ABSTRACT
Cannabis sativa is a member of the family Cannabinaceae. Delta-9-tetrahydrocannabinol is thought to be the principal psychoactive ingredient present in Cannabis and may have important implications for various neurobehavioral processes, including learning, memory, motivation, motor control, etc. Cannabis use is associated with negative health outcomes, psychosocial and cognitive impairments and societal acceptance of cannabis use for medicinal applications continues to increase. Hence, the concerns for this present study. The objectives of the study were to establish the effect of Cannabis sativa on learning and spatial memory; to investigate some neurochemical changes; and to investigate the effects of Cannabis sativa on oxidative stress markers in the brain associated with Cannabis sativa administration. The study used 75 mice, grouped in to three, of 25 mice each. Three standard neurobehavioural test models (EPM, MWM and YM) each for a group were used to assess learning and spatial short term memory. Each of the three groups was further subdivided in to five (5) groups, containing five (5) mice each. Group one and five, the negative and positive control were respectively administered with normal saline and piracetam 100mg/kg, while groups two, three and four were administered with 25mg/kg, 50mg/kg and 75mg/kg of Cannabis sativa extract respectively. All regimens were orally, twice daily and lasted for 21 days. Brain homogenates were used to evaluate acetylcholinesterase, (AchE) activity, dopamine concentration, brain sialic acid concentration, malondialdehyde, (MDA) concentration, Superoxide dismutase, (SOD), and catalase, (CAT). The data obtained from these studies were analysed with statistical package for social science (SPSS) software, version 20 (IBM, USA), using one-way analysis of variance (ANOVA) and Tukey’s multiple comparison post hoc test. All data were expressed as mean ± SEM and p ≤ 0.05 were considered statistically significant. The
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results of the research showed impairment in learning and spatial short term memory with groups 3 and 4 in the EPM model and MWM model. There was increased concentration of AchE in the cannabis treated groups of the EPM model and groups 3 and 4 of MWM model, but, no significant changes in concentration of AchE in the Y maze model. Dopamine and Total brain sialic acid concentrations decrease in group 4 of all the models. Also, there was increase MDA concentration and decrease in catalase and SOD activities, suggesting oxidative stress. Thus, cannabis sativa in high doses is associated with learning and spatial short term memory impairment via some of the neurochemicals involved in learning and memory.
TABLE OF CONTENTS
Cover page…………………………………………………………………………………..i
Declaration…………………….……………………………………………………………ii
Certification……..…………………………………………………………………………iii
Dedication………..…………………………………………………………………………iv
Acknowledgements….……….……….…………………………………………..…………v
Abstract…………………………..…………………………………………………………vii
Table of Contents…………………..……….…………..…………………………………..ix
List of Tables……………………………………………………………………………..xiv
List of Figures…………………………………………………..……………………….…xv
List of Plates ……………………………………………………..………………………..xvi
Abbreviations………………………………………………………………………..……xvii
CHAPTER ONE
1.0 INTRODUCTION.………………………..….……..…..……………………………..1
1.1 Statement of the Research Problem…..……….………………..…………………….3
1.2 Justification of the Study………….……………………………………..……………4
1.3 Aim and Objectives…….………………………………………………………………5
1.4 Research Hypothesis…….……………….……………………..……………………..6
CHAPTER TWO
2.0 LITERATURE REVIEW……………………………………..………………..……..7
2.1 Learning and Memory…..…………………………………….……………………….7
2.2 Amnesia…………………………………………………………………………..……13
2.2.1 Types of amnesia….…………………………………………………………………13
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2.3 Electrical and Biochemical Mechanism of Memory……..……….…..…………….14
2.4 Neurotransmitters involved in Learning and Memory………..……….….……..…17
2.4.1 Cholinergic signaling……………………………………………………………..…18
2.4.2 Monoaminergic signaling……………………….……………………………………19
2.4.3 Sialic acid……………………………………….……………………………………20
2.4 Effects of Oxidative Stress on the Physiological Function of Neuronal Membrane……………………………………………………………………………………………………………23
2.5 Piracetam…..…………………………………………………………………………25 2.5.1 Piracetam and oxidative stress………….……………………………………………28
2.6 Cannabis sativa Plant………..…………………………………………………………29 2.6.0 Cannabinoids…………………………………………………………………………32 2.6.1 Endocannabinoid system……………………………………………………………..32
2.6.2 Cannabinoid receptors and cannabinoid pharmacology……………………………33
2.6.3 Medical benefits of Cannabis sativa………..……………………………………….39
CHAPTER THREE
3.0 MATERIALS AND METHODS…………………………………….…………..…..40
3.1 Materials………………………………………………………………………………40
3.1.1 Experimental animals……………………………………….………………………..40
3.1.2 Drugs…………… ……………………………………………………………………40
3.1.3 Chemicals, reagents and solvents……..………………………………………………40
3.1.4 Equipment and other materials…..………………………………………………….41
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3.1.5 Plant material…………………………………………………………………………41
3.2. Methods…………………………………………………………………………..…..42
3.2.2 Extraction of Canabis sativa leaves…………….…………………………………….42 3.2.2 Acute toxicity (LD50) study………………………………………………………….42 3.3 Grouping of Research Animals and Experimental Designed..………….…………45
3.4 Surgical Removal of the Brain and Brain Tissue Preparation for Assay.………..47
3.5 Detection of Oxidative Stress Indicators….…………………………………………47
3.5.1 Effect of treatments on brain lipoperoxidation……..……………………………….47 3.5.2 Determination of uperoxide dismutase activity……………………………………..48
3.5.3 Determination catalase (CAT) activity………..……………………..………………49
3.6 Determination of Brain Sialic Acid………..…..……………………………………49
3.6.1 Procedure for determination of total sialic acid of brain tissue of mice…….………49
3.7 Acetylcholinesterase Assay ….………..………………………………..………………50
3.8 Learning and Memory Assessments…..…..…………………………………………51
3.8.1 Assessment of learning and spatial memory using morris water maze..…….………51
3.8.2 Assessment of learning and short term memory using elevated plus maze……..…54
3.8.3 Assessment of learning, short term and spatial memory using Y-maze…….………57
3.9. Statistical Analysis..……….………………………………………………..………..59
CHAPTER FOUR
4.0 RESULTS…………………………………………..……..……………….…..………60
4.1 Assessment of Learning and Short Term Memory Using Elevated Plus Maze……60
4.1.1 Acquisition and retention session…..…….………………………………………….60
4.2 Assessment of Learning, Short term and Spatial Memory Using Y-maze………..60
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4.3 Assessment of Learning and Spatial Memory Using Morris Water Maze………..63
4.3.1 Latency to locate escape platform……..………….………………….………………63
4.3.2 Frequency of platform crossings………..……………………………………………63
4.3.3 Time spent in target quadrant (Q4)…..………………..…………………………….63
4.4 Acetylcholinesterase Enzyme Activity………..………………………….…………67
4.4.1 Acetylcholinesterase enzyme activity of mice used in elevated plus maze for memory…………………………………………………………………………………….67
4.4.2 Acetylcholinesterase enzyme activity of mice used in Y maze for memory..………67
4.4.3 Acetylcholinesterase enzyme activity of mice used in morris water maze for memory…………………………………………………………………………………….67
4.5 Level of Dopamine concentration (nmol/L)…………………………………………69
4.6. Concentration of Brain Sialic Acid…..………………………….………………….69
4.6.1 Concentration of free sialic acid……..………………………………………………69
4.6.2 Concentration of bound sialic acid……..………………………………..……….…69
4.6.3 Concentration of total sialic acid……..………………………………………….….69
4.7 Indicators of Oxidative Stress…….…..………………………………….…………..74
4.7.1 Level of TBARS……………………………………………………………………..74
4.7.2 Catalase………………………………………………………………………………74
4.7.3 Superoxide Dismutase (SOD)……………………………………………………….74
CHAPTER FIVE
5.0 DISCUSSION…………………………………………………………….….……….78
CHAPTER SIX
6.0 CONCLUSION AND RECOMMENDATIONS…………………….……………..84
REFERENCES………..…………………………………………………………………..86
CHAPTER ONE
1.0 INTRODUCTION In the last twenty years, significant advances have been made in understanding the endogenous cannabinoid system and how cannabis may affect its functioning, particularly as it pertains to the brain. The abundance of cannabinoid receptors in the hippocampus, amygdala, basal ganglia, and prefrontal cortex (Piomelli 2003; Mackie 2005) suggests that disruption of the cannabinergic system by administration of exogenous cannabinoids, like delta-9-tetrahydrocannbinol (THC; the main psychoactive compound in cannabis), may have important implications for various neurobehavioral processes, including mood and anxiety regulation (Crippa et al., 2009; Crippa et al., 2011). Also, learning, memory, motivation, motor control, reward processing, and executive functions might be disrupted (Gonzalez 2007; Solowij and Pesa 2010; Crean et al., 2011). Indeed, it seems frontal-limbic neurocircuitry is most prominently affected by cannabis use ( Martin-Santos et al., 2010), while other structures in the brain, including the brain stem, occipital lobe, and parietal lobe may be less affected. Not surprisingly, the neurobehavioral effects of cannabis use have been intensely studied over several decades and summarized in numerous published reviews (Grant et al., 2003; Iversen 2003; Ranganathan and D’Souza 2006; Gonzalez 2007;Jager and Ramsey 2008; Schweinsburg et al., 2008; Solowij and Battisti 2008; Solowij and Pesa 2010; Crean et al., 2011). However, many of these reviews conclude that the findings for several neurocognitive domains are equivocal, speaking to the importance of further research in this field (Crean et al., 2011).
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Research on the neurocognitive effects of cannabis continues to grow rapidly. This is an especially pertinent area of research because cannabis continues to be the most widely used illicit substance worldwide (UNODC, 2011). In the United State, cannabis use has recently risen and now surpasses that of cigarette use among adolescents (Johnston et al., 2012). These statistics are particularly concerning, given that cannabis use is associated with negative health outcomes (Kalant 2004), psychosocial and cognitive impairments (Kalant 2004; Solowij and Pesa 2010), and other neurobehavioral consequences such as an increased risk of an automobile crash when driving while intoxicated (Drummer et al., 2003; Mura et al., 2003; Ramaekers et al., 2004; Asbridge et al., 2012; Li et al., 2012). Furthermore, some have argued that the potency of cannabis, as indexed by THC content, has increased in recent years (Burgdorf et al., 2011) and may account for increases in cannabis use disorder diagnoses (Compton et al., 2004), as well as cannabis-related psychosis (Malone et al., 2010). On the other hand, societal acceptance of cannabis use for medicinal applications continues to increase (Fairfield et al. 1998; O’Connell and Bou-Matar; 2007Cohen 2010) and there is a growing body of literature suggesting medical benefits from cannabis use (Watson et al., 2000; Ware et al., 2005; Elikkottil et al., 2009; Ellis et al., 2009) However, not all studies have reported memory impairments in heavy and early-onset cannabis users (Kanayama et al., 2004; Padula et al., 2007; Becker et al., 2010; Ashtari et al., 2011). As indicated by pharmacological challenge studies, an important determinant of the long-term effects of cannabis use may be the precise mixture of the ingredients of the consumed cannabis. Although a naturalistic study reported that cannabis users who smoked cannabis high in CBD and low in Δ9-THC were protected from the memory-
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impairing effects associated with cannabis strains high in Δ9-THC (Morgan et al., 2010), most of the observational studies to date did not include the Δ9-THC/CBD ratio (e.g, by employing hair analysis as a proxy estimate of the amount of Δ9-THC or CBD in the cannabis used) as a potential confounder in statistical reporting. Given the impact of the Δ9-THC/CBD ratio on brain structures implicated in memory function and executive functions in cannabis users while taking into account the variability of this ratio in street cannabis (Chen et al., 2005; Hermann et al., 2007; Demirakca et al., 2011), consideration of this factor in the design and analysis of studies may improve the replicability and interpretability of the results, particularly because simple and valid methods are now applicable for the quantitative detection of Δ9-THC and CBD (Morgan and Curran, 2008; Salomone et al., 2012).
1.1 Statement of the Research Problem
Cannabis is the most widely used illicit drug in many developed and developing societies. Marijuana smoking is a serious public health problem among adolescents in Zaria environment. In a study involving 350 secondary school students in Zaria, a prevalence of 9.43% was recorded (Shehu and Idris, 2008)
Cannabis has both psychological and physiological effects on the human body (Osborne and Fogel 2008). Its health and psychological effects are not well understood and remain the subject of much debate, with opinions on its risks polarised along the lines of proponents’ views on what its legal status should be. An unfortunate consequence of this polarisation of opinion has been the absence of any consensus on what health information the medical profession should give to patients who are users or potential users of cannabis (Osborne and Fogel 2008).
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Also, cannabis receptors are scattered around the brain. The brain regions in which cannabinoid receptors are very abundant are the basal ganglia, associated with movement control; the cerebellum, associated with body movement coordination; the hippocampus, associated with learning, memory, and stress control; the cerebral cortex, associated with higher cognitive functions; and the nucleus accumbens, regarded as the reward center of the brain. Other regions where cannabinoid receptors are moderately concentrated in the hypothalamus, which regulates homeostatic functions; amygdale, associated with emotional responses and fears; the spinal cord, associated with peripheral sensations like pain; the brain stem, associated with sleep, arousal, and motor control; and the nucleus of the solitary tract, associated with visceral sensations like nausea and vomiting (pertwee, 1997). Societal acceptance of cannabis use for medicinal applications continues to increase and there are growing bodies of literature suggesting medical benefits from cannabis use (Cohen, 2010). Hence, the concern for this study.
1.2 Justification of the study
In many western societies, cannabis has been used by a substantial minority, and in some a majority, of young adults, even though its use is prohibited by law (Hall et al., 1998). Debate about the justification for continuing to prohibit cannabis use has polarised opinion about the seriousness of its adverse health effects (Hall et al., 1994). Societal acceptance of cannabis use for medicinal applications continues to increase (Fairfield et al. 1998; O’Connell and Bou-Matar; 2007Cohen 2010) and there is a growing body of literature suggesting medical benefits from cannabis use (Watson et al., 2000; Ware et al., 2005; Elikkottil et al., 2009; Ellis et al., 2009). However, many of these
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reviews conclude that the findings for several neurocognitive domains are equivocal, speaking to the importance of further research in this field (Crean et al., 2011).
In addition, the possible therapeutic effects of cannabinoids have become entangled in the debate about prohibition of recreational cannabis use. The health effects of cannabis use, especially of long-term use, remain uncertain because of the little epidemiological data and also due to disagreements about the interpretation of the limited epidemiological and laboratory evidence (Hall et al., 1994).
1.3 Aim and Objectives
Aim of the Study
The aim of this study is to investigate any neurochemical, neurobehavioural, learning and memory changes associated with Cannabis sativa extract administration on mice.
Objectives of the Study
The objectives of the study were:
I. To establish the effect of Cannabis sativa extract administration on learning and spatial short term memory in mice. II. To investigate some neurochemical changes in the brain such as acetylcholelinerase activity, dopamine levels and brain sialic concentration associated with Cannabis sativa extract administration. III. To investigate the effects of Cannabis sativa extract administration on oxidative stress markers such as superoxide dismutase (SOD), Catalase (CAT) and lipid peroxidation using biochemical techniques.
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1.4 Research Hypothesis
Repeated Cannabis sativa administration has no negative effect on the learning and spatial short memory, brain oxidative biomarker, learning, memory and neurotransmitter release in Adult Mice.
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