Project Summary: In the mammalian central nervous system, two main types of synapses – glutamatergic and GABAergic – play opposing roles in exciting or inhibiting the postsynaptic cell. While it is critical that upon cell-cell contact, new synapses form the correct postsynaptic specialization (excitatory or inhibitory), the molecular pathways specifying this identity remain a fundamental mystery in developmental neurobiology. This process is presumably regulated by trans-synaptic ligand/receptor partners that belong to protein families (e.g. Neuroligins/Neurexins, Ephrins/Ephs, Semaphorins/Plexins) which have been demonstrated to regulate both excitatory and inhibitory synapse formation. Semaphorins (Semas) and Plexins are families of widely expressed and functionally versatile transmembrane or secreted ligands and their transmembrane receptors that first gained attention for their roles in axon guidance during nervous system development. Class 4 Semas and Plexin-B receptors are expressed in mammalian hippocampus (in excitatory and inhibitory neurons and in glia) and promote synapse formation both during development and in adulthood. Our previous studies revealed Sema4D to be one of few molecules having synaptogenic function restricted to inhibitory synapses; the extracellular domain of Sema4D induces inhibitory synapse formation on a rapid timescale (~30 mins) through the Plexin-B1 receptor. Additionally, Sema4A promotes formation of both inhibitory and excitatory synapses in hippocampus via the Plexin-B1 or Plexin-B2 receptors, respectively. The ability to rapidly drive synapse formation by application of Sema4A or Sema4D, as well as the observations that these Semas regulate inhibitory and excitatory synapse formation in unique ways requiring different Plexin-B receptors, are central to our proposed strategy to identify the distinct signaling conformations that instruct synapse identity (excitatory or inhibitory) downstream of Plexin-B receptor engagement. Further, we previously showed that Sema4D protein application increases resilience to seizure in adult mice in vivo, a finding that supports the potential clinical impact of this work. The proposed research uses a combination of transgenic mice, organotypic hippocampal slice culture, gene knockdown, and expression of mutant forms of Plexin-B receptors to examine the divergent roles of 1) unique Plexin-B signaling domains, 2) a novel mechanism gating Plexin-B1 signaling in cis, and 3) contributions from Plexin-B coreceptors, in excitatory and inhibitory synapse formation promoted by class 4 semaphorins.