L'addiction est un comportement compulsif de recherche et de prise de drogues alternant des phases d'abstinence et de rechute malgré les conséquences négatives sur la vie de l'individu. Les êtres humains ne sont pas égaux devant l'addiction et les mécanismes moléculaires sous jacents sont encore mal compris. De nombreuses structures cérébrales, telles que l'aire tegmentale ventrale (VTA), le cortex préfrontal ou l'amygdale convergent sur le noyaux accumbens (NAc) pour réguler les circuits de la " récompense ". Les neurones GABAergiques épineux de taille moyenne (MSN) sont à la fois la voie d'entrée et de sortie majeure du NAc. Les MSNs sont régulés de façon dynamique par les fibres dopaminergiques provenant de la VTA ainsi que par les interneurones cholinergiques locaux (TANs). La destruction sélective des TANs entraine une importante modification des propriétés renforçantes des psychostimulants tel que la cocaïne. En 2002 nous avons découvert que, de façon surprenante, ces neurones expriment à la fois le transporteur vésiculaire de l'acétylcholine (VAChT) et le transporteur vésiculaire du glutamate de type 3 (VGLUT3). Plus récemment nous avons établi que VGLUT3 augmentait le stockage vésiculaire ainsi que la libération d'acétylcholine (ACh) par un mécanisme que nous avons appelé " synergie vésiculaire ". De plus, il a été observé que VGLUT3 confère aux TANs la capacité d'utiliser le glutamate aussi bien qu'avec l'ACh pour communiquer. De façon surprenante, des souris ayant perdu la capacité de libérer l'ACh dans le NAc ne présente que très peu d'altération de leurs réponses comportementales à la cocaïne. Ce résultat suggère que l'ACh n'est pas indispensable à la régulation des comportements de « récompense ». 1.2 Afin de déterminer le rôle de la signalisation VGLUT3-dépendante par les TANs nous avons utilisé une souris n’exprimant plus VGLUT3. Au cours de cette thèse j’ai pu établir que l’absence de VGLUT3 exacerbe les effets comportementaux induit par la cocaïne. Il semble donc que les TANs utilisent l’ACh ou le glutamate pour réguler différentiellement la libération de DA. Nous avons des résultats préliminaires suggérant que le glutamate libéré par les TANs va activer des mGluR qui exercent un contrôle inhibiteur sur la libération de DA. De plus j’ai observé que l’augmentation de libération de DA chez les souris VGLUT3-KO entraine une activation des cascade de signalisation DR1-dépendantes. De plus les MSNs du NAc des souris VGLUT3-KO présentent des augmentations morphologiques et synaptiques de l’activité glutamatergique du NAc. Finalement une augmentation de la fréquence des mutations du gène codant pour VGLUT3 a été trouvée dans une cohorte de sujets souffrants de formes sévères d’addictions. L’ensemble de ces résultats suggère que la régulation concomitante de la signalisation DAergique et glutamatergique dans le NAc agit comme un filtre protecteur contre les effets renforçant de la cocaïne. / Drug addiction is a compulsive pattern of drug-taking/drug-seeking behavior with alternate phases of abstinence and relapse despite adverse consequences. Human beings are not equally susceptible to addictions and molecular mechanisms underlying addiction are still poorly understood. Numerous brain structures such as the ventral tegmental area (VTA), the prefrontal cortex, the amygdala or the hippocampus converge onto the nucleus accumbens (NAc) to regulate reward. GABAergic medium spiny neurons (MSN) are the major input target as well as output pathway of the NAc. MSNs are dynamically regulated by dopaminergic fibers originating from the VTA and by local tonically active cholinergic interneurons (TANs). The selective destruction of TANs modulates rewarding properties of psychostimulant such as cocaine. Twelve years ago we made the surprising discovery that these neurons express both the vesicular acetylcholine transporter (VAChT) and the vesicular glutamate transporter type 3 (VGLUT3). We recently established that VGLUT3 increases the acetylcholine (ACh) vesicular accumulation (and release) by a mechanism named vesicular synergy. Furthermore, the presence of VGLUT3 confers to TANs the ability to release glutamate in addition to ACh. Unexpectedly, mice that have lost the ability to secrete ACh in the NAc show minimal alteration of their behavioral response to cocaine. This result suggests that ACh is not sufficient to modulate reward.To investigate the role of VGLUT3-mediated signaling by TANs we used a mouse line that no longer expressed VGLUT3. During this PhD I established that silencing VGLUT3 in mice dramatically exacerbated cocaine-induced behaviors. Furthermore, we found that VAChT-KO and VGLUT3-KO mice showed a decreased and increased DA release (respectively) in the NAc. Therefore, TANs use ACh and glutamate to differentially regulate DA release. We have preliminary data suggesting the glutamate released by TANs activate mGluR that negatively control DA release. I further observed that in VGLUT3-KO mice the increased DA release enhanced DR1-signaling cascades. In addition, MSNs from the NAc of VGLUT3-KO mice had increased morphologic and synaptic glutamatergic activity in the NAc. Finally, we report non-synonymous mutations in the gene encoding VGLUT3 in patients with severe addictions. Our results suggested that the concomitant regulation of the dopaminergic and glutamatergic tone by VGLUT3 in the NAc acted as a protective filter against reinforcing properties of cocaine.

Die Glutamat-vermittelte Exzitotoxizität gilt als einer der wichtigsten neuropathologischen Faktoren der HIV-Demenz: Während Glutamat in physiologischer Konzentration als exzitatorischer Neurotransmitter fungiert, wirkt es in zu hoher Konzentration neurotoxisch. In vorliegender Arbeit wurde mittels Western Blotting die Proteinexpression der exzitatorischen Aminosäuretransporter EAAT1 und EAAT2 gemessen, die vor allem für den Abtransport von Glutamat aus dem synaptischen Spalt sorgen. Hierzu wurden Gehirne von mit dem simianen Immundefizienz Virus (SIV) infizierten chinesischen und indischen Rhesusaffen verwendet. SIV verursacht im SIV-Rhesusaffenmodell ähnliche Schäden wie das humane Immundefizienz Virus (HIV) beim Menschen. Zur Entstehung der SIV-Enzephalitis tragen, wie auch bei der HIV-Demenz, aktivierte Monozyten und Mikroglia bei, die u.a. das Neurotoxin Tumornekrosefaktor-alpha (TNF-alpha) sezernieren. Dessen Protein- und Genexpression wurde mittels ELISA und Real-Time-PCR ausgewertet. Für die vorliegende Arbeit wurden zwei für die HIV-Demenz besonders relevante Gehirnregionen ausgewählt: das Putamen, das als Teil der Basalganglien für die extrapyramidale Steuerung der Motorik zuständig ist, und der Nucleus Accumbens, der affektives und motivationales Verhalten in Bewegungsabläufe integriert. Als potentielle Pharmaka wurden der MAO-B-Hemmer Selegilin, der NMDAR-Antagonist Memantin sowie die Antioxidantien N-Acetylcystein (NAC) und Melatonin getestet. Es gelang in vorliegender Arbeit erstmals, eine Störung der Proteinexpression der glutamatergen Transporter EAAT1 und EAAT2 im Putamen mit zunehmender Dauer der SIV-Infektion und ihren dramatischen Verlust bei Entwicklung von AIDS nachzuweisen. Im Nucleus Accumbens fand sich eine relativ konstante Proteinexpression des EAAT1 und EAAT2 im Verlauf der SIV-Infektion. Weiterhin konnte ein Anstieg des TNF-alpha mit fortschreitender Infektionsdauer hinsichtlich der Genexpression im Putamen und der Proteinexpression im Nucleus Accumbens nachgewiesen werden. Die fehlende Eignung von Selegilin als neuroprotektive Substanz im Rahmen der SIV-Enzephalitis wurde repliziert. Memantin, NAC und Melatonin hingegen verbesserten in weiten Teilen die Expression von EAAT1 und EAAT2 und wirkten immunstimulierend, was sie zu interessanten Kandidaten für eine neuroprotektive Medikation macht. In beiden Hirnregionen zeigte sich bei den indischen Rhesusaffen eine höhere TNF-alpha-Expression als bei den chinesischen Tieren. Dies entspricht der Beobachtung, dass die SIV-Infektion bei indischen Rhesusaffen meist schneller und schwerer verläuft. / Glutamate-mediated excitotoxicity is considered one of the major neuropathological factors inducing HIV dementia: serving as an excitatory neurotransmitter in physiological concentration, glutamate exerts neurotoxic effects if secreted excessively. In the present study, the protein expression level of the excitatory amino acid transporters EAAT1 and EAAT2, which are responsible for the removal of glutamate from the synaptic cleft, was analyzed via Western Blotting. For this purpose, brains of Chinese and Indian macaques infected with the simian immunodeficiency virus (SIV) were used. SIV causes similar symptoms in the SIV/macaque model as the human immunodeficiency virus (HIV) does in humans. Similar to HIV-dementia, the development of SIV-encephalitis is triggered by activated monocytes and microglia, which secrete – among other things - the neurotoxin tumor necrosis factor-alpha (TNF-alpha). TNF-alpha protein and gene expression was examined using ELISA and real-time-PCR. For the present study, two brain regions were chosen due to their specific relevance for HIV-dementia: first the putamen, which is part of the basal ganglia and exerts influence on extrapyramidal motion sequences, and second the nucleus accumbens, which integrates affective and motivational behavior in motor activity. The MAO-B-inhibitor selegiline, the NMDAR-antagonist memantine and the antioxidants N-acetyl-cysteine (NAC) and melatonin were tested as potential pharmaceuticals. For the first time ever, the present study shows a disruption of protein expression of the glutamatergic transporters EAAT1 and EAAT2 in the putamen during an SIV infection, and a dramatic loss of EAATs associated with the development of AIDS. In the nucleus accumbens, a relatively constant protein expression of EAAT1 and EAAT2 was found during the progression of the SIV infection. Additionally it has been proved that TNF-alpha gene expression in the putamen and TNF-alpha protein expression in the nucleus accumbens increase in the course of an SIV infection. It was replicated that selegiline is unsuitable as a neuroprotective agent regarding SIV encephalitis. Memantine, NAC and melatonin, on the other hand, largely improved the expression of EAAT1 and EAAT2, and stimulated the immune system, so that these substances can be taken into consideration as possible neuroprotective pharmaceuticals. In both brain regions, the Indian macaques showed a higher TNF-alpha expression level than the Chinese macaques. This finding corresponds to the fact that the course of an SIV infection is faster and more severe in Indian macaques.

Fine temporal regulation of dopamine transmission is critical to its effects on behaviour. Dopamine can be cleared from the synapse either by recycling via the dopamine transporter (DAT) or by enzymatic degradation involving catechol-O-methyltransferase (COMT). DAT recycling predominates in striatum and contributes to dopaminergic regulation of reward-guided behaviour, while COMT degradation predominates in cortex and modulates executive functions. However, human functional imaging studies demonstrate interactive effects of DAT and COMT genotype, suggesting that the traditional division between DAT and COMT is not so clear-cut. Given the interdependence of mesolimbic and mesocortical circuitry and the presence of COMT in the striatum, it is possible that DAT and COMT interact to a greater extent than previously thought. We investigated the contributions of DAT and COMT to regulation of dopamine transmission and reward-guided behaviour by combining in vivo electrochemical recording, pharmacology, and behavioural testing in mice. Using fast scan cyclic voltammetry to record evoked dopamine release in anaesthetised animals, we found that systemic DAT blockade increased the size of dopamine transients in the nucleus accumbens (NAc) but not in the medial frontal cortex (MFC), demonstrating that DAT regulates phasic striatal dopamine release and confirming that DAT makes little contribution to regulation of cortical dopamine transmission. Unexpectedly, COMT inhibition did not affect evoked dopamine transients in either the NAc or the MFC. In agreement with these findings, systemic administration of a DAT blocker, but not of a COMT inhibitor, increased motivation to work for reward in a progressive ratio paradigm. COMT inhibition also had little effect on reinforcement learning (RL) strategies during reward-guided decision making. Intriguingly, however, we found that DAT blockade both decreased the influence of model-free RL and increased the influence of model-based RL on behaviour. Our study confirms that DAT regulates dopamine transmission in striatum but not in cortex and indicates that sub-second changes in dopamine transmission in both regions are largely insensitive to COMT. However, our behavioural data reveal the importance of striatal dopamine in multiple components of reward-guided behaviour, including both motivational aspects traditionally associated with striatum as well as cognitive aspects heretofore mainly associated with cortical function. Together, these findings emphasise that reward processing occurs across corticostriatal circuits and contribute to our understanding of how striatal dopamine transmission regulates reward-guided behaviours.

The ability to take effective decisions is fundamental for successful environmental adaptation and survival. In this thesis, I investigated situations in which decisions appear irrational, at least from certain standpoints. I conducted a behavioural decision-making experiment in two groups of macaques: controls and a group with ventromedial prefrontal cortex/medial orbitofrontal cortex (vmPFC/ mOFC) lesions. Some choices lead to compound outcomes composed of different constituent parts. Control macaques' decisions suggested their estimates of the value of the compound were biased away from the sum of the values of the constituents and towards their mean. Lesions of vmPFC/mOFC diminished the size of the effect so that macaques in some ways appeared to make more rational decisions. Based on the results of this experiment I devised a similar Functional Magnetic Resonance Imaging (fMRI) paradigm with the control animals. This demonstrated strong vmPFC/mOFC activity when similar decisions were made and suggested a value comparison process. In addition, I investigated the role of dopamine in learning using Fast-Scan Cyclic Voltammetry (FSCV), while rats performed a simple decision-making task. Theories about the role of dopamine in learning have focused on the possibility that it codes scalar reward value prediction errors. Less consideration has been given to the possibility that dopamine might reflect prediction errors about reward identities regardless of value. I measured dopamine in the nucleus accumbens when unexpected changes in reward value or identity occurred while rats executed a two-choice two-reward instrumental task. Dopamine levels in the nucleus accumbens reflected reward value prediction errors. In addition, however, they also reflected some information about reward identity under some circumstances. Further investigation suggested that this might be due to differences in the nutritional value of different reward types that did not have clear measurable impacts of behaviour in the tasks that I used.

The current study analyzed the effects of environmental enrichment versus isolation housing on the behavioral sensitization to nicotine in the neonatal quinpirole (NQ; dopamine D2-like agonist) model of dopamine D2 receptor supersensitivity, a rodent model of schizophrenia. NQ treatment in rats increases dopamine D2 receptor sensitivity throughout the animal's lifetime, consistent with schizophrenia. Animals were administered NQ (1 mg/kg) or saline (NS) from postnatal day (P)1 to P21, weaned, and immediately placed into enriched housing or isolated in wire cages throughout the experiment. Rats were behaviorally sensitized to nicotine (0.5 mg/kg base) or saline every consecutive day from P38 to P45, and brain tissue was harvested at P46. Results revealed that neither housing condition reduced nicotine sensitization in NQ rats, whereas enrichment reduced sensitization to nicotine in NS-treated animals. The nucleus accumbens (NAcc) was analyzed for glial cell line-derived neurotrophic factor (GDNF), a neurotrophin important in dopamine plasticity. Results were complex, and revealed that NAcc GDNF was increased in animals given nicotine, regardless of housing condition. Further, enrichment increased GDNF in NQ rats regardless of adolescent drug treatment and in NS-treated rats given nicotine, but did not increase GDNF in NS-treated controls compared to the isolated housing condition. This study demonstrates that environmental experience has a prominent impact on the behavioral and the neural plasticity NAcc response to nicotine in adolescence.

Cocaine use disorder is a significant health problem, negatively impacting individuals afflicted. While preclinical self-administration research has provided invaluable insight into the neurobehavioral mechanisms that underlie cocaine abuse, cocaine use outside of the laboratory occurs within an environment where other goods are also available ubiquitously. Although there is an ever-increasing literature investigating drug vs. non-drug choice in rodent models and how alternative goods can compete with the subjective value of cocaine, the neurobiological mechanisms that are associated with cocaine preference remains largely unknown. Additionally, current drug vs. non-drug choice studies use procedures that confound preference with intake, such that preference measures are directly reflective of individual experience with drug and non-drug reinforcers earned through the choices that are made; simply, preference and intake are the same. Moreover, differences in cocaine experience can result in differential neural adaptations, thus making it difficult to determine if the neurobiological mechanisms underlying choice are related to preference or drug intake. Herein a novel choice procedure, which controls for reinforcer intake (controlled reinforcer ratio; CRR), was used to explore how certain reinforcer dimensions (i.e., magnitude and frequency) influence cocaine preference. In addition, neuronal activity, measured via c-fos expression, in the orbitofrontal cortex and nucleus accumbens, areas associated with decision-making and valuation, for cocaine and food were independently targeted and labeled using fluorescent in situ hybridization and fluorescent immunohistochemistry. First, unlike prototypical choice procedures where preference and intake are confounded, the CRR choice procedure was able to dissociate the two. Under the CRR choice procedure, it was revealed that both magnitude and frequency, independent dimensions of reinforcement, greatly influence preference for cocaine. Furthermore, the CRR choice procedure was sensitive to manipulations known to influence cocaine preference while keeping reinforcer intake constant. When neuronal activity was examined after CRR training, the number of cocaine activated cells, relative to food activated cells, did not correlate with individual preferences for cocaine despite overall reinforcer intake being held constant. Instead, results suggest neuronal activity for cocaine was related to overall cocaine intake. Overall, these results give impetus for utilizing the CRR choice procedure to better investigate how drug and non-drug reinforcers are afforded differential subjective value and compete for preference. Moreover, use of a CRR choice procedure may lead to identification of specific neurobehavioral mechanisms and lead toward future development of more effective pharmacological and behavioral treatments to ameliorate substance use disorders.

Multiple interactions between dopamine (DA), glutamate, and nitric oxide (NO) in mesolimbic and corticostriatal circuits suggest that NO may play a critical role in cocaine-induced behavioral and neural plasticity. Clinical and preclinical studies have revealed that females and adolescents display unique vulnerabilities to the behavioral and neurochemical effects of cocaine as a result of sex-dependent and ontogeny-dependent differences in dopaminergic systems. Thus, my research objectives were to investigate the contributions of the neuronal nitric oxide synthase (nNOS) gene, ontogeny, and gender on the rewarding and sensitizing effects of cocaine. I found that nNOS significantly influences the rewarding aspects of cocaine in adolescent mice and adult male mice (i.e., major deficits in several phases of cocaine conditioned place preference (CPP) were detected in nNOS knockout (KO) adolescent mice and nNOS KO adult male mice). However, the contribution of nNOS was sex-dependent as CPP phases were normal in KO adult females. In contrast to CPP, I found a major ontogeny-dependent contribution of nNOS to the sensitizing effects of cocaine. Namely, while nNOS is essential for the development of behavioral sensitization in adult males, this type of behavioral plasticity develops independently of nNOS during adolescence. The contribution of nNOS was once again sex-dependent as behavioral sensitization was normal in adult KO females. Together, this line of investigation has revealed that the NO-signaling pathway has a) a sex-dependent role in the neuroplasticity underlying cocaine CPP and b) a sex-dependent and ontogeny-dependent influence on cocaine-induced behavioral sensitization. Stereological and western blot analysis revealed that a sensitizing regimen of cocaine resulted in an increase in nNOS and tyrosine hydroxylase (TH) immunoreactivity in the dorsal striatum (dST) of adult, but not adolescent, wild-type (WT) male mice. In the absence of nNOS, dopaminergic neurons in the ventral tegmental area (VTA) were severely reduced and cocaine caused a downregulation of dST TH suggesting that nitrergic levels modulate TH. Thus, the finding that nNOS is essential for the development of sensitization in adulthood, but not adolescence, together with the fact that cocaine upregulated nNOS and TH in the dST in adult, but not adolescent mice, strongly suggest that the nitrergic system underlies behavioral sensitization through modulation of the dopaminergic system in adulthood. These findings suggest different approaches in the clinical treatment of drug craving and drug-seeking behavior in adolescent and adult patients.

Binge alcohol consumption is a rising concern in the United States, especially among adolescents as during this developmental period alcohol use is usually initiated and has been shown to cause detrimental effects on brain structure and function. These findings have been established through the use of binge models in animals, where animals are repeatedly administered high doses of ethanol typically over a period of three or four days. While such work has examined the effects of a four-day and repeated three-day binge, there has been almost no work conducted aimed at investigating the long-term behavioral and neurochemical and/or functional consequences of repeated binge pattern administration during adolescence relative to adulthood on later ethanol-induced behavior and neurochemistry in adulthood. The present set of experiments aimed to examine the dose-response and age-related differences induced by repeated binge pattern ethanol administration during adolescence or adulthood on voluntary ethanol consumption (Aim 1), changes in ethanol metabolism following ethanol pretreatment (Aim 2) and mesolimbic dopamine functionality (Aim 3) in adulthood. In both experiments, adolescent and adult male rats were intragastrically administered ethanol (0.5, 1.0 or 2.0 g/kg/ig) or isovolumetric water on postnatal days (PND) 28-31, PND 35-38 and PND 42-45 for adolescent rats and PND 60-64, PND 67-70 and PND 74-77 for adult rats. In both experiments all rats underwent fourteen days of abstinence (PND 46-59 or PND 78-91, respectively). Subsequently, in Experiment 1, all rats underwent voluntary ethanol consumption procedures, in which animals were exposed to 10% ethanol combined with decreasing saccharin concentrations across days from PND 60-82 for adolescent-exposed rats and PND 92-114 for adult-exposed rats. Finally, on PND 83 and PND 115, respectively, all animals were challenged with 2.0 g/kg ethanol and trunk blood samples were collected at 60 and 240 minutes post-injection. Results indicate there was a significant increase in voluntary ethanol intake in adolescent ethanol-exposed rats pretreated with 2.0 g/kg relative to their adult ethanol-pretreated counterparts. Faster ethanol metabolism was observed in adolescent rats pretreated with 2.0 g/kg during adolescence relative to adolescent-exposed rats pretreated with 0.5 g/kg and adults pretreated with 2.0 g/kg. For Experiment 2, all rats underwent surgery (PND 60 for adolescent-exposed and PND 92 for adult-exposed rats). From PND 61-64 for adolescent-exposed and PND 93-96 for adult exposed rats, all animals underwent recovery from surgery. Finally, all rats underwent in vivo microdialysis on PND 65 for adolescent-exposed and PND 97 for adult-exposed rats, with K+ (100 mM) infused into the ventral tegmental area and accumbal dopamine overflow assessed in the nucleus accumbens septi. The results from Experiment 2 indicate lasting changes in mesolimbic dopamine functionality with a trend for decreased potassium-stimulated dopamine overflow in the nucleus accumbens septi in adolescent-ethanol pretreated rats and a trend for increased potassium-stimulated dopamine overflow in adult ethanol-pretreated rats. The results from the present set of experiments show the dose-dependent impact of binge-pattern ethanol exposure during adolescence on subsequent ethanol consumption and ethanol metabolism in adulthood. These findings indeed determine adolescence as a period of vulnerability to the long-term changes in ethanol consumption relative to similarly-exposed adult male rats. Importantly, the results of Experiment 2 indicate an alteration in the functionality of the mesolimbic pathway in adulthood following adolescent binge pattern ethanol exposure, which demonstrates a long-term depression in mesolimbic dopamine functionality following adolescent binge pattern ethanol exposure.

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Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.