Abstract
1. It is postulated that dynorphin inhibits the cocaine-induced increase in striatal dopamine levels, that may contribute to dynorphin’s ability to prevent cocaine-induced conditioned place preference and to attenuate an increase in locomotor activity. In this thesis interactions between dynorphin and the dopaminergic system in cocaine addiction have been addressed using preprodynorphin gene knockout mice treated with a chronic “binge” cocaine protocol. Dopamine D1, D2 receptor and dopamine transporter binding have been mapped by quantitative autoradiography in the brains of knockout mice treated with a chronic “binge” cocaine protocol. A significant decrease in D2 receptor binding was observed in the mediolateral part of the caudate putamen of chronic “binge” cocaine-treated knockout mice compared to knockout saline controls. Moreover, a significant decrease in overall D2 receptor binding was observed in the brains of chronic cocaine-treated preprodynorphin knockout mice compared to knockout controls and to cocaine treated wild-type animals. However, no significant difference in D2 receptor binding was observed in any of the regions of chronic cocaine-treated wild-type mice compared with saline controls. In contrast to D2 receptor binding, no significant differences in D1 receptor and dopamine transporter binding were observed between any of the groups in any of the regions analysed. These results suggest that dynorphin-derived endogenous opioid peptides can modulate chronic cocaine-induced alteration of D2 receptors but not Dl receptors or dopamine transporters. 2. A large body of evidence suggests that the endogenous opioid system directly or indirectly regulates dopaminergic tone in the central nervous system. Triple opioid receptor knockout and wild-type mice brains from congenic C57BL/6 and coisogenic 129S6 backround have been used to address whether the dopaminergic system could be influenced not only by the deletion of all classical opioid receptors but also by genetic background. The absence of all three classical opioid genes does not affect D1, D2 or dopamine transporter binding in either the C57BL/6 or 129S6 background strain suggesting that the endogenous opioid system may not modulate the dopaminergic system and this may not be dependent on the background strain of the knockout mice. 3. In order to investigate the prime receptor target of oxycodone in the brain, quantitative autoradiographic binding of [3H] oxycodone was carried out in the brains of mice lacking either μ-, κ- or δ-opioid receptor as well as in mice lacking all three classical opioid receptors (triple opioid receptor knockout mice). The distribution of [3H]oxycodone labelling was similar to the known distribution of μ-opioid receptor binding sites. Moreover μ-opioid receptor knockout mice had significantly lower [3H]oxycodone binding than wild-type mice across regions suggesting that oxycodone mainly binds to μ-opioid receptors in the brains of mice. In contrast, the binding levels of [3H]oxycodone were significantly increased in the brains of κ-opioid receptor and δ-opioid receptor knockout mice compared to wild-type mice suggesting [3H]oxycodone may also exert its effect on different opioid receptor sites such as μ/δ and/or μ/κ-opioid heterodimers. No [3H]oxycodone binding was detected in brains of triple opioid receptor knockout mice indicating that the ligand can only bind to all three classical opioid receptors. 4. Despite the development of several methods including biochemical, biophysical and pharmacological methods which have been used to investigate the existence of heterodimer/hetero-oligomer forms of the δ-opioid and κ-opioid receptor in vitro and in vivo there has not been direct evidence showing whether heterodimerisation occurs in physiological systems. A novel method combining knockout mice with radioligand binding which is called KSA (knockout Subtraction Autoradiography) has been developed and is proposed to provide further evidence for the existence of heterodimer/hetero-oligomer forms of the δ- and κ-opioid receptors in mouse brain tissue. Quantitative autoradiography of [3H]bremazocine and [3H]nor-binaltorphimine binding was carried out in single (μ) and double (μ and δ or μ and κ) opioid receptor knockout mice. The principle of the method relies on quantification of receptor binding in knockout brain tissue where dimers can (single knockout’s) or cannot (double knockout’s) be formed. Both [3H]bremazocine and [3H]nor-BNI binding fitted the heterodimer/hetero-oligomer model (μ-opioid receptor knockout - μ-opioid receptor/κ-opioid receptor knockout = μ-opioid receptor/δ-opioid receptor knockout) in some regions and the monomer/homo-oligomer model (μ-opioid receptor knockout - μ-opioid receptor/κ-opioid receptor knockout = μ-opioid receptor/δ-opioid receptor knockout) in other regions suggesting that the presence of heterodimer/heterooligomer forms of the δ- and κ-opioid receptors is region-specific. The KSA could prove a useful tool to identify heterodimers in vitro and test binding properties of pharmacological ligands to heterodimers.