Title

Pharmacological modulation of learning and memory in mice and zebrafish: Implications for Alzheimer’s disease animal models

Date of Award

2013

Degree Type

Thesis

Degree Name

Doctor of Philosophy

School

School of Medical Science

Faculty

Computing, Health and Science

First Advisor

Professor Ralph Martins

Second Advisor

Professor Mathew Martin-Iverson

Abstract

Learning and memory impairments are the clinical manifestations of several neurodegenerative disorders including Alzheimer’s disease (AD), the most common form of dementia affecting millions of people worldwide. Current AD treatments target cognitive decline or failure and provide only minor benefits across the array of clinical symptoms. The development of numerous treatment strategies have been trailed in pre-clinical, or human clinical trials. One of the potential candidates to be used for cognitive enhancement is CB1 cannabinoid receptor antagonist. CB1 cannabinoid receptor antagonists have been shown to improve learning and memory in rodents, whereas CB1 cannabinoid receptor agonists have been shown to impair memory. The project has overall aims of (1) investigating the role of CB1 receptors in learning and memory, and assessing the potential of a CB1 receptor antagonist (SR141716A) as a novel cognitive enhancer in mice models; (2) Assessing the potential of zebrafish as an alternative in vivo model for the high-through put assessment of learning and memory. The involvement of cannabinoid CB1 (CB1) receptors in associative learning in two strains of mice (i.e., C57BL/6J and C3H/HeJ) were first investigated. Here, the effects of the cannabinoid CB1 receptor agonist (i.e., CP55940) and antagonist (i.e., SR141716A) on associative learning were assessed in mice using eight-arm radial maze. The results showed that the CB1 receptor agonist impaired reference memory in C57BL/6J but not in C3H/HeJ strains, and the former effect was probably mediated by CB1 receptors since the selective antagonist, SR141716A, reversed these effects. The CP55940 agonist also impaired working memory selectively in the C57BL/6J mice, which was not affected by SR141716A. This study provided evidence for a strain- specific effect of CP55940 on working memory that is likely to be independent of the CB1 receptors. Cannabinoid receptor effects were also evaluated on non-associative learning in mice by measuring habituation of acoustic startle reflex (ASR). CB1 receptor agonist and antagonist's effects were assessed in two different strains of mice (i.e., C57BL/6J and C3H/HeJ). The findings further supported the strain-specific effects of CP55940. This agonist decreased habituation of the ASR in C57BL/6J, but increased habituation in C3H/HeJ. Both of these measures were reversed by SR141716A. This indicates that the cannabinoid agonist exerts its effects on non-associative learning and memory in both strains of mice via CB1 receptors. The advantages of zebrafish in comparison to other common vertebrate models including high fecundity, low maintenance cost, transparent embryos, and rapid development are some reasons for its popularity in genetics, pharmacological and behavioural research. As vertebrates, zebrafish share considerable sequence similarity with humans and are being used as an animal model for various human disease conditions. Here a video and written protocol is presented for the regular care and maintenance of a zebrafish laboratory and the optimisation of breeding conditions to enable efficient embryo production. In order to establish learning and memory procedures for a novel animal model, it is also important to assess potential confounds that may affect the results of the experiments. One such confound is the natural preference of zebrafish towards a particular colour if zebrafish are to be trained for a colour discrimination learning task. Therefore, the natural colour preference of zebrafish towards specific colours was evaluated using a place preference procedure and a T-maze with coloured sleeves. The results from this study showed a strong aversion towards blue colour compared to red, green, and yellow, with yellow being less preferred relative to red and green. Hence, the findings from this study may underpin any further designing of colour-based associative learning and memory paradigms in the zebrafish. The potential of zebrafish to be used for non-associative learning in a high-throughput manner was also evaluated in a 96-well plate by measuring habituation of the acoustic startle reflex in zebrafish larvae. It was found that larvae can be used for assessing non-associative learning and memory in a 96-well plate because zebrafish larvae startled significantly after the presentation of acoustic stimuli and this response decreased when the stimuli were presented continuously over a period of time. The reduction in response over time was consistent with the phenomenon of habituation, a form of non-associative learning. The non-associative learning and memory in zebrafish larvae was further validated by modulating with several pharmacological agents that are known to alter learning and memory in rodents and in humans such as donepezil and scopolamine. Donepezil was found to reduce the ASR and habituation of ASR in zebrafish larvae, consistent with some reports in rodents. Scopolamine treatment of zebrafish larvae produced a biphasic effect on non-cognitive measures such as ASR and locomotion such that lower doses decreased locomotion and ASR whereas the highest dose showed no effects. Similarly, scopolamine impaired non-associative learning with a lower dose but not with the higher dose. Therefore, the use of this drug for cognitive deficits model cannot be supported in this animal model before the reasons for the biphasic dose effect of scopolamine are elucidated. One hypothesis is suggested, that the differences in the effects of scopolamine are due to different affinities of scopolamine for different muscarinic acetylcholine receptor subtypes. The biphasic dose effect of the antagonist may be related to differential activation of these receptors by the endogenous agonist, rather than pharmacological properties of scopolamine itself. Finally, the effect of CP55940 was evaluated on non-associative learning and non-cognitive measures such as locomotor activity in zebrafish larvae. The solubility of CP55940 is a pragmatic issue when used for zebrafish research, as frequently used solvents are toxic to zebrafish. Therefore, CP55940 was dissolved in several different solvents and studied for its toxicity, locomotion effects, ASR and finally habituation of ASR. It was found that CP55940 was successfully dissolved in two solvents (i.e., ethanol + PBS and captisol) that were not toxic to zebrafish larvae, so CP55940 dissolved in these two solvents was tested for its effects on ASR and habituation of ASR. The results of these experiments showed that CP55940 dissolved in these two solvents produced differing effects on ASR and habituation. For instance, CP dissolved in ethanol+PBS had no effects on ASR but impaired habituation; however, CP dissolved in captisol reduced ASR but had no effects on habituation. CP dissolved in both these solvents decreased locomotor activity in zebrafish larvae, a well known effect of CP in rodents. In conclusion, the cannabinoid CB1 agonist may be used as a pharmacological model for inducing memory impairments with a caveat that effects can be strongly influenced by mouse strain. This project also validated the use of zebrafish for high-throughput screening of the effects of pharmacological agents on non-associative learning and showed potential confounds that may impact on zebrafish behaviour. Furthermore, similar to mice, cannabinoid CB1 agonist can induce habituation deficits in zebrafish. Overall, this project has evaluated novel CB1 receptor agonists and antagonists in modulating learning and memory in mice, and has established and validated non-associative memory tasks in zebrafish that can now be utilised in high-throughput screening for modulators of memory.

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