Studies have shown that the number of gamma-aminobutyric acid receptors (GABAARs) present at the plasma membrane controls GABAergic transmission. In fact, a number of neurological and neuropsychiatric disorders have been associated with deficits in the expression and activity of GABAARs. Although of great importance, the exact mechanism by which these receptors accumulate on the neuronal plasma membrane, enabling them to mediate inhibitory neurotransmission, has not been elucidated. By implementing established tools and through the development of novel techniques, work proposed aims to investigate the processes that underlie the assembly and maintenance of GABAergic synapses as well as their modulation under physiological and diseased states. Through the use of super-resolution imaging techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), we aim to study the number and distribution of GABAARs at synapses and to provide a structural picture of the scaffolding protein gephyrin. To aid in the elucidation of the role of gephyrin in the stabilization of GABAARs at synaptic sites, we propose the use of dual-color super-resolution imaging to study the interaction between these proteins. For more accurate and detailed three-dimensional structural studies, we will couple super-resolution microscopy with astigmatic imaging. Importantly, our objectives also encompass the development of novel tools that, once established, can be applied to the study of inhibitory synapses as well as many other biological systems. These techniques involve the incorporation of unnatural amino acids that can later be tagged with organic fluorophores via click chemistry. Because of the presence of a single fluorophore per receptor, single particle super-resolution imaging can be achieved.