the computational power of dendrites.synapse heterogeneity | synaptic strength | astrocyte | hippocampal neuron | heterosynaptic plasticity A n enduring challenge in neurobiology is to understand how neurons set the strengths of their numerous synapses to efficiently process and store different information while maintaining network homeostasis. Electrophysiology and imaging approaches have revealed that synapses display a high degree of functional heterogeneity, even for those sharing the same axon or dendrite (1-3). The observation that synaptic strengths are heterogeneous, in turn, suggests that synapses can operate independently from one another. Accordingly, many studies have demonstrated the input-specificity of Hebbian and also of homeostatic forms of synaptic plasticity, where synaptic changes are restricted to inputs whose activity is altered (4-6). Nevertheless, such a synapse-autonomous behavior could potentially compromise the global network homeostasis by biasing the overall activity toward excitation or depression, and to overcome this issue, it has been proposed that distinct inputs cooperate by coordinating their relative strengths through heterosynaptic interactions (7-9). In support of the idea that synapses behave as interdependent rather than isolated functional units, the restriction of synaptic strength changes to active inputs has been demonstrated to break down at times, with the induction of synaptic plasticity in the stimulated input accompanying either synaptic depression or potentiation of the nonstimulated inputs (10-13). In a highly studied plasticity paradigm of long-term potentiation (LTP) at hippocampal Schaffer collateral-CA1 synapses, tetanic stimulation that induces LTP is often accompanied by presynaptic long-term depression (LTD) of nonstimulated Schaffer collat...