Rapid recycling of glutamate transporters on the astroglial surface.

Glutamate uptake by astroglial transporters confines excitatory transmission to the synaptic cleft. The efficiency of this mechanism depends on the transporter dynamics in the astrocyte membrane, which remains poorly understood. Here, we visualise the main glial glutamate transporter GLT1 by generating its pH-sensitive fluorescent analogue, GLT1-SEP. Fluorescence recovery after photobleaching-based imaging shows that 70-75% of GLT1-SEP dwell on the surface of rat brain astroglia, recycling with a lifetime of ~22 s. Genetic deletion of the C-terminus accelerates GLT1-SEP membrane turnover while disrupting its surface pattern, as revealed by single-molecule localisation microscopy. Excitatory activity boosts surface mobility of GLT1-SEP, involving its C-terminus, metabotropic glutamate receptors, intracellular Ca2+, and calcineurin-phosphatase activity, but not the broad-range kinase activity. The results suggest that membrane turnover, rather than lateral diffusion, is the main 'redeployment' route for the immobile fraction (20-30%) of surface-expressed GLT1. This finding reveals an important mechanism helping to control extrasynaptic escape of glutamate

Super-resolution imaging to reveal the nanostructure of tripartite synapses.

Even though neurons are the main drivers of information processing in the brain and spinal cord, other cell types are important to mediate adequate flow of information. These include electrically passive glial cells such as microglia and astrocytes, which recently emerged as active partners facilitating proper signal transduction. In disease, these cells undergo pathophysiological changes that propel disease progression and change synaptic connections and signal transmission. In the healthy brain, astrocytic processes contact pre- and postsynaptic structures. These processes can be nanoscopic, and therefore only electron microscopy has been able to reveal their structure and morphology. However, electron microscopy is not suitable in revealing dynamic changes, and it is labour- and time-intensive. The dawn of super-resolution microscopy, techniques that 'break' the diffraction limit of conventional light microscopy, over the last decades has enabled researchers to reveal the nanoscopic synaptic environment. In this review, we highlight and discuss recent advances in our understanding of the nano-world of the so-called tripartite synapses, the relationship between pre- and postsynapse as well as astrocytic processes. Overall, novel super-resolution microscopy methods are needed to fully illuminate the intimate relationship between glia and neuronal cells that underlies signal transduction in the brain and that might be affected in diseases such as Alzheimer's disease and epilepsy