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Title: Cannabinoid receptor signalling in the central nervous system
Author: Smith, Imogen
ISNI:       0000 0004 2721 2652
Awarding Body: University of Reading
Current Institution: University of Reading
Date of Award: 2010
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The GPCRs CB1R and CB2R are targets for endocannabinoids, exogenous synthetic agents and phytocannabinoids derived from Cannabis plants. However, the pharmacological properties of many phytocannabinoids remain to be elucidated. The present work focused on activity of cannabinoids at CB1R, and potentially other targets, in brain membrane preparations and a cell culture model of epileptiform activity. The synthetic cannabinoids WIN55,212-2 (CB1/2R agonist) and AM251 (CB1R antagonist), and the phytocannabinoids fl9_THCV, CBO and CBG were investigated using radioligand binding and [35SjGTPyS assays to assess pharmacological actions, and patch-clamp electrophysiology to study functional effects. Radioligand competition binding assays using the CB1R antagonist [3HjSR141716A demonstrated high affinity binding of AM251 and WIN55,212-2, moderate affinity of fl9_ THCV, and weak affinity of CBO and CBG. [35SjGTPyS binding assays were used to construct concentration response curves for all compounds, and showed potent efficacious agonism by WIN55,212-2, whilst fl9_THCV, CBO, and CBG showed no agonist activity. AM251 and fl9_ THCV were used in Schild analyses, and demonstrated potent antagonism of CB1R at submicromolar concentrations. At higher concentrations, AM251 and fl9_THCV caused depression of [35SjGTPyS binding. For AM251, but not fl9_THCV, further investigations demonstrated an adenosine Al receptor component of this depression. To enable functional studies, a novel cell culture model of Mg2+-free pre-treatment induced epileptiform activity in mouse cortical neurones was successfully developed. In electrophysiological investigations WIN55,212-2 and fl9 -THCV reduced action potential firing in epileptiform neurones. The effects of WIN55,212 were blocked by AM251, suggesting a CB1R-mediated mechanism. fl9_THCV, CBG and AM251 reduced peak action potential amplitude, potentially via a non-CBR mechanism. Further investigations showed fl9_THCV and CBG reduced peak Na+ conductance suggesting functional, potentially therapeutic, effects via voltage-gated Na+ channels. These data demonstrate novel forms of cannabinoid signalling in the CNS, show that phytocannabinoids have a range of CBR affinities, and may have additional targets in the CNS.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available