This proposal focuses on examining the mechanism of cannabidiol (CBD), a non-psychoactive component of cannabis with documented anti-seizure effects in several pre-clinical studies(1, 2) and a recent double-blinded, multicenter phase III clinical trial in patients with treatment-resistant epilepsy(3, 4). Despite centuries of clinical usage, the mechanism of action of CBD remains ambiguous. Further exploring the pharmacology of CBD may provide benefit to the 1/3 of epilepsy patients with seizures resistant to current treatments (10). In one leading hypothesis, CBD acts as a competitive antagonist at the G-protein coupled receptor, GPR55, inhibiting binding of a membrane phospholipid, lysophosphatidylinositol (LPI). CBD blocks LPI- mediated increases in presynaptic Ca2+ and vesicular release, thus reducing excitability at axon terminals (12). However, the function of LPI-GPR55 signaling at inhibitory synapses, as well as potential postsynaptic effects, remains to be explored. Using a multidisciplinary approach with ex vivo whole cell patch clamp electrophysiology, and in vitro immunocytochemistry and molecular biology, I discovered that LPI had unique effects at excitatory and inhibitory synapses, both at the pre- and postsynapse. At 5-10 minutes post application, LPI increased the frequency of vesicular release in CA1 hippocampus, consistent with previous reports of presynaptic locus of GPR55 action(12). However, contrary to prior studies, I identified prominent GPR55 staining in the cell bodies of CA1 pyramidal neurons, and a postsynaptic LPI effect of increased GluA1 AMPAR expression at 50-60 minutes post application. I also determined that LPI reduced inhibitory postsynaptic strength, in part by reducing GABAAR γ2 subunit expression. Taken together, these results suggest that LPI increases the excitatory / inhibitory ratio in hippocampal neuronal networks by a dual mechanism: enhancing excitatory transmission and attenuating inhibition. This predicts that CBD, by opposing LPI action, may exert its beneficial anti-seizure effects on both excitatory and inhibitory synapses through actions at GPR55. I plan to explore these possibilities by repeating these experiments with CBD pre-treatment and GPR55-/- mice as controls. To supplement these experiments at the circuit level, I plan to investigate how LPI and CBD regulate evoked transmission and short-term plasticity in CA3-CA1 hippocampal circuits. At the in vivo level, I will use both acute and chronic seizure models to determine seizure susceptibility in GPR55-/- mice. Based on surprising preliminary results, I discovered that seizure activity upregulates GPR55 membrane expression, producing a biomarker for prolonged seizures and potentiating the effects of LPI. In all, as part of this fellowship, I propose to combine my clinical training and scientific experience to elucidate the mechanisms of this intriguing therapeutic target for epilepsy, and help stratify subpopulations for targeted treatm...