DescriptionInteractions between EphA receptors and ephrin-A ligands have been implicated in the regulation of postsynaptic spine density, astrocytic glutamate transport, and synaptic plasticity. Nonetheless, many aspects of the bidirectional EphA/ephrin-A signaling remain elusive. The goal of this dissertation was to characterize expression of EphA and ephrin-A proteins in hippocampal cultures and to examine effects of ephrin-A activation and EphA inhibition on the development of excitatory synapses in vitro. Affinity probe method utilizing EphA-Fc and ephrin-A-Fc chimeric protein constructs was used to detect endogenous ephrin-A and EphA proteins, respectively, on the surface of living hippocampal cells in culture. Additionally, IIIA4 anti-EphA3 antibody, in combination with ephrin-A3-Fc affinity probe, was employed to characterize expression of surface EphA3 receptor and its ligand-binding ability. Finally, to test how ephrin-A activation and EphA inhibition affect excitatory synapse development, a combination of chronic treatments with clustered and unclustered EphA3-Fc chimeras was utilized along with quantitative immunofluorescence microscopy. Data presented in this dissertation showed that EphA and ephrin-A proteins were present on the surface of hippocampal astrocytes and in peridendritic areas of hippocampal neurons. In addition, EphA3 receptor, expressed on the surface of hippocampal cells, displayed differential ability for ligand binding. Lastly, activation of ephrin-A reverse signaling increased the expression of postsynaptic protein PSD-95 and counteracted the repulsive effects of EphA receptor inhibition on synapse formation. These results indicate that EphA/ephrin-A signaling plays attractive and repulsive roles in the development of excitatory synapses and might be regulated by the EphA receptor differential ability for ligand binding.