Acute reorganization of postsynaptic GABAA receptors reveals the functional impact of molecular nanoarchitecture at inhibitory synapses.

Neurotransmitter receptors partition into nanometer-scale subdomains within the postsynaptic membrane that are precisely aligned with presynaptic neurotransmitter release sites. While spatial coordination between pre- and postsynaptic elements is observed at both excitatory and inhibitory synapses, the functional significance of this molecular architecture has been challenging to evaluate experimentally. Here we utilized an optogenetic clustering approach to acutely alter the nanoscale organization of the postsynaptic inhibitory scaffold gephyrin while monitoring synaptic function. Gephyrin clustering rapidly enlarged postsynaptic area, laterally displacing GABAA receptors from their normally precise apposition with presynaptic active zones. Receptor displacement was accompanied by decreased synaptic GABAA receptor currents even though presynaptic release probability and the overall abundance and function of synaptic GABAA receptors remained unperturbed. Thus, acutely repositioning neurotransmitter receptors within the postsynaptic membrane profoundly influences synaptic efficacy, establishing the functional importance of precision pre-/postsynaptic molecular coordination at inhibitory synapses.

AMPA and GABAA receptor nanodomains assemble in the absence of synaptic neurotransmitter release.

Postsynaptic neurotransmitter receptors and their associated scaffolding proteins assemble into discrete, nanometer-scale subsynaptic domains (SSDs) within the postsynaptic membrane at both excitatory and inhibitory synapses. Intriguingly, postsynaptic receptor SSDs are mirrored by closely apposed presynaptic active zones. These trans-synaptic molecular assemblies are thought to be important for efficient neurotransmission because they concentrate postsynaptic receptors near sites of presynaptic neurotransmitter release. While previous studies have characterized the role of synaptic activity in sculpting the number, size, and distribution of postsynaptic SSDs at established synapses, it remains unknown whether neurotransmitter signaling is required for their initial assembly during synapse development. Here, we evaluated synaptic nano-architecture under conditions where presynaptic neurotransmitter release was blocked prior to, and throughout synaptogenesis with tetanus neurotoxin (TeNT). In agreement with previous work, neurotransmitter release was not required for the formation of excitatory or inhibitory synapses. The overall size of the postsynaptic specialization at both excitatory and inhibitory synapses was reduced at chronically silenced synapses. However, both AMPARs and GABAARs still coalesced into SSDs, along with their respective scaffold proteins. Presynaptic active zone assemblies, defined by RIM1, were smaller and more numerous at silenced synapses, but maintained alignment with postsynaptic AMPAR SSDs. Thus, basic features of synaptic nano-architecture, including assembly of receptors and scaffolds into trans-synaptically aligned structures, are intrinsic properties that can be further regulated by subsequent activity-dependent mechanisms.

Dually innervated dendritic spines develop in the absence of excitatory activity and resist plasticity through tonic inhibitory crosstalk.

Dendritic spines can be directly connected to both inhibitory and excitatory presynaptic terminals, resulting in nanometer-scale proximity of opposing synaptic functions. While dually innervated spines (DiSs) are observed throughout the central nervous system, their developmental timeline and functional properties remain uncharacterized. Here we used a combination of serial section electron microscopy, live imaging, and local synapse activity manipulations to investigate DiS development and function in rodent hippocampus. Dual innervation occurred early in development, even on spines where the excitatory input was locally silenced. Synaptic NMDA receptor currents were selectively reduced at DiSs through tonic GABAB receptor signaling. Accordingly, spine enlargement normally associated with long-term potentiation on singly innervated spines (SiSs) was blocked at DiSs. Silencing somatostatin interneurons or pharmacologically blocking GABABRs restored NMDA receptor function and structural plasticity to levels comparable to neighboring SiSs. Thus, hippocampal DiSs are stable structures where function and plasticity are potently regulated by nanometer-scale GABAergic signaling.

miRNA-mediated control of gephyrin synthesis drives sustained inhibitory synaptic plasticity.

Activity-dependent protein synthesis is crucial for many long-lasting forms of synaptic plasticity. However, our understanding of the translational mechanisms controlling inhibitory synapses is limited. One distinct form of inhibitory long-term potentiation (iLTP) enhances postsynaptic clusters of GABAARs and the primary inhibitory scaffold, gephyrin, to promote sustained synaptic strengthening. While we previously found that persistent iLTP requires mRNA translation, the precise mechanisms controlling gephyrin translation during this process remain unknown. Here, we identify miR153 as a novel regulator of Gphn mRNA translation which controls gephyrin protein levels and synaptic clustering, ultimately impacting GABAergic synaptic structure and function. We find that iLTP induction downregulates miR153, reversing its translational suppression of Gphn mRNA and allowing for increased de novo gephyrin protein synthesis and synaptic clustering during iLTP. Finally, we find that reduced miR153 expression during iLTP is driven by an excitation-transcription coupling pathway involving calcineurin, NFAT and HDACs, which also controls the miRNA-dependent upregulation of GABAARs. Overall, this work delineates a miRNA-dependent post-transcriptional mechanism that controls the expression of the key synaptic scaffold, gephyrin, and may converge with parallel miRNA pathways to coordinate gene upregulation to maintain inhibitory synaptic plasticity.

zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways.

Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.

The differential actions of clozapine and other antipsychotic drugs on the translocation of dopamine D2 receptors to the cell surface.

Most clinically available antipsychotic drugs (APDs) bind dopamine D2 receptors (D2R) at therapeutic concentrations, and it is thought that they suppress psychotic symptoms by serving as competitive antagonists of dopamine at D2R. Here, we present data that demonstrate that APDs act independently of dopamine at an intracellular pool of D2R to enhance transport of D2R to the cell surface and suggest that APDs can act as pharmacological chaperones at D2R. Among the first- and second-generation APDs that we tested, clozapine exhibited the lowest efficacy for translocating D2R to the cell surface. Thus, our observations could provide a cellular explanation for some of the distinct therapeutic characteristics of clozapine in schizophrenia. They also suggest that differential intracellular actions of APDs at their common G protein-coupled receptor (GPCR) target, D2R, could contribute to differences in their clinical profiles.