Abstracts

Rationale: Accruing clinical and pre-clinical evidence suggests that cannabidiol (CBD) reduces seizure activity. However, the exact mechanism by which CBD restrains excitability remains obscure. Of the multimodal pharmacological profile of CBD, one promising target is the G-protein coupled receptor 55 (GPR55). CBD blocks the pro-excitatory effects of an endogenous GPR55 lipid agonist, lysophosphatidylinositol (LPI), at GPR55 on presynaptic excitatory terminals. In this study, we explored how the LPI-GPR55 axis regulates excitatory-to-inhibitory coordination in the basal state, and how this system changes following seizure activity, providing a target for CBD blockade. Methods: Using whole cell patch clamp electrophysiology, we determined the effects of LPI (4µM) and pure CBD (1-10µM) on evoked (e) and miniature (m) excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) recorded in acute ex vivo hippocampal brain slices taken from healthy mice. We also assayed the effect of LPI using whole cell patch clamp recordings in ex vivo hippocampal brain slices of rats experiencing spontaneous recurrent seizures following lithium-pilocarpine induced epileptogenesis (127 mg/kg LiCl s.c., 25 mg/kg pilocarpine s.c.). Furthermore, we measured expression of GPR55 following acute pentylenetetrazole (PTZ, 105 mg/kg, i.p.)-induced status epilepticus. Results were analyzed using unpaired or paired t test and one-way ANOVA with post-hoc Holm-Sidak test for multiple comparisons.  Results: Using a multidisciplinary approach, we found that LPI produces distinct pre- and post-synaptic effects at excitatory and inhibitory synapses in the CA1 region of the hippocampus. LPI transiently increased mEPSC frequency (156.1±25.8% vs. vehicle controls, p=0.0054, n=8) and evoked EPSC amplitude (121.6±6.2%, p=0.0214, n=5), whilst attenuating inhibitory postsynaptic strength (mIPSC amplitude: 76.5±5.6%, p=0.0123, n=10; evoked IPSC amplitude: 36.9±5.4%, p=0.0006, n=6) via decreased postsynaptic GABA_AR ?₂ subunit expression (65.3±3.9%, p=0.0001, n=8). The effects of LPI at both excitatory and inhibitory synapses were fully blocked by CBD pretreatment (eEPSCs: p=0.0057 vs. LPI alone, n=5; eIPSCs: p=0.0134, n=4) and absent in GPR55 KO mice (eEPSCs: p=0.0277 vs. WT, n=5; eIPSCs: p=0.0027, n=5). Furthermore, acute PTZ seizures elevated GPR55 expression (204.3±34.2% vs. non-epileptic controls, p=0.03, n=6), consistent with a 2.8±0.6x potentiation of the LPI effect on mEPSCs frequency in epileptic rats brain slices (p=0.0476, n=9 vs. non-epileptic controls). CBD pretreatment (200 mg/kg, i.p., 1 hour prior to PTZ administration or 10µM in ex vivo recordings) reversed the seizure-induced elevation of GPR55 expression (n=6, p=0.0069 vs. seizure+vehicle) and blocked the effect of LPI in epileptic rats brain slices (n=6). Conclusions: Collectively, we predict that CBD may exert its beneficial anti-seizure effects by opposing LPI action at GPR55 in both excitatory and inhibitory synapses, thus restoring excitatory/inhibitory coordination. Further, we posit that the LPI-GPR55 axis may represent a potential biomarker of prolonged seizure activity, and potentially predict CBD responsiveness.  Funding: Research funding provided by NINDS (ECR, F30 NS100293), NIMH (RWT, 5R37MH071739), NIDA (RWT, DA040484-01), and the Simons Foundation (RWT). We thank GW Pharmaceuticals for generous support of study materials (purified CBD).