Study on synthesis and release of endocannabinoids in rat central nervous system

Endocannabinoids (ECs) are proposed to be extracellular retrograde messengers that regulate the excitability of neurons in the brain by CBi receptor-dependent inhibition of neurotransmitter release. They play physiologically essential roles in diverse biological systems and are widely believed to be synthesized in cell membranes, on demand, via calcium-sensitive phospholipases. Endocannabinoid (EC) levels are governed by the balance between synthesis, metabolism and trans- membrane transport. Thus, it is important to understand the factors regulating intracellular and extracellular EC concentrations, in order to understand mechanisms involved in synthesis (turnover) and release of ECs. The main aims of this thesis were to develop a sensitive and accurate analytical method to measure EC levels in rat cortical slices and the incubation medium, and then apply this method to investigate synthesis (turnover) and release of ECs through measurement of EC concentrations following pharmacological interventions in rat cortical slices and incubation medium in vitro with the special focus on "on- demand" dogma of ECs formation. This method was also applied to study release of ECs in vivo in freely moving rats using the microdialysis technique. Initially a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed allowing the measurement of anandamide (AEA), oleoyl ethanolamide (OEA), palmitoylethanolamide (PEA) and 2-arachidonoyl glycerol (2-AG) in rat cortical slices and incubation medium. In vitro experiments designed to increase EC levels either by stimulating synthesis using depolarising stimuli, G- protein-coupled receptor activation and Ca=-moblhsing stimuli or inhibiting hydrolysis, using FAAH and MAGL-inhibitors. Incubation of slices with glutamate receptor agonists, depolarizing concentrations of KCl, or ionomycin failed to alter tissue levels of ECs, while ECs in the medium were unaltered by elevated KC!. URB597, an inhibitor of fatty acid amide hydrolase, significantly enhanced tissue levels of AEA, OEA and PEA, without altering 2-AG levels, while evoking proportional elevations of AEA in the medium. Removal of extracellular calcium ions failed to alter tissue levels of AEA, OEA and PEA but significantly reduced 2- AG levels in the tissue by 90 % and levels in the medium below the detection limit. Supplementation of the medium with 50 IlM OEA failed to alter either tissue or medium levels of ECs. The data presented indicate little evidence for Ca2+ driving and tonic regulation of synthesis of ECs by CB! or TRPV1 receptors in rat brain slices. The effects of URB597 suggest a high turnover rate of AEA, PEA and OEA in this preparation and that 2-AG is not subject to catabolism by FAAH. These results highlight the ongoing turnover of endocannabinoids, and the importance of calcium ions in maintaining 2-arachidonoylglycerol levels in this tissue. The release of ECs from cortical slices appears, in the main, to be passively driven by the trans-membrane concentration gradient. The cortical slice preparation provides, therefore, a suitable in vitro model for the investigation of EC synthesis, metabolism and release in the brain. Furthermore, we adopted a specific and sensitive method to measure extracellular AEA, OEA and PEA in distinct brain regions in vivo combining our analytical technique to in vivo microdialysis. Due to low levels and the lipophilic nature of ECs in extracellular fluid, approaches such as inclusion of cyclodextrin to the perfusion fluid and use of the most suitable probe; CMA (12)- Polycarbonate are necessary to increase the relative recovery of ECs during the micro dialysis process. Further refinements are necessary to increase the sensitivity and detection of other ECs, including 2-AG, in extracellular fluid of distinct brain regions. Measurement of extracellular brain levels of ECs by the in vivo microdialysis, could provide useful information on the role of neurally released ECs.EThOS - Electronic Theses Online ServiceGBUnited Kingdo