Vitamin E TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate), the water-soluble derivative of the naturally occurring d-a-tocopherol, was used initially to overcome malabsorption and correct severe vitamin E deficiency in cholestatic children. The observation that its administration increased the absorption of vitamin D focused attention on its amphiphilic properties as nonionic surfactant and applications in solubilizing lipophilic and poorly soluble compounds and enhancing their absorption and bioavailability. Further research has evaluated its safety and efficacy which combines solubilization and formation of micelle-like particles with inhibition of P-glycoprotein-mediated efflux, the key mechanism to the development of drug resistance. These properties expanded its applications in pharmaceuticals and dietary, disease-specific, supplements for malabsorbing patients. Emerging research on major nutraceuticals and the developing field of cannabinoids have shown that poor water solubility and extensive first-pass metabolism cause poor absorption and bioavailability. This chapter describes the properties, safety, and efficacy of vitamin E TPGS with a focus on its applications in nutraceuticals and cannabinoids.

The development and implementation of biosensors as an integral and growing part of our modern world has prompted the push for precision health as the next step in medicine. Adapted from aircraft engine monitoring, where an array of sensors is used to build a digital twin to preemptively predict problems, precision health requires an increase in molecular monitoring. Organic thin-film transistors (OTFTs), as sensitive, low-cost, and adaptable devices are well suited to meet this need. Phthalocyanines (Pcs), as an organic semiconducting layer for OTFTs, are easily synthesized and highly tunable small molecules which can be deposited through both solution and physical vapor deposition techniques, enhancing their utility. This work presents Pc-based OTFTs for cannabinoid sensing and chemotyping to meet the quality control needs of a growing Canadian and International cannabis industry, and to broadly demonstrate the sensitivity and selectivity attainable with Pc-based OTFTs incorporating molecular analyte sensors. Spectroelectrochemistry is established as a screening technique for Pc-based OTFT sensors and, in combination with thin-film characterization, is used to propose a mechanism for Pc-cannabinoid interactions and OTFT cannabinoid sensitivity with and without a cannabinoid-sensitive chromophore. Thin-film morphologies and polymorphs, pre- and post-analyte exposure, are demonstrated as key drivers of Pc-based OTFT sensing responses and are further explored through controlled deposition conditions and post deposition annealing techniques. Through material screening and thin-film engineering, part-per-billion cannabinoid sensitivity is achieved with Pc-based OTFTs. This report documents several strategies for sensitizing Pc-based OTFT sensors to organic analytes, and the results herein serve as a basis for continued development of Pc-based OTFT biosensors.

No description available.

Rationale. For centuries, medications derived from the marijuana plant have been used for therapeutic purposes across numerous cultures. In 1964, the primary psychoactive ingredient in cannabis, delta-9-tetrahydrocannabinol (-9-THC) was defined. This, followed by the discovery of the endocannabinoid system, marked the beginning of comprehensive research into the beneficial exploitation of this system. The cannabis plant contains various other cannabinoids besides -9-THC. Most of the effects of cannabinoid-based therapies are based on the agonistic action of -9-THC through cannabinoid receptors. Alternatively, some of these effects are caused by the actions of other cannabinoids, like cannabidiol, which does not have high affinity for cannabinoid receptors. Like -9-THC, cannabidiol (CBD), the non-psychoactive phytocannabinoid in Cannabis sativa, has been hypothesized to ameliorate adverse effects of -9-THC. Cannabidiol possesses neuroprotective, antiemetic, and anti-inflammatory properties. Sativex, a 1:1 ratio of CBD and -9-THC, is currently an approved medication in Europe for the treatment of conditions such as neuropathic pain, and has been fast tracked by the USFDA for late stage clinical trials for a host of disorders, ranging from epilepsy to irritable bowel disease. Additionally, increasing preclinical evidence demonstrates that treatment with Cannabidiol alone produces efficacy on a variety of nervous system injuries, including neuropathic pain, schizophrenia and anxiety disorders. Furthermore, there is mounting evidence of an “entourage effect” in cannabinoid-based pharmacotherapies. This effect occurs when treatment with a combination of cannabinoids derived from the plant produce more efficacy than treatment with a single cannabinoid (1). As cannabinoid-based treatments continue to develop and clinical data increases, further investigation of the entourage effect is necessary to facilitate the appropriate future treatment regimens for nervous system disorders. Hypotheses. We hypothesized that treatment with the non-psychoactive cannabis compound cannabidiol would be as effective as the psychoactive cannabis compound -9-THC, or a combination of the two, in mitigating neuropathic pain in a mouse model of chemotherapy-induced peripheral neuropathy. We additionally hypothesized that cannabidiol would not affect classic cannabinoid-agonist induced cognitive impairment in rodent models of learning and memory. Methodology. Neuropathic pain was induced by repeated injections of the chemotherapeutic agent Paclitaxel. Mechanical hypersensitivity to Paclitaxel was assessed using the Von Frey assay. Cognition was assessed using three rodent models of learning and memory: 1) Conditional Discrimination, 2) Conditional Discrimination with a reversal component, and 3) Barnes Maze. Results. Cannabidiol was found to be more potent and more effective than -9-THC in attenuating neuropathic pain in a dose dependent manner. Combinations of CBD+-9-THC revealed that lower, ineffective doses of CBD and -9-THC display supra-additive effects when given in combination while higher, individually effective doses exhibit sub-additive effects in combination. Cognitively, no deficits were observed over a range of doses of any cannabinoid tested in the conditional discrimination tasks, although a slight trend was observed in animals administered the synthetic mixed CB1/CB2 agonist WIN55,212-2. In the Barnes Maze task, treatment with -9-THC alone dose-dependently decreased number of entries and total time spent in the target zone. Cannabidiol did not produce any effects in the Barnes Maze alone, nor did it attenuate the effects seen in animals treated with -9-THC alone. Lastly, -9-THC did not affect total distance traveled or average speed, whereas combination treatment increased both locomotor measurements at all but the highest combination dose. Conclusions. The results of these studies indicate that cannabidiol is more potent than -9-THC in attenuating neuropathic pain. Results of cognitive testing indicate subtle impairment in animals treated with -9-THC and WIN55,212-2 that were not reversed by CBD. / Biomedical Sciences

Submitted in fulfillment of the requirement for the degree of Doctor of Philosophy in the Faculty of Health Sciences at the University of the Witwatersrand, Johannesburg / Renewed interest in cannabinoid compounds arose since the discovery of the endocannabinoid system in the early 1990’s and its role in mediating the body’s energy balance. The aim of this study was to investigate the effect of an organic Cannabis sativa (hereafter referred to as C. sativa) extract on β-cell secretory function using an in vivo diet-induced obese rat model and an in vitro isolated rat pancreatic islet model and to determine the associated molecular changes within the pancreatic tissue. Materials and methods: Diet-induced obese Wistar rats and rats fed on standard pellets were subcutaneously injected, over a 28 day period, with an organic C. sativa extract or the vehicle (1% Tween 80® in saline). The effect of diet and treatment was evaluated using the intraperitoneal glucose tolerance tests (IPGTTs) and quantitative polymerase chain reaction (qPCR) analysis on rat pancreata. In vitro studies were conducted using isolated rat islets exposed to 11.1 (representative of normoglycemic conditions) and 33.3 mM glucose levels (representative of hyperglycemic conditions) over a 24-(D1; acute) and 96-hour (D4; chronic) period, and treated with C. sativa extract containing an equivalent of 2.5 (T1) and 5 ng/mL (T2) tetrahydrocannabinol (THC). Glucose-stimulated insulin secretion (GSIS), immunohistocytochemistry for apoptosis and proliferation detection and western blotting for detection of cannabinoid receptor type 1 (CB1), CB2 receptors and specific transduction factors were undertaken. Antagonist studies were conducted using AM251 (A1) and AM630 (A2) to block CB1 and CB2, respectively, to determine the role of cannabinoid receptors in insulin secretion. Results: The overall increase in body weight in the experimental groups occurred at a significantly slower rate than the control groups (P < 0.01), irrespective of diet. In the lean group, the area under the curve for glucose (AUCg) was significantly higher compared to the diet-induced obese group (P < 0.001), while C. sativa treatment significantly improved the AUCg in the lean rats (P < 0.05). The cafeteria diet did not induce hyperglycemia and insulin resistance in the obese rats and C. sativa treatment maintained a plasma glycemic profile similar to the obese control rats. The lower AUCg values in the obese group may, in part, be due to the inclusion of milk products (shown to be beneficial in reducing diabetes) in the cafeteria diet. qPCR analysis showed that the cafeteria diet induced down-regulation of the following genes in the obese control group, relative to lean controls: UCP2 (P < 0.01), c-MYC (P < 0.05) and FLIP (P < 0.05), and upregulation of CB1 (P < 0.01), GLUT2 (P < 0.001), UCP2 (P < 0.001) and PKB (P < 0.05), relative to the obese control group, while c-MYC levels were down-regulated (P < 0.05), relative to the lean control group. In the in vitro study, results showed C. sativa treatment decreased chronic insulin secretion in islets cultured under normoglycemic condition for D1 (P < 0.05), but not for D4. In islets cultured under hyperglycemic conditions, C. sativa treatment for the D4 period showed a significant increase in their chronic insulin secretion (HD4T1, P = 0.07; HD4T2, P < 0.001), increase in basal insulin secretion (HD4T1, P < 0.001; HD4T2, P < 0.001), increase in GSIS (HD4T1, P < 0.05; HD4T2, P < 0.001), reduction in glucose-stimulated:basal insulin production (HD4T1, P < 0.05; HD4T2, P < 0.05), reduction in insulin content (HD4T1, P < 0.001), increase in the percentage basal : content ratio (HD4T1, P < 0.001; HD4T2, P < 0.01) and increase in the percentage GSIS : content ratio (HD4T1, P < 0.001; HD4T2, P < 0.05), relative to ND4C islets. In antagonist studies, A2 preconditioning did not affect suppress the stimulatory effect of C. sativa treatment on chronic insulin secretion under normo- and hyperglycemic conditions, relative to the NC and HC islets, respectively. qPCR studies showed that C. sativa exposure induced a 2.2-fold increase in CB1 gene expression, relative to normoglycemic control islets (P < 0.05), while c-MYC and FLIP expression was significantly reduced by 12% (ND4T1, P < 0.05) and 37% (HD4T1, P < 0.05), respectively. C. sativa treatment also induced increased secretion of anti-inflammatory cytokines/chemokines under hyperglycemic conditions. Conclusion: These results suggest that C. sativa protects pancreatic islets against the negative effects of obesity (in vivo studies) and hyperglycemia (in vitro studies). In light of these findings, further investigation into the potential of C. sativa as a complementary therapeutic agent in the treatment of the deleterious effects of hyperglycemia in diabetic patients is warranted. In addition, the significant effect of C. sativa treatment on adipose tissue in experimental rats needs further investigation to determine how the cannabinoids affect the mechanisms of adipogenesis and lipolysis in diet-induced obesity. Keywords: Diet-Induced Obesity, Cannabinoids, C. sativa, THC, β-cell, AM251, AM630.

Το ενδογενές σύστημα των κανναβινοειδών αποτελεί ένα πολύπλοκο ενδογενές σύστημα μεταγωγής σήματος το οποίο επηρεάζει ένα σημαντικό αριθμό φυσιολογικών διεργασιών και μεταβολικών μονοπατιών του οργανισμού (Cota and Woods, 2005). Απαρτίζεται από τους διαμεμβρανικούς υποδοχείς των κανναβινοειδών (CBR), τα ενδοκανναβινοειδή και τις πρωτεΐνες που είναι υπεύθυνες για την βιοσύνθεση και την αποικοδόμηση των δεύτερων. (Petrocellis et al., 2004). Τα κανναβινοειδή παρουσιάζουν ποικιλία επιπτώσεων, όπως δυσλειτουργία στη μάθηση και μνήμη, διαφοροποίηση των συναισθηματικών καταστάσεων, μείωση κινητικού ελέγχου και αναλγησία. Αναστέλλουν τη συναπτική διαβίβαση σε διάφορες περιοχές του εγκεφάλου όπως ο ιππόκαμπος, ο επικλινής πυρήνας και ο προμετωπιαίος φλοιός κυρίως μέσω προσυναπτικών μηχανισμών. Το γλουταμινικό οξύ είναι ο κύριος διεγερτικός νευροδιαβιβαστής στο Κεντρικό Νευρικό Σύστημα των θηλαστικών. Οι γλουταμινεργικοί νευρώνες διανέμονται ευρέως στο κεντρικό νευρικό σύστημα και παίζουν ρόλο σε πολλές βιολογικές διεργασίες, συμπεριλαμβανομένης της κωδικοποίησης των πληροφοριών, το σχηματισμό και την ανάκτηση των μνημών, τη χωρική αναγνώριση και τη διατήρηση της προσοχής (McEntee και Crook, 1993). Λόγω του ρόλου του στη συναπτική πλαστικότητα, το γλουταμινικό εμπλέκεται σε γνωσιακές λειτουργίες του εγκεφάλου, όπως η μάθηση και η μνήμη (McEntee και Crook, 1993). Το γλουταμινικό δρα στα μετασυναπτικά κύτταρα σε τρεις οικογένειες ιοντοτροπικών υποδοχέων τους NMDA, τους AMPA και τους καϊνικούς υποδοχείς, ενώ υπάρχουν, επίσης, και γλουταμινικοί μεταβολοτροπικοί υποδοχείς που συνδέονται με G πρωτεΐνες. Η αμυγδαλή εκτελεί ένα πρωταρχικό ρόλο στην επεξεργασία των συναισθηματικών αντιδράσεων και τη μνήμη, ενώ ο ιππόκαμπος έχει σημαντικό ρόλο στην εδραίωση των πληροφοριών από βραχυχρόνια μνήμη σε μακροχρόνια μνήμη και στη χωρική πλοήγηση. Οι δύο αυτές περιοχές είναι πλούσιες σε CB1 και NMDA υποδοχείς, ενώ τα τελευταία χρόνια, πληθώρα ερευνών υποδεικνύει ότι η έκθεση πειραματοζώων σε κανναβινοειδή επιφέρει σημαντικές αλλαγές σ’ αυτές τις δύο περιοχές. Στόχος της ερευνητικής εργασίας ήταν η μελέτη της επίδρασης των κανναβινοειδών στο γλουταμινεργικό σύστημα, στην αμυγδαλή και τον ιππόκαμπο επίμυων. Η μελέτη επικεντώθηκε στους NMDA υποδοχείς του γλουταμινικού οξέος με τη χρήση των μεθόδων της in situ υβριδοποίησης για το mRNA των NR1, NR2A και NR2B υπομονάδων του υποδοχέα και της ποσοτικής αυτοραδιογραφίας υποδοχέων, σε επίμυς που τους χορηγήθηκε WIN55212-2, ένας συνθετικός αγωνιστής του CB1 κανναβινοειδικού υποδοχέα, ο οποίος εμφανίζει παρόμοια δράση με την Δ9-τετραϋδροκανναβινόλη (THC), το φυσικό συστατικό του φυτού Cannabis sativa. Μελετήθηκαν τέσσερις ομάδες επίμυων όπου σε δύο από αυτές χορηγήθηκε WIN και στις υπόλοιπες δύο χορηγήθηκε ο διαλύτης του WIN, Vehicle. Στις δύο ομάδες που χορηγήθηκε WIN55212- 2 1mg/kg για 20 ημέρες, οι επίμυς της μίας ομάδας (ομάδα WIN) θανατώθηκαν 2 ώρες μετά από την τελευταία δόση ενώ οι επίμυς της άλλης ομάδας (ομάδα WIN+WITHD) θανατώθηκαν 7 ημέρες μετά την τελευταία δόση. Κατά το διάστημα αυτό δεν έγινε καμία χορήγηση ώστε να μελετηθεί αν οι επιπτώσεις της χρόνιας χορήγησης του WIN ήταν μόνιμες. Τα αποτελέσματά μας έδειξαν ότι μετά τη χρόνια χορήγηση του WIN τα επίπεδα έκφρασης των υπομονάδων του NMDA υποδοχέα μειώθηκαν στο ιππόκαμπο και την αμυγδαλή των επίμυων, ενώ μετά την διακοπή της χορήγησης του WIN, τα επίπεδα έκφρασης επανέχρονται. Είναι πιθανό ότι οι αλλαγές στην έκφραση και λειτουργία των υποδοχέων του γλουταμινικού που παρουσιάζονται ως προσαρμοστικές αλλαγές στο νευρικό σύστημα, να είναι ένας κοινός μηχανισμός με τον οποίο τα εθιστικά ναρκωτικά επηρεάζουν την νευρική λειτουργία. / The endocannabinoid system is a complicated endogenous signaling system that affects a variety of physiological processes and metabolic routes in human body (Cota and Woods, 2005). The endocannabinoid system includes the transmembrane cannabinoid receptors (CBR), the endocannabinoids and the enzymes that synthesize and degrade the endocannabinoids (Petrocellis et al., 2004). Cannabinoids have a variety of effects, such as impairment in learning and memory, modulation of emotional states, reduced motor control and analgesia. Cannabinoids inhibit synaptic transmission in several brain regions such as the hippocampus, nucleus accumbens and the prefrontal cortex mainly via presynaptic mechanisms. Glutamate is the most abundant excitatory neurotransmitter in the mammalian central nervous system. The glutamatergic neurons are widely distributed in the central nervous system and play a role in many biological processes, including the coding of information, the formation and recovery of memories, the spatial recognition and maintaining the attention (McEntee και Crook, 1993). Because of its role in synaptic plasticity, glutamate is involved in cognitive functions like learning and memory in the brain. (McEntee και Crook, 1993). Glutamate activates three families of ionotropic receptors in postsynaptic cells, those are NMDA, AMPA and kainate receptors, while there are also metabotropic G proteins coupled glutamate receptors. Amygdala has a primary role in the processing of emotional reactions and memory, whereas hippocampus has an important role in the consolidation of informations from short term memory into long term memory and spatial navigation. These two brain regions contain a large number of CB1 and NMDA receptors, while recently, many studies suggest that animals treated with cannabinoids display significant changes in these two areas. The aim of this research was to study the changes in glutamatergic system in the amygdala and hippocampus of rats treated with cannabinoids. We focused on NMDA glutamate receptors, using in situ hybridization for studying the expression of NR1, NR2A and NR2B subunits and quantitative receptor autoradiography, in rats treated with WIN55212-2, a synthetic agonist of the CB1 cannabinoid receptor, which shows similar effects with delta-9-tetrahydrocannabinol (THC), a natural component of the plant Cannabis sativa. Adult rats were injected with WIN55212-2 (1mg/kg) and Vehicle. Animals received repeated administrations of WIN55212-2 1mg/kg once a day for 20 days. Animals in group WIN were sacrificed 2 hours after the last administration whereas in group WIN + WITHD were sacrificed 7 days after the last administration. During this time there was no administration of WIN so we could study whether the effects of chronic exposure were permanent. Our results demonstrate that chronic exposure to WIN55212-2 produced significant decreases in the expression of NMDA receptor's subunits in hippocampus and amygdala. These changes were reversed one week after abstinence. These adaptive synaptic changes may share common mechanisms with addictive drugs in modifying neural circuitry.

The Endocannabinoid system has been found in the gastrointestinal tract, where it plays an important role in gut under both physiological and pathological conditions. Although the major effects of cannabinoids in the gut are mediated through effects on enteric neurons, the role of cannabinoids in the enteric nervous system is poorly understood. In the present study, we have used the primary cultures of myenteric ganglia and a newly developed fetal enteric neuronal cell line to identify whether the endocannabinoid, anandamide, affects ganglionic and neuronal survival and the pathways involved. Anandamide had a biphasic effect on ganglionic survival, increasing survival at low concentrations (1nM-0.1uM) and decreasing survival at high concentrations (1-10uM). Maximal survival (68% increase in number of ganglia surviving) occurred at 0.1uM and the ED50 was 3nM. This effect on promoting survival was inhibited by the CB1 antagonist AM251 (1uM) and by AraC (10uM), but not the CB2 antagonist AM630 (1uM). AM630 (1uM) significantly blocked the decreased survival induced by high concentration anandamide (10uM). The enteric glia was involved in anandamide-induced ganglion survival. Anandamide had no effect on the number of neurons/ganglion in the presence of enteric glia, but decreased the number of neurons/ganglion by 15-20% in absence of enteric glia. This effect was partially reversed by CB1 antagonist, AM251 (1uM) (20%-145% at 1nM-10uM) and by CB2 antagonist AM630 (1uM) (40%-185% at 1nM-10uM). In the fetal enteric neural cell line (IM-FEN), anandamide decreased enteric neuronal survival in a concentration-dependent manner at both 39 and 33 degree (11-45% and 10-22%decrease in survival at 1nM-10uM, respectively). Coculture of astrocytes with the enteric neuronal cells was not able to reverse anandamide-mediated neuronal death. Immunocytochemistry and western blot confirmed that the presence of both CB1 and CB2 receptors in enteric neurons (primary cultures and IM-FEN) and glia (primary cultures). In addition, the PLC-beta inhibitor U73122 (1uM) inhibited anandamide induced ganglia survival significantly. Anandamide also induced increased expression of phospho-P44/42MAPK (13-48% at 1nM-10uM) and phospho-AKT (1-28% at 1 nM-10uM) in IM-FEN. We conclude that anandamide has a differential effect on survival of enteric ganglia and neurons. It promotes ganglionic and neuronal survival by CB1 receptors in the presence of glia and this involves the PLC-beta pathway. Conversely, anandamide promotes neuron death in absence of glia as a result of effects on both the MAPK and PI-3K/AKT pathways. Since the endocannabinoid system is upregulated in inflammatory bowel diseases, these effects may play a role in the pathogenesis of the response to inflammation as well as the recovery and reinnervation of the gut following the acute phase of inflammation. The further significance of this work could contribute to developing new therapeutic methods for treatment of inflammatory bowel disease and related symptoms in clinic practice.

Vascular calcification is strongly correlated with the clinical manifestations of atherosclerosis, heart attacks and strokes. The calcification process resembles bone formation and involves the osteogenic trans-differentiation of smooth muscles cells within the arterial wall. Cannabinoid receptors are known to modulate bone formation and are present in atherosclerotic vessels, suggesting they may also play a role in modulating calcification. Therefore, we evaluated the effects of cannabinoids on the expression of osteogenic proteins by vascular smooth muscle cells undergoing calcification.

Doctor of Philosophy (PhD) / Abstract Olfactory tasks are used very often with laboratory animals in studies of the neurobiology of learning and memory. Rats and mice are extremely sensitive in their detection and discrimination of odours, learn olfactory tasks rapidly, and can display higher order cognitive functions in olfactory tasks. This cognitive capacity may rival the ability of primates to learn analogous tasks with visual cues and most likely reflects strong anatomical connections between the olfactory bulbs and higher brain regions such as the piriform cortex, orbitofrontal cortex and hippocampus. The current thesis explored olfactory discrimination learning and performance in rats and had two principal aims. The first part of the thesis was oriented around odour masking phenomena in rats: the ability of one odour in a mixture to suppress detection of a second odour in that mixture. A specialized behavioural paradigm was developed to allow the study of odour masking in the rat. The second part of the thesis was pharmacological and determined whether the acquisition, reversal and performance of olfactory discriminations, and analogous auditory discriminations, are affected by two commonly used classes of drugs (benzodiazepines and cannabinoids). Together, these studies attempt to gain a better understanding of the nature of olfactory discrimination learning in rats, by using both psychophysical and pharmacological approaches, and to develop behavioural paradigms which may be used in future psychophysical and pharmacological studies. Following an introduction and review of olfactory and auditory studies in rat (Chapter 1), odour masking phenomena were studied in Chapter 2. The aliphatic aldehydes butanal (C4) and heptanal (C7) were used in the study. Aldehydes were of interest as this class of odorants abound in nature and may be important for rodents’ species-specific communication. Thirsty rats were initially trained to discriminate C4 and C7 in the olfactometer, using a go/no-go olfactory discrimination task. This involved rats learning to nose poke in an odour port and to lick a tube for a water reward on presentation of the rewarded component S+, while withholding licking at the tube when the other, unrewarded, aldehyde (S-) was presented. Odour mixtures (C4C7 or C7C4) were then introduced into the task as an additional non-rewarded condition (mixture S-). The concentration of the non-rewarded aldehyde in the mixture was then systematically decreased, while the concentration of the rewarded aldehyde was kept constant. When the non-rewarded aldehyde reached a critical low level in the mixture, rats started to make responses to the non-rewarded mixture (false alarms) showing that the S+ odour was suppressing the S- odour in the mixture, so the mixture was being responded to in the same manner as the S+ odour presented alone. Results also showed asymmetric suppression in the mixture condition, such that butanal suppressed detection of heptanal at a much lower concentration than vice versa. A second experiment demonstrated that when both butanal and heptanal were present in a binary mixture at the same concentration (10-6 volume %), rats responded to the mixture as if only butanal was present. Our findings are in agreement with human studies showing component interactions in binary mixtures of aldehydes. The molecular feature of carbon chain length appears to be a critical factor in determining the outcome of interactions between aldehydes at peripheral olfactory receptors, with smaller chain aldehydes better able to compete for receptor occupancy. Subsequent chapters explored the effects of two classes of commonly used drugs - benzodiazepines and cannabinoids - on olfactory and auditory discrimination in rats. Animal models such as the radial arm maze, Morris water maze and object recognition test are routinely used to test adverse and facilitatory effects of drugs on cognition in rodents. However, comparatively few pharmacological studies employ olfactory or auditory go/no-go paradigms. Thus, an important part of the present thesis was to assess the viability of using such paradigms in detecting pharmacological effects, and to identify whether such effects may be modality specific (i.e. whether a drug has a greater effect on olfactory or auditory tasks). In Chapter 3, the effects of benzodiazepines on olfactory discrimination tasks were explored. Rats were injected with the benzodiazepine drugs midazolam or diazepam and tested on discrimination tasks involving either the auditory and olfactory modality. Results showed that midazolam (0.5–2 mg/kg sc) did not affect the performance of a well-learned two-odour olfactory discrimination task, and moderately facilitated the performance of a go/no-go auditory discrimination task. On the contrary, midazolam (1 mg/kg) impaired the acquisition of a novel go/no-go olfactory discrimination task, as well as the reversal of a previously well-learned olfactory discrimination. However, midazolam did not affect the acquisition or reversal of an equivalent auditory discrimination task. The olfactory bulb and the piriform cortex are intimately involved in associative learning and behavioural aspects of olfactory performance, and have high concentrations of benzodiazepine receptors. These may therefore be possible neural substrates for the disruptive effects of benzodiazepines on olfactory learning. Findings from Chapter 4 indicated that the prototypical cannabinoid agonist delta-9-tetrahydrocanabinol (Δ9 THC) (0.3, 1 and 3 mg/kg) impairs auditory discrimination performance, but had no effect on equivalent olfactory discriminations. This is in marked contrast to the effects of benzodiazepines. Residual effects were observed, such that auditory discrimination performance was still impaired on the day following Δ9 THC administration. Delta-9-tetrahydrocanabinol effects were prevented by co-administration of the cannabinoid antagonist rimonabant (3 mg/kg). In addition, the anandamide hydrolysis inhibitor URB597 (0.1 and 0.3 mg/kg), which boosts levels of endogenous cannabinoids in the synapse, also impaired auditory discrimination performance, and this effect was also reversed by rimonabant. This study also assessed the effects of Δ9 THC (0.3, 1 and 3 mg/kg) and URB597 (0.1 and 0.3 mg/kg) on acquisition and reversal of novel olfactory discriminations. Results showed that Δ9 THC impairs olfactory reversal learning without affecting acquisition of the original discrimination. It is argued that this reversal deficit may be part of a wider capacity for cannabinoids to impair cognitive flexibility. The final Chapter (General Discussion) discusses the relevance and implications of the combined findings. The results add significantly to our current understanding of perceptual, learning and memory processes involving the olfactory modality in rats. With respect to olfactory perception, this thesis introduced a new behavioural paradigm, which can be used to assess component suppression in mixtures, and may be of use in future psychophysical studies involving rodents or other species. With respect to learning and memory, the thesis provides novel information on the disruptive effects of benzodiazepines and cannabinoids on olfactory and auditory tasks. It is concluded that go/no-go olfactory and auditory discrimination tasks in rats can provide a useful platform for assessing the disruptive and modality-specific effects of drugs on learning, performance and cognitive flexibility. Future studies might expand the range of drugs tested on these paradigms and might consider chronic as well as acute drug effects.

In both vertebrates and invertebrates, including leeches, microglia are rapidly activated by central nervous system (CNS) damage and migrate to the lesions. Adenosine triphosphate (ATP), nitric oxide (NO) and endocannabinoids have been implicated in controlling activation and migration, but details of the mechanisms are uncertain. This dissertation tests the hypothesis that endocannabinoids coordinate and influence the microglial response to nerve cord crushing. Chapter 1 reports that application of endocannabinoids to nerve cords at concentrations as low as 100 nM for arachidonylethanolamide (anandamide, or AEA) reduced the number of migrating microglia, but not when cords were pretreated with 10 µM of the CB2 cannabinoid receptor (CB2R) antagonist SR144528. In addition, immunoblots confirmed the expression of CB1-like and CB2-like receptors and immunohistochemistry showed that they were concentrated at lesions, where microglia accumulated. Benzoyl ATP (BzATP) also reduced microglia accumulation, an effect blocked by pretreatment of nerve cords with SR144528, whereas the G-protein coupled P2YR agonists uridine triphosphate (UTP) and methylthio-ATP (MeSATP) at 100 µM did not reduce accumulation. This result suggested that P2X7R activation elicited production and release of a CB2R agonist that influenced microglia movement. Chapter 2 reports that extracellular ATP levels were highest in the CNS within the first 30 min of injury and remained above unharmed controls for at least 2 hours. Application of 10 units (U) of the ATPase apyrase to nerve cords reduced accumulation of microglia at lesions, another indication that microglia require extracellular ATP to accumulate. Chapter 3 reports that AEA stopped ATP-induced movement of microglia, and that this effect was blocked by pretreatment of nerve cords with the CB1R antagonist SR141716A (10µM), the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L NAME, 2 mM) or the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-oxyl-3-oxide (carboxy-PTIO or cPTIO, 1 mM). Thus the migration of microglia to lesions is regulated not only by ATP acting on P2YR and by NO acting on soluble guanylate cyclase (sGC), but also potentially by ATP binding to P2X7-like receptors to increase the production of cannabinoids. Cannabinoids, binding to the CB1R and CB2R cause production of NO, which suppresses microglia movement.