Arc and homer 1a expression following intravenous administration of heroin and cocaine : a novel application of the catFISH technique

This thesis applies catFISH, a variant of the standard fluorescence in situ hybridisation technique, to study the neuronal ensembles activated by heroin and cocaine across brain structures involved in motivated behaviour, in the Sprague-Dawley rat. The first chapter reviews the pharmacology of heroin and cocaine, rodent models of drug-related behaviours, and heroin and cocaine’s ability to trigger immediate-early gene expression when administered acutely or chronically. It is suggested that the empirical evidence points towards a significant separation between the neuronal systems engaged by the two drugs. The main goal of this thesis was to test whether this separation can be seen within brain areas playing a key role in motivation and reward (e.g. the nucleus accumbens). Since immediate-early genes serve as markers of neuronal activity, and catFISH is a technique which can detect the expression of such genes in response to two separate stimuli, the technique was chosen as the best method to test if heroin and cocaine activate the same neuronal ensembles when administered acutely. The second chapter summarises the methods used across experiments described in following chapters. The third chapter presents an experiment addressing the methodological issues associated with using catFISH to study pharmacological stimuli. The technique was originally used to study the hippocampus and brain activity triggered by stimuli with well-controlled on- and offset (e.g. exposure to a novel environment or discrete cues). Arguably, acute drug injections comprise a stimulus with an on- and offset which can only be controlled indirectly – they depend on the drug dose and route of administration, among other factors. It was shown that acute intravenous injections of heroin and cocaine at doses usually self-administered by animals are suitable stimuli to use with catFISH. Chapter four describes an experiment showing that, across the striatum, the neuronal ensembles activated by an injection of cocaine followed by an injection of heroin overlap significantly less than the neuronal ensembles activated by two consecutive injections of cocaine. This is interpreted as direct evidence for a significant separation between the neuronal pathways activated by heroin vs. cocaine, even in brain areas often considered a common neurobiological substrate for the effects of the two drugs. It must be noted that the magnitude and the nature of this separation depends on the particular part of the striatum and the order in which drugs are administered. Chapter five describes a pilot experiment attempting to incorporate the logic of the catFISH technique into a rodent drug self-administration paradigm. It was found that the rats preferred self-administering heroin over cocaine, and that there was no detectable expression of the homer 1a gene across the striatum in response to acute heroin and cocaine after extended experience with the two drugs. There was also no change from baseline expression of the homer 1a and arc genes in areas of the prefrontal cortex. Finally, chapter six summarises the main findings and the key methodological considerations from all three experiments. As a whole, it is suggested that the experiments in this thesis provide a proof of concept that heroin and cocaine are processed differently by the brain, even within brain areas considered to be common substrates for the reinforcing and addictive properties of the two drugs. Future studies should take this separation into account, as it may have important implications for understanding drug addiction as a whole. The appendices contain representative fluorescence microscopy images of brain tissue analysed for catFISH.