Centre for Neuroscience - Theses
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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.