MiR-186-5p inhibition restores synaptic transmission and neuronal network activity in a model of chronic stress.

Chronic stress exerts profound negative effects on cognitive and emotional behaviours and is a major risk factor for the development of neuropsychiatric disorders. However, the molecular links between chronic stress and its deleterious effects on neuronal and synaptic function remain elusive. Here, using a combination of in vitro and in vivo approaches, we demonstrate that the upregulation of miR-186-5p triggered by chronic stress may be a key mediator of such changes, leading to synaptic dysfunction. Our results show that the expression levels of miR-186-5p are increased both in the prefrontal cortex (PFC) of mice exposed to chronic stress and in cortical neurons chronically exposed to dexamethasone. Additionally, viral overexpression of miR-186-5p in the PFC of naïve mice induces anxiety- and depressive-like behaviours. The upregulation of miR-186-5p through prolonged glucocorticoid receptor activation in vitro, or in a mouse model of chronic stress, differentially affects glutamatergic and GABAergic synaptic transmission, causing an imbalance in excitation/inhibition that leads to altered neuronal network activity. At glutamatergic synapses, we observed both a reduction in synaptic AMPARs and synaptic transmission, whereas GABAergic synaptic transmission was strengthened. These changes could be rescued in vitro by a miR-186-5p inhibitor. Overall, our results establish a novel molecular link between chronic glucocorticoid receptor activation, the upregulation of miR-186-5p and the synaptic changes induced by chronic stress, that may be amenable to therapeutic intervention.

MicroRNA-186-5p controls GluA2 surface expression and synaptic scaling in hippocampal neurons.

Homeostatic synaptic scaling is a negative feedback response to fluctuations in synaptic strength induced by developmental or learning-related processes, which maintains neuronal activity stable. Although several components of the synaptic scaling apparatus have been characterized, the intrinsic regulatory mechanisms promoting scaling remain largely unknown. MicroRNAs may contribute to posttranscriptional control of mRNAs implicated in different stages of synaptic scaling, but their role in these mechanisms is still undervalued. Here, we report that chronic blockade of glutamate receptors of the AMPA and NMDA types in hippocampal neurons in culture induces changes in the neuronal mRNA and miRNA transcriptomes, leading to synaptic upscaling. Specifically, we show that synaptic activity blockade persistently down-regulates miR-186-5p. Moreover, we describe a conserved miR-186-5p-binding site within the 3'UTR of the mRNA encoding the AMPA receptor GluA2 subunit, and demonstrate that GluA2 is a direct target of miR-186-5p. Overexpression of miR-186 decreased GluA2 surface levels, increased synaptic expression of GluA2-lacking AMPA receptors, and blocked synaptic scaling, whereas inhibition of miR-186-5p increased GluA2 surface levels and the amplitude and frequency of AMPA receptor-mediated currents, and mimicked excitatory synaptic scaling induced by synaptic inactivity. Our findings elucidate an activity-dependent miRNA-mediated mechanism for regulation of AMPA receptor expression.

An open-source microscopy framework for simultaneous control of image acquisition, reconstruction, and analysis.

We present a computational framework to simultaneously perform image acquisition, reconstruction, and analysis in the context of open-source microscopy automation. The setup features multiple computer units intersecting software with hardware devices and achieves automation using python scripts. In practice, script files are executed in the acquisition computer and can perform any experiment by modifying the state of the hardware devices and accessing experimental data. The presented framework achieves concurrency by using multiple instances of ImSwitch and napari working simultaneously. ImSwitch is a flexible and modular open-source software package for microscope control, and napari is a multidimensional image viewer for scientific image analysis. The presented framework implements a system based on file watching, where multiple units monitor a filesystem that acts as the synchronization primitive. The proposed solution is valid for any microscope setup, supporting various biological applications. The only necessary element is a shared filesystem, common in any standard laboratory, even in resource-constrained settings. The file watcher functionality in Python can be easily integrated into other python-based software. We demonstrate the proposed solution by performing tiling experiments using the molecular nanoscale live imaging with sectioning ability (MoNaLISA) microscope, a high-throughput super-resolution microscope based on reversible saturable optical fluorescence transitions (RESOLFT).