Previous studies have revealed that cannabinoids have detrimental effects on learning and memory in humans and animals. Infusion of the exogenous Cannabinoid agonists including D9-tetrahydrocannabinol (D9-THC), HU210 and WIN-55, 212-2 (WIN-2) in animals has consistently been shown to induce deficits in spatial learning and memory. CB1 receptors are evident in the hippocampus and prefrontal cortex; brain areas involved in spatial learning and memory processes. The main aim of this study was to determine the mechanisms underlying these Cannabinoid-induced effects on spatial learning and memory. Lister Hooded rats were used to study the effects of cannabinoids in a sequence of experiments using the water maze. A novel paradigm to test place preference in the water maze was developed and it was revealed that D9-THC induced place aversion whereas WIN-2 had no effect. These aversive properties of D9-THC along with the non-spatial, non-CB1receptor effects of HU210 in reference memory may confound the results obtained for spatial learning and memory. By contrast, WIN-2 deficits would be a genuine result of spatial impairments. WIN-2 induced differential effects on working and reference memory. Reversal with the CB1 antagonist AM281 and vanilloid receptor (VR1) antagonist Iodo-RTX suggest that the WIN-2 effects are mediated via a non-CB 1/non-VR1 receptor located in the hippocampus. The WIN-2 effects could also be mediated via a possible interaction with the cholinergic system, as the cholinesterase inhibitor Rivastigmine was able to reverse the deficit. In addition to exogenous cannabinoids, using the CB1 antagonist SR141716A (SR) observed that endocannabinoids also impaired spatial learning and memory, with SR acting like an inverse agonist. In conclusion both cannabinoids and endocannabinoids impair spatial learning and memory in the water maze, and these deficits may occur via interactions with other neurotransmitter systems. However, whether these actions are mediated via the CB1 or a non-CB1 mechanism still remains unclear.

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.

This project determined whether non-A'tetrahydrocannabinol phytocannabinoids (non-L'l9THC pCBs) had any effect on feeding patterns in rats and, if so, to characterise such effects. It is well described that L'l9THC administration induces cannabinoid type 1 receptor-mediated hyperphagia by increasing short- term food consumption due to reductions in the latency to feed. Yet, at the outset of this work there was little data available concerning whether or not other pCBs had any effectJs on feeding patterns. Using a pre-satiated paradigm, groups of male rats were administered one of either a range of purified pCBs, two standardised cannabis extracts, two extract analogues or a synthetic L'l9THC. Feeding microstructure data were then examined and analysed for hourly intakes and critical meal parameters. It has been demonstrated for the first time that cannabinol administration increases appetitive (behaviours which govern the latency to feed) and consummatory behaviours (those which control the length and size of meals) and the amount of food consumed. Also for the first time, L'l9THC-mediated effects on meal patterns have been observed. Furthermore, cannabidiol has been shown to induce reductions in food intake over a four hour period. By contrast, cannabigerol and fl9tetrahydrocannabivarin were ineffective feeding modulators in this paradigm. Given the lack of psychotropic side effects observed following the administration of the pCBs, both here and in reported literature, these data suggest that cannabinol and cannabidiol could form the basis of two therapeutic agents to control feeding patterns without psychotropic side effects. These data form the basis for future experiments, potentially clinical, in the field.

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.

CB₁ receptors are confined to the nervous system; their expression within the hippocampus is localised to the presynpatic terminals of a subset of cholecystokinin containing GABA-ergic neurones, where they inhibit GABA release. Hippocampal glutamate release is also inhibited by cannabinoids but at the site of action and receptor involved is still to be identified. Studies of K⁺-evoked release of [³H]D-Aspartate from rat hippocampal synaptosomes suggest anadamide may not just be acting on presynpatic cannabinoid receptors, but also directly on glutamate uptake transporters. The current study aims to characterise cannabinoid-modulation of excitatory amino acid (EAA) release and uptake from hippocampal glutamatergic nerve terminals. Glutamate release from synaptosomes was quantified using spectrofluorimetry and uptake was quantified with [³H]EAA’s. Both the cannabinoids, (WIN55,212-2 and anandamide) inhibited evoked glutamate release in the absence of calcium. Interestingly, WIN55,212-2-inhibition was blocked by SR141716A (CB₁ antagonist), whereas the response to anandamide was not. The non-selective EAA transporter (EAAT) blocker T3MG, inhibited anandamide-attenuation of evoked release. Anandamide reduced basal glutamate levels, where again, neither SR141716A nor capsazepine altered the response to anandamide, indicating these effects are not mediated by either CB₁ or TRPV1 receptors. The action of anandamide was neither mimicked by arachidonic acid, nor affected by PMSF, (FAAH, fatty acid amide hydrolase) inhibitor, suggesting anandamide metabolism to arachidonic acid by FAAH is not required. Anandamide also stimulated [³H]L-glutamic acid uptake; this was attenuated by the non-selective EAAT blocker, DL-TBOA. We suggest that the effects of anandamide are mediated by interaction of the endocannabinoid with glutamate receptors.

-  This study was directed at exploring the method(s) by which cannabidiol is capable of antagonizing the cannabinoid CB₁/CB₂ receptor agonist (<i>R</i>-(+)-WIN55212 in the mouse isolated vas deferens tissue preparation. - The mechanism appeared not to involve competition at the cannabinoid CB₁ receptor, since the CB₁ K_i binding potency (4.9 <span style='font-family:Symbol'>mM) was significantly less than the apparent K_B antagonizing potency (71.4 nM) for the antagonism of <i>R</i>-(+)-WIN55212 in the isolated mouse vas deferens. Additionally, the ability of cannabidiol to antagonize <i>R-</i>(+)-WIN55212 in the mouse isolated van deferens was not reproduced in the guinea-pig myenteric plexus-longitudinal muscle (MPLM) preparation; a tissue that like the mouse vas deferens expresses cannabinoid CB₁ receptors. -  Evidence was generated that suggested the antagonism of <i>R</i>-(+)-WIN55212 by cannabidiol was not through a non-specific mechanism, since the pharmacological properties investigated displayed structural-dependence. Furthermore cannabidiol exhibited stereoselectivity as an antagonist of <i>R-</i>(+)-WIN55212. -  The cannabidiol analogue, 6”-azidohex-2”-yne-cannabidiol (O-2654) displayed a higher affinity than cannabidiol for cannabinoid CB₁ receptors (K_i = 114.4 nM) and cannabinoid CB₂ receptors (K_i = 54.4 nM). Unlike, cannabidiol, O-2654, appeared to antagonize <i>R</i>-(+)-WIN55212 in the mouse isolated vas deferens by behaving as a neutral cannabinoid CB₁ receptor antagonist, since the apparent K_B value (85.7 nM) for this antagonism was not significantly different from the determined CB₁ K_i value. -  Another analogue of cannabidiol, 7-OH-DMH-CBD, was observed to potently inhibit electrically evoked contractions of the mouse isolated vas deferens (EC₅₀ = 13.3 nM) at a putative site that was demonstrated to be a non-CB₁, non-CB₂, non-TRPV1, non-<span style='font-family:Symbol'>a₂-adrenergic and non-opioid molecular target that is sensitive to antagonism by cannabidiol. -  Together these data have identified a novel target for cannabinoids and a cannabidiol analogue that behaves as a neutral cannabinoid CB₁ receptor antagonist.

The use of a whole plant cannabis extract, containing <span style='font-family:Symbol'>D⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD) as the principal constituents, showed statistically significant improvements in the management of multiple sclerosis. Inhibition studies (e.g. IC₅₀ and K_i determinations) using phenotyped human liver microsomes and cDNA expressed human P450s (Supersomes<span style='font-family:Symbol'>â demonstrated that CBD competitively inhibits the principal P450s involved in the THC biotransformation, CYP2C9 (K_i = 0.5 <span style='font-family:Symbol'>mM), CYP2C19) (K_i = 0.4 <span style='font-family:Symbol'>mM) and CYP3A4 (K_i = 0.07 <span style='font-family:Symbol'>mM.  CBD inhibition of CYP3A4 was mechanism-based, which suggests that a CBD metabolite (e.g. CBD-hydroxyquinone) is involved in CYP3A4 inhibition. CBD differentially induced rat P450s, whereas THC had no discernible effects on rat P450s.  CBD significantly increased CYP1A2 protein at 150 mg kg^-1, but showed no change in mRNA expression. In addition, CYP1A-dependent activity was inhibited by < 80 % by CBD. These results suggest that CBD may bind tightly to and modify the CYP1A2 active site, thereby stabilising the protein but preventing substrate interaction. The significant increase in CYP2B1 mRNA implies that CBD transcriptionally regulates CYP2B, perhaps by activating CAR or through “cross-talk” by PXR. The 4-fold increase in CYP3A23 mRNA level suggests that CBD may be a weak ligand for PXR or that CBD is acting via CAR, which can also bind to response elements on the CYP3A23 gene. CBD is a potent inhibitor of P450-catalysed THC metabolism <i>in vitro</i> however pharmacokinetic modelling predicted that the therapeutic level of CBD (low nM range) after sublingual co-administration of THC and CBD (10 mg of each) was insufficient to inhibit THC metabolism of other human volunteers. This does not rule out the potential for CBD to inhibit the metabolism of other co-administered drugs <i>in vivo</i>. CBD may also induce the human orthologues of rat P450s, mainly CYP2B6 and CYP3A4, following extended periods of administration at high doses.

The research detailed in this thesis consisted of three individual investigations: Firstly, the characterisation of the novel eicosanoid, leukotriene B₄ ethanolamide (LTB₄ ethanolamide), with leukotriene BLT and vanilliod TRPV1 receptors using rat TRPV1-transfected CHO cells, neonatal rat cultured dorsal root ganglion neurones, guinea-pig lung parenchyma and human neutrophils. LTB₄ ethanolamide acts as an antagonist/partial agonist, depending upon receptor expression, at BLT receptors, and as a weak partial agonist at TRPV1 receptors. Secondly, the characterisation of novel compounds at BLT and TRPV1 receptors. These compounds were designed to exhibit antagonism of both these receptors. O-3367and O-3383 act as competitive BLT receptor antagonists with IC₅₀ values of ~ 60 nM and ~ μM, respectively. O-3367 acts as a competitive antagonist at TRPV1 receptors with an IC₅₀ values of ~ μM. Thirdly, the effect of cannabinoids have on human neutrophil migration was investigated. Anamdamide, <i>N</i>-arachidonyl dopamine, virodhamine, CP55940, cannabidiol, (+)-cannabidiol and abnormal-cannabidiol all inhibited <i>N</i>-formylmethyionylleucylphenylalainine (fMLP) – induced neutrophil migration in a concentration-dependent manner. Sphingosine-1 –phosphate, a compound structurally related to anandamide was also capable of inhibiting induced migration, whereas palmitoylethanolamide, 2-arachidonylglycerol, Δ⁹tetrahydrocannabinol, O-2654, SR141716A, SR144528, capsazepine and AM630 was not. O-2654 induced a concentration-dependent stimulation of neutrophil migration with an EC₅₀ of 16.7 nM. The findings suggest cannabinoids function as anti-inflammatory or immunosuppressive agents, by interfering with chemoattractant-induced directional migration of neutrophils.

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