Download THESIS OUTLINE

THESIS OUTLINE
Outline
The sensitivity to the reinforcing effects of drugs of abuse constitutes a risk factor for drug
dependence. The overall objective of this thesis was to investigate the neurobiological
mechanisms of cocaine reinforcement and in particular the role of µ-opioid receptors in
cocaine reinforcement.
General Introduction - In Chapter 1 of this thesis, an introduction to drug addiction and
preclinical addiction research is provided. Existing theories for mechanisms of addiction
processes are discussed, with emphasis on the role of neurotransmitter systems, including
endogenous dopamine and opioid systems, in drug reinforcement. In addition to
pharmacological approaches, more recent studies have used gene knockout strategies to
determine the role of specific genes in amongst others drug reinforcement. An overview of
genetic approaches in addiction research, particularly preclinical, is provided in Chapter 2.
Endogenous opioid systems - The primary aim of the studies described in this thesis was to
establish the role of the endogenous opioid system and, more specifically, of µ-opioid
receptors in cocaine reinforcement. The studies in Chapter 3 were designed to establish the
involvement of µ-opioid receptors in cocaine reinforcement, which was determined with
acquisition of cocaine self-administration by µ-opioid receptor knockout mice as a measure. It
appears from previous studies with the opioid antagonist naltrexone (NTX), that opioid
receptors in the ventral tegmental area (VTA) are of particular importance in endogenous
opioid modulation of cocaine reinforcement (Ramsey et al., 1999). We therefore focused on
the VTA and performed electrophysiology studies in horizontal slices of the VTA of µ-opioid
receptor knockout and wild-type mice. Inhibitory post-synaptic current (IPSC) recordings
were made from dopamine neurons in order to assess GABA mediated neurotransmission in
the VTA in absence of µ-opioid receptors (Chapter 3). In addition, signal transduction
pathways coupled to µ-opioid receptors in the VTA were explored in an in vitro study as
described in Chapter 4. Slices of the VTA were treated with a specific µ-opioid receptor
agonist, fentanyl, and were processed for immunohistochemistry with antibodies for different
phospho-specific proteins for quantification of phospho-protein immunoreactivity.
Long-term exposure to drugs such as cocaine causes sensitization to that particular drug,
which is manifested both by increased locomotor stimulant effects and augmented reinforcing
properties of the drug and is considered to reflect neuroadaptations, which contribute to the
actual development of drug dependence. In Chapter 5, the role of µ-opioid receptors in the
acute locomotor response and in cocaine-induced behavioural sensitization was studied using
µ-opioid receptor knockout mice, as a model for ectopic absence of µ-opioid receptors, and
chronic NTX pre-treated mice, which is a model for transient opioid receptor over-expression
(Chapter 7).
Besides endogenous opioid systems, dopamine is generally regarded to be a common factor in
the effects of different drugs of abuse. Previous anatomical and electrophysiology studies
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Outline
suggested interactions between opioid and dopamine systems in amongst others the
mesolimbic system. Such interaction may be of relevance to µ-opioid receptor-induced
modulation of drug reinforcement. In Chapter 6 we therefore quantified levels of mRNA
encoding the rate-limiting enzyme in dopamine synthesis, TH, and levels of dopamine
receptor binding for mice with reinforcement relevant changes in µ-opioid receptor levels: µopioid receptor knockout mice and chronic NTX treated mice. In addition, locomotor activity
in the open field and spontaneous climbing, behaviours which both involve dopamine were
assessed for these mice.
In Chapter 7, the effects of chronic treatment with the opioid antagonist NTX upon opioid
receptor levels were determined using quantitative autoradiography. Chronic NTX treatment
has been shown to induce supersensitivity to morphine’s analgesic effects and is known to
increase opioid receptor numbers as assessed using whole brain homogenates. Interestingly,
this same treatment is also effective in potentiating the reinforcing effects of both drugs of
abuse, such as cocaine (Ramsey & Van Ree, 1990) and alcohol (Phillips et al., 1997). We
performed a full quantitative mapping study for the three main opioid receptor subtypes: µ-, δand κ-opioid receptors. The chronic NTX model was used to investigate the role of (µ-)opioid
receptors in cocaine-induced sensitization (Chapter 5) and in the study of opioid-dopamine
interactions (Chapter 6).
Endogenous Cannabinoid systems – Recently, the endogenous cannabinoid system has been
implicated in addiction processes. The studies described in Chapter 8 were designed to
further investigate the role of endogenous cannabinoids, through interactions with the CNS
cannabinoid type 1 (CB1) receptor, in cocaine reinforcement and cocaine-induced behavioural
sensitization. CB2 receptor expression is restricted to the periphery. For this purpose we used
the selective CB1 receptor antagonist SR141716A, which was administered prior to cocaine
self-administration or before the repeated intermittent cocaine injections for sensitization.
The results described in this thesis demonstrate an important and specific role of µ-opioid
receptors in cocaine reinforcement, thus suggesting that variations in µ-opioid receptor levels
might alter an individual’s proneness to develop cocaine dependence. Further the
neurobiological and behavioural findings are combined to a proposed mechanism through
which µ-opioid receptors might modulate cocaine reinforcement in the General Discussion
(Chapter 9).
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