Optical dissection of glycine and D-serine signalling using newly-developed sensors

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Persistent changes of the strength of excitatory synaptic connections are thought to underlie learning processes. The induction of these changes often relies on a subtype of glutamate receptor, the so-called N-methyl-D-aspartate receptor (NMDAR). Activation of NMDARs also requires the binding of an NMDAR co-agonist, either D-serine or glycine. Therefore, their availability controls to what degree synaptic plasticity can occur. There is, however, an ongoing intense debate about the spatial extracellular distribution of D-serine and glycine, the mechanisms that control their extracellular concentration and the differential roles of neurons and astroglia in their supply.To directly study NMDAR co-agonists signalling, we previously designed novel optical NMDAR co-agonist sensors. We have succeeded in generating an optical FRET-based glycine sensor (GlyFS) and a D-serine sensor prototype (DSerFS). Using GlyFS we could recently verify important predictions about the distribution and development of resting glycine concentrations in the hippocampus and discovered that plasticity-inducing activity governs the extracellular glycine concentration. The exact mechanisms and the cellular sources of glycine remained, however, unclear. In the current project, we will further develop and refine optical sensors for glycine (GlyFS) and D-serine (DSerFS) while at the same time dissecting the mechanisms that control NMDR co-agonist levels. For glycine imaging, we will refine the current generation of GlyFS by exchanging the FRET-pair (fluorescent proteins) and by optimization of FRET donor/acceptor orientation, distance and interaction. We will also modify the sensor so it can be targeted to the surface of specific cell types and subdomains. Using the original GlyFS and improved versions we will establish how the frequency and strength of network activity determines extracellular glycine levels, which mechanisms contribute and what the relative role of neurons and astroglial are (in acute hippocampal slices). By optical recording of synaptic and extrasynaptic glycine concentrations we will visualize how the glycine concentration at the corresponding NMDAR populations is changed by activity. In parallel, we will test the current D-serine sensor prototype DSerFS and obtain first experimental results regarding D-serine signalling. For instance, it will be tested if direct and isolated stimulation of neurons or astrocytes increases extracellular D-serine levels. At the same time, we will improve the optical properties of DSerFS and its versatility by using the same techniques as outlined for GlyFS.Our work will provide new and important insights into NMDR co-agonist signalling and thus into NMDAR-dependent synaptic plasticity in the hippocampus. We will further add two optical sensors to the neuroscientist’s toolbox that will be important for studying memory formation and other NMDAR-dependent processes in the brain.