Centre for Neuroscience - Theses
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ItemRelaxin-3 systems in brain: effects on feeding, anxiety, depression and addiction( 2012)The neuropeptide, relaxin-3 (RLN3), and its major endogenous receptor, RXFP3 (relaxin family peptide receptor 3), have been postulated to modulate feeding, anxiety- and depressive-like behaviour, and reward based on neuroanatomical and neurochemical data. The major aim of the research described in this thesis, therefore, was to determine the effect of centrally administered RXFP3-selective peptides on these functions in the adult rat. Intracerebroventricular (icv) administration of the RXFP3-selective agonist peptides, R3/I5 and RXFP3-A2, and native rat/mouse relaxin-3 (rmRLN3) significantly increased acute food intake in male Sprague-Dawley rats, and this effect was prevented by prior icv injection of the RXFP3-selective antagonist peptides, RXFP3-A3 and R3(B1-22)R. Icv injection of RXFP3-A2 decreased anxiety-like behaviour in male Sprague-Dawley rats in a range of behavioural tests, including the light-dark box and elevated plus maze, and produced antidepressant-like effects in the repeat forced swim test, but notably only in rats that had previously been tested in anxiety-like paradigms. Icv injection of RXFP3-selective agonists had no significant effect on general activity assessed quantitatively in automated locomotor cells. Icv administration of an RXFP3 antagonist (R3(B1-22)R) decreased alcohol but not sucrose self-administration in male inbred alcohol-preferring (iP) rats in a dose-related manner, and decreased cue-induced reinstatement (a model of relapse) for alcohol but not sucrose, suggesting a possible role for relaxin-3/RXFP3 signalling in alcohol-seeking. In addition, endogenous relaxin-3 mRNA expression in the hindbrain nucleus incertus correlated with daily alcohol and sucrose intake in the two-bottle choice paradigm, suggesting a role for endogenous relaxin-3 in modulating intake of ‘rewarding’ substances. Overall, the results from this research suggest that the endogenous relaxin-3/RXFP3 system promotes ‘motivated’ or ‘goal-seeking’ aspects of behaviour. These studies contribute to a deeper understanding of the neurobiology underlying clinical disorders such as obesity, anxiety, depression and addiction, which may in turn lead to novel diagnostics and therapeutic approaches. These important findings are therefore predicted to have a significant impact within the fields of neuropeptide biology and behavioural neuroscience, and are associated with several published or submitted scientific journal articles and reviews. In addition, the data have helped stimulate interest in this research area, with several major pharmaceutical companies expressing interest in the relaxin-3/RXFP3 system as a possible target for the development of therapeutic treatments for individual and co-morbid psychiatric and metabolic disorders.
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ItemInvestigating the role of CREB and CBP in addiction using conditional knockout mouse models( 2012)Drug addiction is a chronic, relapsing brain disease which represents an enormous social and economic burden to society. The transition from casual to compulsive drug use and the enduring propensity to relapse is thought to be underpinned by long-lasting neuroadaptations within specific brain circuitry. The transcription factor cAMP response element-binding protein (CREB) has been identified as a key molecular substrate involved in drug-induced plasticity. The first broad aim of this thesis was therefore to further elucidate the role of CREB signalling within specific brain reward regions in addiction-related behaviours. To this end, mice were generated with a postnatal deletion of CREB targeted to the striatum, a brain structure critically involved in reward-related learning. While striatal CREB deletion did not appear to alter the reinforcing properties of cocaine, enhanced expression of locomotor sensitization to cocaine was revealed following chronic treatment, in addition to increased amphetamine-induced stereotypies. However in the absence of striatal CREB, upregulation of the related transcription factor cAMP responsive element modulator (CREM) was observed, indicating possible redundancy amongst this family of transcription factors. In support of this, striatal deletion of CREB-binding protein (CBP), a coactivator recruited by both CREB and CREM, resulted in an even more pronounced sensitivity to psychostimulants. This suggests that CREM, acting via CBP, is able to partially compensate in the absence of CREB. In contrast, mice with a postnatal deletion of CBP directed to midbrain dopamine neurons were indistinguishable from controls on a number of addiction-related measures. The second main aim of this thesis was to investigate possible brain nuclei involved in cue-induced morphine- vs. sucrose-seeking in a mouse model of relapse using the neuronal activity marker Fos. This resulted in the identification of putative circuitry common to morphine- and sucrose-seeking as well as circuitry specific to each reinforcer. Structures activated in both relapse groups included the anterior cingulate, nucleus accumbens shell, basolateral amygdala, substantia nigra, ventral tegmental area, anterior periaqueductal gray and locus coeruleus. Structures activated only in morphine-seeking mice included the orbitofrontal cortex, nucleus accumbens core, ventral pallidum, bed nucleus of the stria terminalis, central nucleus of the amygdala and hippocampus. The dorsal raphe was the only structure found to be specifically activated in sucrose-seeking mice. These findings broadly support a cortico-striatal limbic circuit driving opiate-seeking behaviour, and additional circuitry potentially relevant to reward-seeking was identified. In summary, this thesis has contributed to a growing body of literature investigating the role of CREB signalling upon behavioural responses to drugs of abuse. Deletion of CREB/CBP from the striatum confers increased sensitivity to the locomotor activating properties of psychostimulants, without influencing the reinforcing properties of cocaine. In contrast, deletion of CBP from midbrain dopamine neurons does not appear to affect behavioural responses to cocaine under the tested paradigms. Finally, findings from the relapse study have revealed a number of structures not previously explored in the context of reward-seeking that are worthy of further investigation, and concordance with data from human imaging studies supports this model as a relevant tool for studying relapse-like behaviour in mice.
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ItemTargeted knockdown of CREB1 in brain nuclei critically involved in drug-seeking behaviour( 2009)The purpose of this thesis was to characterise the contribution that a specific molecule, CREB1, plays in the many facets of a developing addiction phenotype. Indeed, CREB1 is known to contribute to long term learning and memory, and present an altered activation profile upon exposure to reinforcing substances, in brain regions implicated in addiction. Together, these observations provide a prima facie driver to investigate the specific involvement of CREB1 in brain regions implicated in reinforcement and drug-seeking. Initially, I investigated Sprague Dawley rats whom had undergone behavioural sensitization to the repeated administration of the psychostimulant d-Amphetamine. Detailed in Chapter 3, the aims of this study were to determine the impact that environmental drug-context associations and psychostimulant sensitization makes upon expression of the activated or phosphorylated form of CREB1 (pCREB1). The data presented in the study reveals that many brain nuclei relevant to the behavioural effects of drug exposure show expression of pCREB1 subsequent to enduring amphetamine abuse, as well as upon return to an environment previously paired with amphetamine. The profile of pCREB1 expression within brains was unique to each pattern of drug dosing and context exposure, suggesting that unique sub-circuits underlie these different behavioural repertoires. Using the impetus from this study, I determined to further investigate the contribution of CREB1 from specific brain regions, and the impact of its deletion upon behaviours characteristic of addiction. Indeed, the aims of this section of the project were to firstly employ relevant detection systems and current genetic-engineering technologies in creating appropriate expression animal lines, emphasising reward and reinforcement pathways. In addition, I aimed to understand the signalling systems and pathways which are activated by neurotransmitters, culminating in the phosphorylation of CREB and subsequently altered gene expression and long-term cellular and neuronal adaptation, induced by ongoing exposure to drugs of abuse. Detailed in Chapter 4, I created a novel mutant mouse which was deficient in CREB1 within the dorsal telencephalon. Mice 'floxed' for the Creb1 gene expressed loxP DNA sequence around an exon critical to CREB1 function. These mice were interbred with mice expressing the enzyme Cre recombinase in dorsal telencephalic brain regions. Thus, mice expressing Cre recombinase and floxed for Creb1 demonstrated the deletion of CREB1 protein in these brain regions, which is demonstrated through experiments presented in Chapter 4. Further in vitro characterisation of this mutant mouse was carried out and presented in Chapter 5. As CREB1 is important in synaptic plasticity and growth, it was necessary to evaluate any impact upon ontogeny through stereological analysis of cell number and volume, for relevant brain nuclei. The experiments demonstrate that mutant CREB1 mice were no different to control mice, however, it was possible that this lack of phenotype was partly contributed though changes in the level of other CREB/ATF-1/CREM bZIP family members. To this end, I determined to assay for transcript changes in these and related genes, finding confirmation of the deletion of the Creb1 transcript in the cortex and hippocampus, whilst observing a concomitant increase in Crem transcript. These data suggested that compensatory changes in brain regions receiving a recombination of Creb1 were apparent, contributing to the lack of an obvious phenotype in these mice. Having confirmed the specific deletion of CREB1 in the appropriate brain nuclei, I then moved to examine the impact of the deletion behaviourally, both in terms of general ethology, and in regard to drug-induced phenotypes. Presented in Chapter 6, experiments assaying general ethology of the CREB1 mutant revealed a spontaneous hypoactivity when placed in a small open field environment. As CREB1 is involved in neural plasticity, I wished to assay for the impact on behavioural sensitization, a paradigm which reveals long-lived neural change. Experiments to this effect showed no perturbation of behavioural sensitization to the effects of cocaine in the mutant. In addition, mutant mice also showed a similar response to the rewarding effects cocaine as witnessed in the control mice, however, the CREB1 mutants demonstrated a perturbed drug-environment contextual memory, which was not retained in long-term place preference experiments. Operant conditioning studies for intravenous self administration of cocaine revealed that CREB1 mutants displayed a dose-specific diminished drive to self-administer cocaine, whereas in contrast, self administration of a natural reward was no different to control mice. These data suggest that there is a specific role for CREB1 in telencephalic glutamatergic neurons regulating the motivational and associative properties of cocaine. Together, these data provide evidence that CREB1 functions as a key molecular substrate in long lived drug-context environment associations and neural change underlying the developing addicted state, warranting future investigation for its properties in producing drug related functional and behavioural change.