Cannabinoids (CBs) are compounds that activate the CB1 and CB2 receptors. CB receptors mediate many different physiological functions, and cannabinoids have been reported to decrease tumor cell viability, proliferation, migration, as well as to modulate metastasis. In this thesis, the effects of cannabinoids on human colorectal carcinoma Caco-2 cells (Paper I) and mouse P19 embryonal carcinoma (EC) cells (Paper III) were studied.  In both cell lines, the compounds examined produced a concentration- and time-dependent decrease in cell viability. In Caco-2-cells, HU 210 and the pyrimidine antagonist 5-fluorouracil produced synergistic effects upon cell viability. The mechanisms behind the cytocidal effects of cannabinoids appear to be mediated by other than solely the CB receptor, and a common mechanism in Caco-2 and P19 EC cells was oxidative stress. However, in P19 EC cells the CB receptors contribute to the cytocidal effects possibly via ceramide production. In paper II, the association between CB1 receptor immunoreactivity (CB1IR) and different histopathological variables and disease-specific survival of colorectal cancer (CRC) was investigated. In microsatellite stable (MSS) cases there was a significant positive association of the tumor grade with the CB1IR intensity. A high CB1IR is indicative of a poorer prognosis in MSS with stage II CRC patients. Paper IV focused on the cytotoxic effects of cannabinoids during neuronal differentiation. HU 210 affected the cell viability, neurite formation and produced a decreased intracellular AChE activity. The effects of cannabinoids on embryonic development and survival were examined in Paper V, by repeated injection of cannabinoids in fertilized chicken eggs. After 10 days of incubation, HU 210 and cannabidiol (without CB receptor affinity), decreased the viability of chick embryos, in a manner that could be blocked by α-tocopherol (antioxidant) and attenuated by AM251 (CB1 receptor antagonist). In conclusion, based on these studies, the cannabinoid system may provide a new target for the development of drugs to treat cancer such as CRC. However, the CBs also produce seemingly unspecific cytotoxic effects, and may have negative effects on the neuronal differentiation process. This may be responsible for, at least some of, the embryotoxic effects found in ovo, but also for the cognitive and neurotoxic effects of cannabinoids in the developing and adult nervous system.

Οι έρευνες που αρχικά στόχευαν στην ανίχνευση του μηχανισμού δράσης της Δ9-τετραϋδροκανναβινόλης, του κύριου ψυχοδιεγερτικού συστατικού που εξάγεται από την ινδική κάνναβη, οδήγησαν τελικά στην ανακάλυψη ενός νέου νευρορυθμιστικού συστήματος, του ενδογενούς κανναβινοειδούς συστήματος. Το ενδοκανναβινοειδές σύστημα περιλαμβάνει μια σειρά από ενδογενείς, λιπιδικής φύσεως προσδέτες (κυρίως την ανανδαμίδη και την 2-αραχιδονοϋλγλυκερόλη), μεμβρανικούς υποδοχείς (CB1-brain type, CB2-immune type, αλλά και άλλους) που ανήκουν στην υπεροικογένεια των συζευγμένων με G πρωτεΐνες υποδοχέων, καθώς και τα ένζυμα εκείνα που είναι απαραίτητα για τη βιοσύνθεση και αποικοδόμησή τους. Στον ενήλικο εγκέφαλο τα ενδοκανναβινοειδή λειτουργούν ως παλίνδρομα σηματοδοτικά μόρια: απελευθερώνονται από μετασυναπτικά κύτταρα, ενώνονται με υποδοχείς που εντοπίζονται στους προσυναπτικούς νευρώνες και ρυθμίζουν την απελευθέρωση διαφόρων νευροδιαβιβαστών (κυρίως του ανασταλτικού γ-αμινοβουτυρικού οξέος, αλλά και του διεγερτικού γλουταμινικού οξέος). Οι κύριες επιδράσεις της τετραϋδροκανναβινόλης οφείλονται στη διέγερση αυτών των υποδοχέων και είναι ευφορία, διαταραχή πρόσφατης μνήμης και άλλων νοητικών λειτουργιών, ακινησία, αναλγησία, αύξηση της όρεξης. Στον αναπτυσσόμενο οργανισμό, η εντόπιση νωρίς κατά την εμβρυϊκή ζωή λειτουργικών CB1 υποδοχέων σε θέσεις στις οποίες δεν εκφράζονται στην ενήλικο ζωή (άτυπες θέσεις) αποτέλεσε μία από τις πρώτες ενδείξεις, οι οποίες οδήγησαν στην υπόθεση ότι το ενδοκανναβινοειδές σύστημα παίζει κάποιον/ους ρόλο/ους στην φυσιολογική εμβρυϊκή ανάπτυξη. Το ενδιαφέρον σχετικά με τη σπουδαιότητα της ύπαρξης φυσιολογικής σηματοδότησης μέσω του ενδοκανναβινοειδούς συστήματος προκειμένου να επιτευχθεί όχι μόνο φυσιολογική εμβρυογένεση, αλλά και μεταγεννητική ανάπτυξη, επικεντρώθηκε στο κεντρικό νευρικό σύστημα. Τα πειράματα που έχουν πραγματοποιηθεί μέχρι τώρα έχουν οδηγήσει στο συμπέρασμα ότι τα κανναβινοειδή συμμετέχουν σε όλες τις διαδικασίες της φυσιολογικής εμβρυϊκής ανάπτυξης του νευρικού συστήματος: πολλαπλασιασμό και δέσμευση των προγονικών νευρικών κυττάρων προς συγκεκριμένη κυτταρική σειρά, διαφοροποίηση των δεσμευμένων νευροβλαστών ή σπογγιοβλαστών, μετανάστευση των μεταμιτωτικών νευρώνων, μορφολογική και λειτουργική ωρίμανση των νευριτών, δημιουργία λειτουργικών συνάψεων, απόπτωση και μυελινοποίησηση των νευραξόνων. Σκοπός της εργασίας αυτής είναι η ανασκόπηση της διεθνούς βιβλιογραφίας σχετικά με τον φυσιολογικό ρόλο των κανναβινοειδών στην ανάπτυξη του εμβρύου και ιδιαίτερα στην ανάπτυξη του κεντρικού νευρικού συστήματος. Μετά από μία συνοπτική περιγραφή της φυσιολογικής εμβρυογένεσης του κεντρικού νευρικού συστήματος, ακολουθεί η ανάλυση του ενδογενούς κανναβινοειδούς συστήματος, καθώς και των σηματοδοτικών μονοπατιών που η ενεργοποίηση των υποδοχέων του διεγείρει. Στη συνέχεια αναπτύσσονται διεξοδικά όλες οι απόψεις περί συμμετοχής του συστήματος αυτού στην φυσιολογική εμβρυογένεση. Είναι γνωστό ότι η τετραϋδροκανναβινόλη περνά τον αιματοπλακουντιακό φραγμό, φτάνει μέχρι τον εγκέφαλο του εμβρύου σε σημαντικές συγκεντρώσεις και μπορεί επομένως να διαταράξει τη φυσιολογική σηματοδότηση από τους CB1 υποδοχείς, η οποία φαίνεται να είναι απαραίτητη για την δημιουργία του φυσιολογικού νευρικού συστήματος. Η κατανόηση των μηχανισμών μέσω των οποίων τα κανναβινοειδή συμμετέχουν στη φυσιολογική εμβρυογένεση μπορεί ερμηνεύσει τα νοητικά και κοινωνικά ελλείμματα που παρουσιάζουν απόγονοι γυναικών που κατά τη διάρκεια της κυήσεως έκαναν χρήση κάνναβης, κάποια από τα οποία διαρκούν μέχρι την ενήλικο ζωή. / Recent evidence suggests that the endogenous cannabinoid system emerges and is operative early during brain development and that CB1 receptors are located in areas that do not contain or have a small density of these receptors in adult brain. This was the first hint which led to research about the potential role of the endocannabinoid system during brain development. It is now well accepted that endocannabinoids, acting as epigenetic factors, regulate neural progenitor proliferation, commitment into neuroblasts or spongioblasts, as well as differentiation of commited cells, control of neurite outgrowth, neurotransmitter maturation and establishment of synaptic contacts. There is also a great interest in the role of cannabinoids as neuroprotective agents in the developing, as well as the adult brain, though their role as a general endogenous protection system is yet to be established. In utero exposure to tetrahydrocannabinol (THC) induces behavioral and cognitive deficits enduring into adulthood. Improving our knowledge on the molecular mechanisms of cannabinoid actions during brain development could lead to a better understanding of these deficits.

No description available.

Neuropathic pain (NPP) is a chronic syndrome suffered by patients with multiple sclerosis (MS), for which there is no cure. Underlying cellular mechanisms involved in its pathogenesis are multifaceted, resulting in significant challenges in its management. In addition to its complex pathophysiology, the clinical management of MS-induced NPP is further complicated by the lack of clinical therapeutics trials specific to this population. The primary aim of the work underlying this thesis was to contribute to the evidence-based management of individuals with MS-induced NPP through the completion of two clinical therapeutics trials in this population. A secondary aim was to describe pain variability in this patient population through the development and validation of a pain variability algorithm tool. Resulting from this work, we demonstrated that nabilone – a synthetic oral cannabinoid – represents an effective, well-tolerated and novel treatment for MS-induced NPP. Additionally, we show that the SSRI paroxetine was poorly tolerated in our patient population, with a correspondingly high attrition rate. As a result, we were unable to determine any treatment effect in this trial due to insufficient recruitment due to drop-out. Lastly, we were able to define and describe pain instability in this cohort, noting that approximately 30% of individuals with MS-induced NPP experiencing highly variable daily pain. The results of these projects provide novel information for this patient population. Patients currently living with the daily burden of MS-induced NPP would benefit from additional trials ensuing from this, and other, research in order to initiate a momentum for much-needed clinical research in this complicated patient cohort.

Neuropathic pain (NPP) is a chronic syndrome suffered by patients with multiple sclerosis (MS), for which there is no cure. Underlying cellular mechanisms involved in its pathogenesis are multifaceted, resulting in significant challenges in its management. In addition to its complex pathophysiology, the clinical management of MS-induced NPP is further complicated by the lack of clinical therapeutics trials specific to this population. The primary aim of the work underlying this thesis was to contribute to the evidence-based management of individuals with MS-induced NPP through the completion of two clinical therapeutics trials in this population. A secondary aim was to describe pain variability in this patient population through the development and validation of a pain variability algorithm tool. Resulting from this work, we demonstrated that nabilone – a synthetic oral cannabinoid – represents an effective, well-tolerated and novel treatment for MS-induced NPP. Additionally, we show that the SSRI paroxetine was poorly tolerated in our patient population, with a correspondingly high attrition rate. As a result, we were unable to determine any treatment effect in this trial due to insufficient recruitment due to drop-out. Lastly, we were able to define and describe pain instability in this cohort, noting that approximately 30% of individuals with MS-induced NPP experiencing highly variable daily pain. The results of these projects provide novel information for this patient population. Patients currently living with the daily burden of MS-induced NPP would benefit from additional trials ensuing from this, and other, research in order to initiate a momentum for much-needed clinical research in this complicated patient cohort.

Food intake is regulated by both appetite and orosensory reward systems. Appetite systems stimulate or reduce hunger, while orosensory reward motivates consumption of high fat sweet foods, resulting in food enjoyment. The majority of advanced cancer patients suffer from malnutrition and wasting, which may be caused by a loss of appetite due to physiological changes or a hindered orosensory reward system due to taste and smell (chemosensory) changes or both. Orosensory reward systems were hypothesized to be impaired in advanced cancer. To understand the influence of chemosensory alterations on food intake and enjoyment, the nature (intensity) of chemosensory alterations in cancer patients and their relationship with ingestive behaviour and quality of life (QOL) were investigated (study 1). Advanced cancer patients (n=192) more frequently self-reported tastes and odours to be heightened rather than diminished (p=0.035). Patients with perceived chemosensory alterations had poorer QOL (p=0.0176) and lower caloric intake (p=0.0018) compared to patients with no alterations. Cannabinoids (e.g. -9-tetrahydrocannabinol, -9-THC) increase food intake by stimulating both appetite and orosensory reward systems as well as potentially enhancing chemosensory function. To palliate chemosensory alterations and poor appetite, advanced cancer patients (n=21, study 2) with these symptoms were randomized to receive either -9-THC (2.5mg) or placebo oral capsules twice daily for 18 days. Compared to patients receiving placebo, -9-THC-treated patients reported that food tasted better (p=0.04), they had improved chemosensory perception (p=0.026), increased preference and intake of high protein foods (p=0.008), and improved appetite (p=0.05), quality of sleep (p= 0.025), and relaxation (p= 0.045). Like cancer patients, tumour-bearing rats appeared to experience a loss of orosensory reward, showing tumour-associated anorexia when fed a rewarding diet to the same degree as on a usual diet (study 3). -9-THC significantly increased caloric intake compared to vehicle for both tumour-bearing (p=0.0146) and healthy rats (p=0.0004), suggesting endocannabinoid-mediated appetite systems are functioning in this tumour model. The findings of this thesis suggest orosensory reward systems to be impaired in advanced cancer, decreasing the liking and motivation to eat. -9-THC treatment may help to palliate perceived chemosensory alterations and loss of appetite and food enjoyment in advanced cancer. / Food Science and Technology

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.

Thesis (Ph. D.)--University of Sydney, 2009. / Title from title screen (viewed June 1, 2009) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Pharmacology, Faculty of Medicine. Degree awarded 2009; thesis submitted 2008. Includes bibliography. Also available in print form.

Thesis (M.S.)--State University of New York at Binghamton, Department of Psychology, 2008. / Includes bibliographical references.

Thesis (Ph.D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Pharmacology and Toxicology. Title from title-page of electronic thesis. Bibliography: leaves 109-123.