The behavioural and neural effects of cannabinoids: Studies using Lewis and Wistar strain rats

Cannabis (known in its common forms as Cannabis sativa or Cannabis indica) is the most widely used illicit drug in the world and has been used for thousands of years for medicinal, religious and hedonistic purposes. In the last half of the 20th century the therapeutic uses of cannabis were largely ignored as most Western governments prohibited the use of the drug. Prohibition has come about largely as a result of the view that cannabis is a dangerous drug that poses major risks to both mental and physical health. However, this view is being increasingly challenged in recent years with a major popular movement towards decriminalization of cannabis occurring in some Western countries and a resurgence of interest in the medicinal properties of cannabis. Since Mechoulam and colleagues first isolated delta 9-tetrahydrocannabinol (delta 9-THC) as the main psychoactive constituent of cannabis, considerable advances have been made in the pharmacology of cannabis and cannabis-like drugs (cannabinoids). Central and peripheral cannabinoid receptors have been isolated and two endogenous ligands have been discovered. In addition, two cannabinoid receptor antagonists have been developed. However, our knowledge of the behavioural, neural and emotional effects of cannabis and the cannabinoids has often lagged behind our understanding of basic cannabinoid pharmacology. The present thesis attempts to further the understanding of the behavioural, neural and emotional effects of cannabinoids, using laboratory rats as subjects. A synthetic analogue of delta 9-THC (CP 55,940), is used as the primary pharmacological tool. The thesis offers a broad perspective with three major areas of investigation. These are: 1) the effects of CP 55,940 on anxiety-related behaviour (Chapters 2 and 3); 2) the effects of CP 55,940 on patterns of brain activation as indicated by c-fos expression (Chapter 4) and; 3) the addictive potential of CP 55,940 and its capacity to produce sensitization to the effects of other drugs such as cocaine (Chapters 5 and 6). A recurring theme throughout the thesis is that genetic factors may partially determine the behavioural, neural and emotional response to cannabinoids. To this end, the thesis compares Lewis and Wistar trains of rat in a wide variety of assays. Previous research has isolated Lewis rats as an "addiction-prone" and a "cannabinoid-preferring" strain, as they are more sensitive to the rewarding effects of various drugs of abuse including cannabinoids. Conversely, cannabinoids appear to have aversive effects in Wistar rats. A long-standing puzzle in cannabinoid research has been the question of why rats do not self-administer cannabis or cannabinoids. One likely reason is that cannabinoids have predominately aversive effects in rats. It is proposed here that these aversive effects arise because cannabinoids are anxiogenic agents in most rat strains. However some evidence indicates that the Lewis strain of rat are the only strain to find cannabinoids rewarding. It is hypothesised that Lewis rats may be more susceptible to the rewarding effects of cannabinoids because they are less susceptible to the anxiogenic effects of these compounds. In Chapters 2 and 3 the anxiogenic effects of the synthetic cannabinoid agonist CP 55,940 were compared in Lewis and Wistar rats in several different animal models of anxiety. In Chapter 2, the predatory odour avoidance, open area avoidance and conditioned ultrasonic vocalization (USV) models were utilised. In the predatory odour avoidance model, rats were exposed to cat odour in a rectangular arena and given the opportunity to hide in a small box. Both Lewis and Wistar rats displayed high levels of hiding during odour exposure. In Wistar but not Lewis rats, 50 �g/kg of CP 55,940 (i.p.) enhanced this avoidance response. Unfortunately, Lewis rats showed exceptionally high avoidance of the cat odour making it difficult to discern the effects of CP 55,940. To avoid this problem a second experiment was conducted, where rats were tested in the same arena as in the first experiment but with no cat odour present. Again in Wistar, but not Lewis rats, 25 and 50 �g/kg of CP 55,940 (i.p.) increased the avoidance of the open space. In the third experiment, Lewis and Wistar rats were placed in a chamber in which they had previously received footshock. Wistar but not Lewis rats re-exposed under the influence of 10, 25 or 50 �g/kg CP 55,940 (i.p.) emitted significantly more USVs than vehicle-treated rats. Thus, CP 55,940 clearly increased anxiety-related behaviour in Wistar rats but not Lewis rats, supporting the notion of a genetic predisposition towards cannabinoid-induced anxiety. In Chapter 3 the generality of the findings made in Chapter 2 were tested by utilising two further animal models of anxiety, the social interaction and light-dark emergence tests. From the results of Chapter 2, it could be claimed that Lewis rats were merely subsensitive to the effects of CP 55,940. Therefore a higher dose range (0, 25, 50 and 75 �g/kg i.p.) of CP 55,940 was employed in Chapter 3. In addition, the rotarod test was used to assess whether CP 55,940 has ataxic effects at these doses. In the first experiment, two unfamiliar rats were placed in a large arena and the time the rats spent socially interacting was recorded. CP 55,940 significantly reduced the total time rats spent socially interacting in Lewis (25 and 75 �g/kg) and Wistar rats (50 and 75 �g/kg). However, CP 55,940 has a significantly greater effect in Wistar rats compared to Lewis rats. In the second experiment, rats were placed in a small box within a large open arena and the latency to emerge from this box was measured. CP 55,940 increased emergence latency (at 75 �g/kg) and mean time per entry into the box (at 25 and 75 �g/kg) in Wistar but not Lewis rats. Furthermore, CP 55,940 caused a greater decrease in time spent in the open arena (at 25 and 75 �g/kg) and frequency of emergence (at 75 �g/kg) in Wistar rats in comparison to Lewis rats. In the third experiment, CP 55,940 (at 25, 50 and 75 �g/kg) caused mild incoordination only in Lewis rats as measured by the rotarod test. This finding argues against the assertion that the CP 55,940-induced anxiety-like behaviours in Wistar rats are merely a result of motoric impairment. Furthermore, it illustrates that Lewis rats are not generally subsensitive to the effects of CP 55,940. That is, when compared to other rat strains, Lewis rats may be more or less sensitive to the effects of CP 55,940 depending on what behaviour is being assessed. From the results of Chapters 2 and 3 it can be seen that Lewis rats are less sensitive to the anxiogenic effects of CP 55,940 than Wistar rats. In Chapter 4 it was hypothesised that in Lewis rats the effects of CP 55,940 on neural substrates of reward far outweigh the effects the compound has on neural substrates mediating anxiety. To examine this issue, the effects of CP 55,940 at a moderate (50 �g/kg i.p.) and high (250 �g/kg i.p.) dose were observed on c-fos expression (a measure of neural activation) and behaviour in Lewis and Wistar rats. CP 55,940 dose-dependently inhibited locomotor activity and reduced body temperature with Lewis rats being significantly less affected than Wistar rats. The 250 �g/kg dose caused significant catalepsy in both strains with a significantly greater effect in Wistar rats. These strain differences in the effects of CP 55,940 on body temperature and motor behaviour clearly correlated with c-fos expression in various regions and subregions. In general, Lewis rats showed significantly less Fos-labeled cells in comparison to Wistar rats. These strain differences in the effects of CP 55,940 on c-fos expression appeared unique to cannabinoids, as cocaine (15 mg/kg i.p.) had equivalent effects on c-fos expression in Lewis and Wistar rats. CP 55,940 promoted c-fos expression in areas not previously assessed, such as the median preoptic nucleus (MnPO), medial preoptic nucleus (MPO), anterior hypothalamic area (AH), islands of Calleja (ICjM), periaqueductal gray (PAG) and the pedunculopontine tegmental nucleus (PPTg). The strain differences uncovered in Chapters 2 and 3 correlated well with strain differences in the effects of CP 55,940 on c-fos expression in areas implicated in cannabinoid-induced anxiety, such as the central nucleus of the amygdala, bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus and PAG. However, the effects of CP 55,940 on c-fos expression in a neural circuit which may underlie reward, which includes the shell of the nucleus accumbens (NAS) and PPTg, were also less in Lewis rats in comparison to Wistar rats. Future investigations must address whether the reduced effects of CP 55,940 on the Lewis rat are due to pharmacokinetics or pharmacodynamics. In addition, future studies must reconcile the pattern of c-fos expression observed here with prior reports of the Lewis rat being a unique "cannabinoid-preferring" strain. In Chapter 4, CP 55,940 administration promoted c-fos expression in areas of the brain thought to play a critical role in behavioural sensitization such as the ventral tegmental area and NAS. This is interesting because it is possible that c-fos is involved in promoting neuroadaptations that underlie drug addiction. To examine this idea, Chapter 5 investigated a behavioural assay of the long-term neural adaptations that may occur with the chronic administration of cannabis, namely, behavioural sensitization. This chapter also examined an animal model of the "gateway hypothesis", that is, the hypothesis that prior exposure to cannabis increases an individuals vulnerability to using other drugs. This animal model is known as cross-sensitization. First it was shown that Lewis, but not Wistar rats, given cocaine (15 mg/kg i.p.) every second day over a two week period displayed a progressively greater locomotor response to the drug over days indicating behavioural sensitization. When CP 55,940 (0, 10, 25 or 50 �g/kg i.p.) was administered under a similar regime, no such sensitization was observed in either strain. Rather, the two highest doses of CP 55,940 (25 and 50 �g/kg) caused locomotor suppression that lasted throughout administration. When Lewis or Wistar rats pre-exposed ten times to CP 55,940 were challenged with cocaine (15 mg/kg), no exaggerated locomotor response to cocaine was evident relative to non pre-exposed rats. When these rats were subsequently re-tested with CP 55,940, it continued to produce a dose-dependent suppression of locomotor activity. Finally, when CP 55,940 (50 �g/kg) was co-administered with cocaine in Lewis rats, it significantly reduced the locomotor hyperactivity produced by the drug but did not block the development of behavioural sensitization to cocaine. These results show that CP 55,940 does not sensitize locomotor activity with repeated administration in the same way as cocaine, and that pre-exposure or concurrent exposure to CP 55,940 does not enhance sensitivity to the subsequent behavioural effects of cocaine. Therefore, unlike Chapters 2, 3 and 4 where strain differences were observed in CP 55,940?induced anxiety, hypothermia, catalepsy, c-fos expression and ataxia, there were no strain differences with respect to behavioural sensitization. Landmark studies by Gardner and colleagues showed that Lewis rats are particularly susceptible, in comparison to other rat strains, to the rewarding effects of delta 9-THC on: 1) medial forebrain bundle (MFB) self-stimulation behaviour and; 2) dopamine (DA) efflux in the NAS. However, in Chapter 4 Lewis rats were less susceptible than Wistar rats to CP 55,940-induced c-fos expression in the NAS. Further, Lewis rats showed no behavioural sensitization to the chronic administration of CP 55,940. In light of these findings, Chapter 6 assessed whether CP 55,940 does have a rewarding effect on MFB self-stimulation behaviour in Lewis rats. Lewis rats were trained to self-stimulate the MFB using a rate?frequency paradigm and then administered CP 55,940 (0, 10, 25 and 50 �g/kg i.p.). CP 55,940 had no effect on MFB self-stimulation behaviour as assessed by the M50, the stimulation frequency at which half-maximal response rates were obtained. This result calls into question previous assertions that Lewis rats are a "cannabis-preferring" strain of rat. Previous studies utilising the cannabinoid CB1 receptor antagonist, SR 141716, have shown that the endogenous cannabinoid system may have some involvement in the rewarding effects of cocaine, morphine, sucrose and alcohol. Thus, Chapter 6 also assessed the effects of SR 141716 (0, 1, 3, 10 and 20 mg/kg i.p.) on MFB stimulation in Lewis rats. The role of DA in MFB stimulation reward has already been established, so for comparison purposes the effects of the DA D1 receptor antagonist SCH 23390 (0.06 mg/kg i.p.) was also assessed. Only a very high dose of SR 141716 (20 mg/kg) caused a significant inhibition of the rewarding efficacy of the stimulation with all other doses (1, 3, and 10 mg/kg) being ineffective in modulating the rewarding impact of brain stimulation. This was seen as an increase in M50. By comparison, a relatively low dose (0.06 mg/kg) of SCH 23390 caused a large increase in M50. These results indicate a relatively modest influence of the endogenous cannabinoid system on reward-relevant neurotransmission in the self-stimulation paradigm. Chapter 7 concludes the thesis and discusses the implications of the results obtained. The main findings of the current thesis are: 1) that the suggested "addiction-prone" Lewis strain of rat is less susceptible to cannabinoid-induced anxiety in comparison to Wistar rats; 2) Lewis rats show less cannabinoid-induced c-fos expression in comparison to Wistar rats (including in brain regions implicated in cannabinoid-induced anxiety and reward); 3) cannabinoid-induced c-fos expression exists in a number of brain regions never previously assessed such as the MPO, ICjM and PPTg; 4) behavioural sensitization does not occur with the repeated administration of CP 55,940; 5) cannabinoid pre-exposure or co-administration does not increase the sensitivity of the locomotor-activating effects of cocaine; 6) the endogenous cannabinoid system, at most, only has a minor influence on the neural substrate of brain stimulation reward and; 7) that there are previously unreported strain differences in cannabinoid-induced hypothermia, catalepsy and ataxia. These results add to our understanding of the effects of the behavioural, emotional and neural effects of cannabinoids and the endogenous cannabinoid system

Be rihtre æwe: legislating and regulating marital morality in late Anglo-Saxon England

This thesis examines some projects of moral regulation, implemented by the agents of the church and king in the late Anglo-Saxon period, which sought to modify and govern marital conduct. Theories of moral regulation are analysed in the Introduction, which also examines Germanic marriage practices, as far as they can be recovered, and the Anglo-Saxon church’s inherited attitudes towards marriage. Manuscripts and texts are examined firstly as projects of moral regulation, and secondly as projects which attempted to alter marital behaviour. In Chapter 1, moral regulation is situated within the context of the Benedictine reform through the examination of one manuscript – Cambridge, Corpus Christi College, MS 201 – as a case-study in the cooperative efforts of the church and king to regulate society. In particular, the legislative and penitential texts which are compiled in MS 201 bear witness to the tendency in late Anglo-Saxon England for legislation to be moralised, and for morality to be legislated. MS 201 also includes the unique copy of the Old English translation of Apollonius of Tyre, and the marital morality inscribed therein perhaps accounts for its inclusion in this predominantly Wulfstanian manuscript. In Chapter 2 the riddles recorded in the Exeter Book are interpreted as literary exercises in regulation. This chapter establishes the possible moral and regulatory agenda of the Exeter Book riddles by offering a new interpretation of, and solution to, one riddle. It also analyses the marriages made manifest in some of the so-called ‘double entendre’ riddles, which regulate the moral relationship following Pauline exegesis: emphasis in these riddles is on the sanctity of marriage, wifely obedience, and the payment of the conjugal debt. Conversely, Ælfric, in his Lives of Saints, idealises marriage as characterised by the absence of all sexual relations. In his Life of St Agnes (examined in Chapter 3), and in his Lives of married saints (SS Julian and Basilissa, SS Cecilia and Valerian, and SS Chrysanthus and Daria, examined in Chapter 4), Ælfric makes non-sexual, companionable, and loving marriage morally paradigmatic. Whilst both marriage and morality have been studied by modern critics, neither topic has inspired extended, specific study (with a few, notable, exceptions), and the nexus between these two topics has been hitherto unacknowledged. Although new, and often profound, insight is gained into Anglo-Saxon texts by considering them in the context of moral regulation, the morality they propose, as well as the regulatory process used to impose that morality, varies across context, text, genre, and author. This conclusion is also true for marital morality, Anglo-Saxon perceptions of which differed in each of the texts chosen for evaluation. This thesis does not claim to be comprehensive; nor does it attempt to synthesise attitudes towards marriage and morality, since a synthesis does not do justice to the richness or complexity with which this topic was treated. It is hoped that this thesis will provide insight into not only individual Anglo-Saxon attitudes towards marriage but also processes of regulation and social control, and, indeed, into the intersection between attitudes and processes

Restoration of former agricultural fields on acid sandy soils:Conversion to heathland, rangeland or forest?

Three restoration strategies on agricultural fields with acid sandy soil were evaluated after 18 and 25 years: conversion to heathland, rangeland and forest. Changes in soil microstructure, chemical characteristics, availability of N and P, and vegetation composition were analyzed in agricultural soils, three undisturbed reference sites and five types of restored former agricultural fields. Agricultural soils were characterized by organic slurry without much soil life. Soil nutrients were especially high for P, mostly in mineral form, and P-desorption rates were high. Partial and complete topsoil removal, aiming at heathland restoration, led to (much) lower soil organic matter and nutrients, but not to recovery of soil life, nor to P-limited soils. Heather was accompanied by many grassland species, even with complete topsoil removal. Conversion to rangeland did not decrease nutrient stocks, but led to improved soil life, although different from reference grasslands due to the higher pH. P-availability remained high, but net N-mineralization and plant N-content were clearly lower after 25 than after 18 years. Plant diversity was relatively high, and cover of eutrophic grasses decreased to 8–39% in intermediate and productive rangelands. Nutrient-poor species remained absent, but the slightly higher pH improved conditions for many grassland herbs. Afforestation did also not lead to nutrient-poor conditions, but soil life clearly increased and nutrients were used for rapid tree growth. Undergrowth species however remained eutrophic. It was impossible to retrieve the P-limited reference ecosystems within 25 years of restoration, not even with complete topsoil removal. Differences in plant diversity between expensive topsoil removal and much cheaper conversion to rangeland were also relatively small. For restoration on a landscape scale, it may thus be better to focus on conversion to semi-natural grasslands and afforestation. The half-open, nutrient-, mineral-, and species-rich landscape offers opportunities for large grazers, but also for many insects and birds

Cytoskeletal dynamics of Cytotoxic T cells during migration in the tumour microenvironment

Typically, migrating T cells display an elongated polarized shape with a very dynamic leading edge and a uropod in the rear. This ‘amoeboid’ movement guarantees a fast migration driven by the formation of polarized protrusions at the front. The actomyosin cytoskeleton is responsible for the generation of the forces that are involved in this process. This thesis aims to determine what is the effect of T cell migration when different components of the actomyosin cortex were inhibited using a pharmacological approach. We found that the inhibition of each component of the actomyosin cortex, T cells display different conformation of the actin filaments and produce different type of protrusion. Furthermore, T cell migration is an important feature for the killing and clearance of canner cells. It has been reported that T cells can migrate efficiently in any kind of tissue whilst scanning for cognate antigen. On the other hand, it is known that the tumor microenvironment secretes immunosuppressive cytokines such as TGF-β impairing the antitumor activity of T cells. Therefore, we aim to determine how TGF-β affects the migration behavior of T cells and its consequences in the scanning strategy to search their cognate antigen