GLYT2 SWITCH · Development and maturation of glycine functions in the CNS - role of GLYT2 in the switch from GABAergic to glycinergic transmission

Final Activity Report Summary - GLYT2 SWITCH (Development and maturation of glycine functions in the CNS - role of GLYT2 in the switch from GABAergic to glycinergic transmission)

Fast inhibitory transmission in the spinal cord and brain stem is mediated by glycine and Gamma-aminobutyric acid (GABA). Single neurons may switch from using one inhibitory transmitter to using the other and, in many immature neurons, glycine and GABA are stored and released from the same synaptic vesicles. The mechanism by which their presynaptic and postsynaptic balance is achieved is unknown. In this project we used two approaches, namely a model cell system and cultured spinal cord neurons, to investigate the presynaptic mechanisms that insure robust glycinergic transmission. Knockout studies of the plasma membrane transporter (GlyT2) and the Vesicular inhibitory amino acid transporter (VIAAT) identified these two transporters as essential determinants of glycinergic transmission. Given that previous investigations dependent on fully functional VIAAT were unsuccessful, we developed a model synapse system that allowed us to successfully assay inhibitory transmitter uptake by VIAAT and provide the first positive evidence that glycine uptake by GlyT2 and VIAAT acted synergistically to specify the glycinergic vesicular phenotype. We also demonstrated that GlyT2 could overcome two major limitations of VIAAT for the loading of glycine in synaptic vesicles, i.e. the extremely low glycine affinity for VIAAT and the lack of VIAAT specificity for glycine over GABA, the other inhibitory transmitter. We designed an original double-sniffer patch-clamp technique to unambiguously detect the vesicular release of both inhibitory transmitters from a single vesicle and showed that the level of competition between glycine and GABA for VIAAT was the main determinate of the inhibitory vesicular phenotype, either glycinergic, GABAergic or mixed. In addition, we reported the first evidence that the vesicular transport of glycine and GABA could be differentiated by a single point mutation in a vesicular transporter, thus opening the way to manipulate the glycine and GABA balance in synaptic vesicles. Interestingly, we found that the VIAAT orthologue from caenorhabditis elegans (UNC-47), a species lacking glycine transmission, also supported glycine exocytosis in the presence of GlyT2. Taken together, these results suggested that the increased availability of glycine in VIAAT containing terminals was crucial for the emergence of glycinergic transmission in vertebrates. This work was in review for publication in the Journal of Neuroscience by the time of the project completion. By using culture spinal cord neurons from mice expressing the eGFP fluorescent protein under the GlyT2 promoter we were able to easily identify GlyT2 expressing neurons and we were able to isolate for the first time native GlyT2 currents. We confirmed that GlyT2 had similar characteristics in neurons as those described in recombinant non-mammalian systems and, based on the amplitude of the GlyT2 currents, we also confirmed that GlyT2 was expressed at very high levels in inhibitory spinal cord neurons. We subsequently investigated the functional coupling of GlyT2 with the strength of glycinergic transmission by using the specific GlyT2 inhibitor ORG25543. We showed that long term blockade of GlyT2 was able to decrease the content of glycine in individual vesicles. We were able to conclude that this deficit in glycine transmission was because of an inhibition of GlyT2 and not due to compensatory changes in exocytosis or glycine receptor (GlyR) expression given that, following ORG25543 (ORG) treatment, we did not observe any change in vesicle destaining of FM stryal dyes during exocytosis or in the amplitude of the GlyR current stimulated by extracellular application of glycine. Finally, we showed that short incubation of ORG treated cells in extracellular glycine restored glycinergic transmission to its pretreatment levels. Thus, we demonstrated that the loading of synaptic vesicles with glycine in a neuronal system could be modulated by the activity of GlyT2. This work was carried out in conjunction with France Rousseau, and was prepared for publication by the time of the project completion.

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