Controlling the persistence of drug-evoked plasticity in the mesolimbic dopamine system

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Thesis

Reference

Controlling the persistence of drug-evoked plasticity in the mesolimbic dopamine system

MAMELI, Manuel

Abstract

La consommation répétée et prolongée de drogues est à l'origine de l'addiction, une maladie affectant le fonctionnement du cerveau. L'addiction représente un fardeau économique et social pour la société actuelle, se chiffrant en un coût annuel de plusieurs dizaines de milliards d'euros, en Europe. Cette maladie chronique est caractérisée par des états de tolérance, de manque et d'usage compulsif de drogues, malgré des conséquences négatives certaines, ainsi que d'un risque de rechute à long terme. Afin de développer des outils thérapeutiques performants, il est nécessaire de comprendre les processus physiologiques et pathologiques sous-jacents à l'addiction et à sa persistance. Une hypothèse actuelle propose que l'addiction affecte plus particulièrement les mécanismes d'apprentissage et de mémorisation, lors desquels la prise répétée de drogues provoque des changements à long terme dans le cerveau, avec pour résultat une augmentation de la réponse aux drogues, ou à des stimuli directement rattachés aux drogues. Parmi ces changements provoqués par les drogues, la modification de [...]

MAMELI, Manuel. Controlling the persistence of drug-evoked plasticity in the

mesolimbic dopamine system. Thèse de doctorat : Univ. Genève et Lausanne, 2009, no.

Neur. 34

URN : urn:nbn:ch:unige-21384

DOI : 10.13097/archive-ouverte/unige:2138

Available at:

http://archive-ouverte.unige.ch/unige:2138

Disclaimer: layout of this document may differ from the published version.

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FACULTÉ DES SCIENCES

DOCTORAT EN NEUROSCIENCES des Universités de Genève

et de Lausanne

UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Professeur Christian Lüscher, directeur de thèse

TITRE DE LA THESE

CONTROLLING THE PERSISTENCE OF DRUG-EVOKED PLASTICITY IN THE MESOLIMBIC DOPAMINE SYSTEM.

THESE Présentée à la Faculté des Sciences de l’Université de Genève

pour obtenir le grade de Docteur en Neurosciences

par

Manuel MAMELI de Cagliari (ITALIE)

Thèse N° 34 Genève

Atelier d'impression ReproMail 2009

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RESUMÉ EN FRANÇAIS

La consommation répétée et prolongée de drogues est à l’origine de l’addiction, une maladie affectant le fonctionnement du cerveau. L’addiction représente un fardeau économique et social pour la société actuelle, se chiffrant en un coût annuel de plusieurs dizaines de milliards d’euros, en Europe. Cette maladie chronique est caractérisée par des états de tolérance, de manque et d’usage compulsif de drogues, malgré des conséquences négatives certaines, ainsi que d’un risque de rechute à long terme. Afin de développer des outils thérapeutiques performants, il est nécessaire de comprendre les processus physiologiques et pathologiques sous-jacents à l’addiction et à sa persistance.

Une hypothèse actuelle propose que l’addiction affecte plus particulièrement les mécanismes d’apprentissage et de mémorisation, lors desquels la prise répétée de drogues provoque des changements à long terme dans le cerveau, avec pour résultat une augmentation de la réponse aux drogues, ou à des stimuli directement rattachés aux drogues. Parmi ces changements provoqués par les drogues, la modification de l’activité synaptique semble être un mécanisme clef du stockage de mémoire à long terme, caractéristique de l’addiction.

De nombreuses preuves indiquent que le système dopaminergique, projetant de l’aire ventrale tegmentale (VTA), dans le mésencéphale, jusqu’au noyau accumbens, est fortement impliqué dans l’assimilation de la drogue à une récompense. Les populations neuronales situées dans ces régions, en particulier les neurones dopaminergiques de la VTA et les neurones moyennement épineux du noyau accumbens, reçoivent des afférences excitatrices de nombreuses régions du cerveau. Sachant qu’il a été plusieurs fois suggéré que la plasticité des synapses excitatrices est le corrélat cellulaire des phénomènes d’apprentissage et de mémorisation, il se pourrait que cette plasticité, dans le système de récompense, représente les fondements moléculaires des mécanismes d’apprentissage impliqués dans les comportements liés aux drogues.

Un corollaire unifiant ces théories soutient que l’addiction pourrait être éliminée en inversant les modifications plastiques infligées au cerveau par la drogue.

Notre laboratoire a auparavant démontré que l’activation des récepteurs métabotropiques au glutamate (mGluR) inverse le renforcement précoce de la transmission synaptique excitatrice sur les neurones dopaminergiques de la VTA, induit

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par une seule exposition à la cocaïne. Lors de mon travail de thèse, j’ai voulu explorer en détails les mécanismes cellulaires à l’origine de l’inversion de la plasticité due à la cocaïne par les mGluR. J’ai pu mettre en évidence que la sous-unité GluR2 des récepteurs AMPA joue un rôle crucial dans ce phénomène. De plus, il s’est avéré que le rôle joué par les mGluR in vitro est tout aussi critique in vivo. Cette capacité des mGluR a permis de révéler une autre caractéristique de la plasticité due à la cocaïne, à savoir que si les altérations au niveau de la VTA sont maintenues assez longtemps, elles entrainent alors une adaptation synaptique dans les régions ciblées, comme le noyau accumbens.

Ces résultats contribuent ensemble à une meilleure compréhension des mécanismes cellulaires apparaissant dès les premières prises de drogues, et pouvant finalement mener à leur usage compulsif.

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Abbreviations:

6-OHDA, 6-hydroxydopamine

ABP, AMPA receptor binding protein

AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid A/N, AMPA versus NMDA-mediated currents ratio

BDNF, brain-derived neurotrophic factor BNST, bed nucleus of stria terminalis Ca²⁺, calcium

cAMP, cyclic adenosine monophosphate CB, cannabinoid receptor

CF, climbing fiber

CPP, conditioned place preference D, dopamine receptor

DA, dopamine DAG, diacylglycerol DD, dopamine-deficient

DHPG, d-hydroxyphenylglycine eCB, endocannabinoid

ER, endoplasmic reticulum

EPSC, excitatory postsynaptic current ERK, extracellular signal regulated kinase FMRP, fragile X mental retardation protein

FRAP, fluorescence recovery after photobleaching G, glycine

GABA, g -hydroxybutyrate GHB, g-hydroxybutyric acid GFP, green fluorescence protein

GIRK, G-protein-coupled inwardly rectifying potassium channel GluR, AMPA glutamate receptor subunit

GPCR, G-protein-coupled receptors

GRIP, glutamate receptor interacting protein Hr(s), hour/s

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I-LTP, LTP at inhibitory synapses IP3, inositol triphosphate

IPSC, inhibitory postsynaptic receptor KO, knock out

LTD, long-term depression LTP, long-term potentiation

MAGUK, membrane-associated guanylate kinase MAPK, mitogen-activated protein kinase

Mg²⁺, magnesium

mGluR, metabotropic glutamate receptor mRNA, messenger RNA

MSN, medium spiny neurons

mTOR, mammalian target of rapamycin NAcc, nucleus accumbens

NMDA, N-methyl-D-aspartate NO, nitric oxide

NR, NMDA receptor subunit

NSF, N-Ethylmaleimide-sensitive fusion protein NTD, N terminal domain

OXR, orexin receptor PC, purkinje cell PF, parallel fiber PFC, prefrontal cortex

PICK1, protein interacting C kinase PKA/C, protein kinase A/C

PLC, phospholipase C PSD, post-synaptic density Q, glutamine

R, arginine

RNA, ribonucleic acid SEP, superecliptic pHluorin SPT, single particle tracking

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TARP, transmembrane AMPA receptor regulatory protein THC, tetrahydrocannabinol

V-Glut, vesicular glutamate transporter VGCC, voltage-gated calcium channel VTA, ventral tegmental area

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Summary

Drug addiction is a chronic and relapsing brain disease that represents a prominent social and economical burden to our societies. It is an inevitable consequence of a prolonged and repetitive drug taking. This chronic brain disorder is characterized by tolerance, withdrawal, compulsive use that escapes control, even when serious negative consequences ensue, and by a long-lasting risk of relapse. Drug addiction is a prominent social and economical problem that costs billions of Euros to our society. In order to develop more effective therapeutic interventions, it is important to understand the pathophysiologic processes that underlie addiction and its persistence.

It has been hypothesized that addiction is a disease of learning and memory in which repeated administration of drugs produces long-lasting changes in the brain resulting in a stronger association of the drug with drug-related stimuli. Among these drug-induced changes, modifications in the strength of synaptic activity appear to play an important role in the cellular mechanism undergoing storing the long lasting memories that are typical of addiction.

There is strong evidence that the dopaminergic system that projects from the ventral tegmental area of the midbrain to the nucleus accumbens is a major substrate for the rewarding effects of drugs. Neuronal populations located in these areas, particularly the dopamine neurons in the ventral tegmental area and the medium spiny neurons in the nucleus accumbens receive excitatory inputs from several brain regions. It has been postulated that plasticity at excitatory synapses might be a cellular correlate of memory and learning. Therefore plasticity at excitatory synapses in the reward system may represent the molecular readout of learning mechanisms involved in drug-associated behaviors.

An obvious corollary of these theories is that drug addiction could be eliminated by reversing the neuroplastic changes produced by drugs in the brain.

In our laboratory we previously reported that activation of postsynaptic mGlu receptors reverses the early strengthening at excitatory synapses onto dopamine neurons in the VTA caused by a single exposure to cocaine. During my thesis work, I tried to explore in depth the cellular mechanism underlying the reversal of cocaine-evoked plasticity after activation of mGlu receptors. Interestingly, I could show that the GluR2

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subunit of AMPA receptors had a crucial role during this form of plasticity. In addition the role of mGlu receptors was not important only in vitro, but mGlu receptors function plays a critical role in reversing early forms of drug-evoked plasticity in vivo. This role of mGlu receptors unrevealed another important trademark of cocaine-evoked synaptic plasticity: alterations at the level of the ventral tegmental area, if enough persistent, gate synaptic adaptation in targeted areas, such as the nucleus accumbens. Altogether these results contributed to a better understanding of the cellular mechanisms behind early-drug exposure that may be the first step leading to compulsive drug use.

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1. Drug addiction

The 2007 report of the European Monitoring Center for Drugs and Drug Addiction (EMCDDA) indicates that about 12 million European citizens have used at least once in their life the highly addictive drugs cocaine or amphetamine. The European Brain Council has reported that in 2005 nine million people suffer of compulsive behavior (nicotine addiction not included) in Europe, generating direct and indirect totalling 57 billions Euros per year (European Brain Council, Cost and Utilities of Brain disorders in Europe, report 2005). In 1969, the World Health Organization defined addiction as compulsive drug use despite negative consequences. Since then the definition has evolved. Today the emphasis is put on the narrowing of the goals of an addicted person who focuses exclusively on obtaining, using and recovering from the drug use, despite failures in daily life tasks, medical illness, risk of incarceration, and other problems (Hyman, 2005). An important characteristic of addiction is its persistence (McLellan et al., 2000). Although some individuals can stop compulsive use of drugs on their own, a large part of them, more vulnerable due to genetic and non-genetic factors, continue to take their drug for long time, suggesting that addiction is a chronic and relapsing disease (Kendler et al., 2003). This is one of the reasons why treating addiction is such a difficult task. Even after prolonged drug-free periods, well after the last withdrawal symptom has worn off, the risk of relapse remains high and can be easily triggered by the exposure of the subject to drug-associated cues such as the environment, other drugs (i.e. alcohol or tobacco). In general, despite multiple episodes of treatment, and despite the risk of significant life problems, relapse to addictive drugs is the rule. However, this pathological behavior appears only in a small proportion (15 to 17%) of those using drugs, which highlight the still unresolved question of individual vulnerability (Anthony et al., 1994). An effective therapeutic intervention is needed, but in order to obtain it, it is important to understand the pathophysiological processes that underlie addiction and its persistence (Hyman et al., 2006).

Addictive drugs are both rewarding (i.e. a positive experience) and reinforcing (i.e.

drug-associated behaviors tend to be repeated) (Hyman et al., 2006). The use of addictive substances induces molecular changes in the brain that promote the continued drug

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taking, a situation that the individual find increasingly difficult to control. Repeated drug use may produce serious unwanted effects, including tolerance to some drug effect, sensitization to others and dependence which sets the stage for withdrawal syndrome when the drug use stops.

Tolerance is defined as a decrease of the effects of a drug despite at constant dose, or the need for increased dosage to obtain the very same stable effect. Depending of the pattern of use, some drugs, particularly psychostimulants such as amphetamines and cocaine, can also produce sensitization (increase) of drug response (Kalivas and Stewart, 1991).

Addictive drugs can also induced dependence, which nowadays is used distinctly from addiction. Dependence refers to a state that is defined by the appearance of cognitive, emotional or physical withdrawal symptoms when the substance is suddenly discontinued. It corresponds to an adapted state of circuits or organ system that occurs in response to excessive drug stimulation and it occurs in 100% of users. Dependence (in the past also referred to as “physical dependence”) involves adaptations that occur in brain circuits controlling body function such as heart rate or blood pressure. Ethanol, barbiturates and opiates do produce a very strong dependence while highly addictive drugs such as cocaine and amphetamine induce dependence to a much lesser degree. That dependence and addiction can be dissociated is also apparent when one considers drugs such as beta-adrenergic agonist which produce tolerance, dependence and withdrawal but not compulsive use (Hyman et al., 2006). In contrast to the physical dependence,

“psychological dependence” has now been replaced by addiction. The importance of a clear distinction of dependence and addition is also underlined by the demonstration that they are mediated by different mechanisms acting at different level of the brain.

The recent notion of addiction is very often difficult to perceive and most of the people have problem to discern addiction from dependence.

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2. Hijacking of neural systems

The survival of individuals and species requires that organisms find and obtain needed resources (food and shelter), and opportunities for mating despite costs and risks.

Such survival-relevant natural goals act as rewards. Learning processes will therefore strength a signal that coding for the anticipation of their consumption will produce an expected outcome (it makes feel better). Goal-directed behaviors tend to persist strongly to a conclusion, increase over time and are defined as positive reinforcers (Hyman, 2005).

Internal motivational states such as hunger, thirst and sexual arousal increase the incentive value of goal-related cues and increase the pleasure of consumption (food taste better when you are hungry) (Kelley and Berridge, 2002). External cues reminiscent of the reward (incentive stimuli), such as the sight and the odor of food or the odor of an estrous female, can trigger or further strength motivational states, increasing the rate of success even for difficult tasks such as foraging or hunting. Therefore at one point the behavior involved in obtaining the reward becomes overlearned and systematic, rendering smooth and efficient even a difficult task (Berke, 2003).

Addictive substances promote behaviors that are reminiscent of those induced by natural rewards. As for natural reward, drug taking is rapidly learned as anticipation of positive outcomes, however, when individuals fall deeper into a pathological form of drug use (addiction), drug seeking becomes the only available goal to pursue despite negative consequences (compulsion). As already mentioned, repetitive drug taking produces homeostatic adaptations inducing dependence, which in the case of alcohol and opioids can lead to distressing withdrawal syndromes with sudden cessation of the drug.

However, dependence and withdrawal alone do not explain addiction, or the characteristic persistence of relapse risk long after detoxification (O'Brien et al., 1998;

Berke and Hyman, 2000). Particularly, relapse after detoxification is often precipitated by cues, such as people, places, or body feelings associated with prior drug use (O'Brien et al., 1998) or by stress (Marinelli and Piazza, 2002). Cues, in general activate drug

“wanting” (Robinson and Berridge 2003), drug seeking (Berke and Hyman, 2000) eventually leading to drug consumption. The drug seeking becomes as for natural rewards an overlearned and automatic action that hijacks the reward system in order to obtain the drug. However, drugs tend to be always reinforcing, strengthening

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continuously these maladapted behaviors. Indeed, the cue-dependent activation of automatized drug seeking has been hypothesized to play a major role in relapse (Robbins and Everitt, 1999; Berke and Hyman, 2000).

2.1. Genetic factors

Importantly, Goldman and colleagues (Goldman et al., 2005) demonstrated that the genetic background is primordial for drugs of abuse predisposition. Statistical analysis showed a similar risk of addiction shared by two persons presenting a similar genetic background, like monozygotic twins, in comparison with people genetically different (dizygotic twins). A good correlation between the heritability of addictive disorders and the relative risk of addiction associated with addictive drugs was also observed.

Therefore, more a substance is addictive, the more its addiction is heritable. However, the genes that can be implicated in vulnerability to drug addiction are still unknown (Goldman et al., 2005).

On the other hand, interindividual variations at the genetic level cannot alone explain addiction. Addiction has also been described as a “brain disease” where normal learning and memory processes have been altered (Hyman, 2005) or, more precisely, as

“a mental disease” that affects a restricted pool of brain regions that constitute the brain reward system (Mohn et al., 2004). Despite a wide panel of addictive drugs that evoke a multitude of effects, they all strongly activate the reward system. Contrary to natural rewards, drugs exposure dramatically modifies the functionality of this system and lead, in certain cases, to compulsion and addiction.

2.2. Behavioral paradigms

It is important to know that to measure the reinforcing effects of a reward (natural reward or addictive substance), several animal models are used in order to study selected parts of human syndromes (Koob, 2006). Conditioned place preference (CPP) and self- administration paradigms are the main tools generally employed for this purpose (Bozarth et al., 1980). Briefly, in the CPP test, animals experience two distinct neutral environments that are subsequently paired spatially and temporally with distinct drug states. The animal is later given an opportunity to choose to enter and explore either environment, and the time spent in either environment is considered an index of the

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reinforcing value of the drug. The choice made by the animal to spend more time in an environment is assumed to be an expression of the positive reinforcing experience within that environment. CPP gives information regarding an early stages of drug seeking behavior, however, we can also study late stages of drug seeking behaviors by looking at the extinction of these behavior and how a single re-exposure to the drug reinstate the behavior, a measure of relapse (Bozarth et al., 1980). These techniques allowed researchers to identify the neuronal networks coding for reward-associated processes.

Self-administration paradigm allows to actively measure the reinforcing strength of a reward. The advantage of this technique is to let animals press by themselves a lever to auto-administrate a substance (intravenously for example), implicating a strong motivational component. A direct estimation of the rewarding properties of the substance can be obtained by monitoring the amount of presses. This type of animal model has high prediction of abuse potential and has been suggested to be used as part of a battery for the preclinical assessment of the abuse liability of new agents (Bozarth et al., 1980).

3. The reward system

Investigations using diverse techniques including in vivo neurochemical measurements, microinjections of agonist and antagonists into specific brain regions, and local lesions, have converged on the conclusion that natural rewards and addictive drugs influence behaviors as a result of their ability to increase synaptic dopamine (DA) in the nucleus accumbens (NAcc), the major component of the ventral striatum (Koob and Bloom, 1988; Wise, 1998). DA is released by the DA neurons located in the ventral tegmental area (VTA), a region of the midbrain.

3.1. The mesolimbic system

The reward-activated system or mesolimbic DA system was identified in 1971 (Ungerstedt, 1971) and named in this manner because projection neurons are mainly releasing the neurotransmitter DA (Figure 1). These projecting neurons originate in an area called the VTA. Later studies have identified several DA projection patterns, mainly to the NAcc, prefrontal cortex (PFC), the hippocampus, the amygdala and the olfactory tubercule (Ungerstedt, 1971; Lindvall and Bjorklund, 1974; Fallon and Moore, 1978;

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Swanson, 1982). The major cell type in the VTA is the DA neuron, which receives excitatory inputs from several regions including the PFC, laterodorsal tegmental nuclei, lateral hypothalamus, and bed nucleus of stria terminalis (BNST) (Georges and Aston- Jones, 2001; Georges and Aston-Jones, 2002; Omelchenko and Sesack, 2007). DA neurons also receive GABA inhibitory inputs from local interneurons (source of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA)), projections from the NAcc and the ventral pallidum (VP) (Johnson and North, 1992; Ikemoto and Wise, 2004) (Figure 1).

Several evidences have demonstrated that among these different fiber bundles, the one projecting to the NAcc is the main inputs involved in the reward processes. Indeed, DA receptors antagonist (haloperidol) systemically injected in rodents blocks rewarding effects of food, amphetamine and cocaine, suggesting the implication of DA in these phenomena (Spyraki et al., 1982a; Spyraki et al., 1982b; Spyraki et al., 1987).

Furthermore, death of DA neurons induced by injections of the neurotoxin 6-OHDA (6- hydroxydopamine) suppresses rewarding effects of cocaine or amphetamine (Roberts et al., 1980; Spyraki et al., 1982a). Thus, this evidence demonstrates that the DA connection from VTA to NAcc is crucial for reward effects mediation. In addition, direct administration of DA receptors agonists in the NAcc elicits reinforcing effects revealed by induction of CPP (White et al., 1991). Finally, rewarding action of morphine can also be observed by CPP after direct injection not in the NAcc but in VTA (Phillips and LePiane, 1980), suggesting the existence of distinct classes of drugs with different way of action (Table 1).

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Figure 1. Principal afferences and efferences projections of the VTA. Direct connections to and from the VTA are shown in black or color. Other connections are shown in gray. The color scale indicates an estimation of projecting neurons that are cytochemically identified as DA. On the other hand, DA neurons receive GABA inhibitory inputs mainly from the VP ad the nucleus accumbens and excitatory inputs from the prefrontal cortex and the PPTg. Abbreviations: LDT, laterodorsal tegmental nucleus; PPTg, pedunculopontine tegmental nucleus; LH, lateral hypothalamus; VP, ventral pallidum; SC, superior colliculus, Modified from Fields, Hjelmstad et al. 2007.

Recent advance in the description of the anatomical layout of the midbrain DA system suggest that the VTA and the NAcc are part of a spiraling network, which is hijacked by drug use. DA neurons from the medial part of the VTA project to the medial part of the NAcc, the shell. Inhibitory medium spiny neurons (MSN) located in the shell, backproject to the lateral VTA via the intermediate action of an interneuron. Lateral DA neurons send their axon to the more lateral core of the NAcc. MSNs from the core project to the substantia nigra compacta. They send their axons to the dorsal striatum (Haber et al., 2000; Ikemoto, 2007). The recruitment of the dorsal part of the DA system has been recently implicated in cocaine-seeking habits important for addiction (Belin and Everitt, 2008). In this in vivo study, the NAcc core was surgically lesioned on one side and DA

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receptors were instead blocked in the controlateral striatum. In this condition, cocaine seeking was decreased, as if the DA antagonist were injected bilaterally in the striatum.

These data, suggest that dorsal parts of the DA system are implicated in cocaine-seeking habits. Importantly, the connection between areas implicated in the reward system provides the idea of a spiraling network that suggests a potential role for each single brain area of the reward system at a certain time point (Belin and Everitt, 2008). However, many questions are still unresolved and fundamental issues such as the early stages of this spiraling network are still unclear.

3.2. Experimental results after DA depletion

A general idea is therefore that DA is necessary in mediating the rewarding effect of a drug. Interestingly, another line of thinking claims that DA is not fully implicated in reward and that other DA-independent mechanisms can be involved. Indeed, in 2005, it has been demonstrated that CPP to morphine was still present in DA deficient (DD) mice (Hnasko et al., 2005). These animals generated by inactivating the tyrosine hydroxylase gene, coding for an essential DA synthesis enzyme, are unable to synthesize DA while the DA circuitry remained intact (Zhou and Palmiter, 1995). A regular CPP for morphine

Protocol Results Behavioral test

Systemic injection of DA antagonists

Abolishment of reinforcing

effects by food, cocaine CPP Spyraki et al., 1982a, 1982b, 1987

Systemic injections of

morphine Reinforcement CPP Phillips and LaPiane 1980

Systemic injections of cocaine Reinforcement CPP Spyraki et al., 1982

Injection of DA antagonsists in NAcc

effects of cocaine Self-administration Samson et al., 1993 Injection of DA agonists in

NAcc Reinforcement CPP White et al., 1991

Injection of morphine in the

VTA Reinforcement CPP Phillips and LaPiane 1980

Disruption DA neurons by neurotoxins (6-OHDA)

effects of amphetamine CPP Spyraki et al., 1982; Roberts et al., 1980

Table 1. Behavioral experiments demonstrating the important role of DA in mediating rewarding effects.

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in these animals suggests that DA is not implicated, at least for the induction of this behavioral response. In addition, a more recent paper showed a normal CPP to cocaine in DD mice (Hnasko et al., 2007). Such evidence tends to prove, again, that DA release is not essential for this behavior. Nevertheless, in the same paper, the authors clearly showed that serotonin mediates cocaine CPP in DD mice but not in wild-type animals. In other words, adaptive or compensatory changes in DD mice, probably during development, allow another monoamine, serotonin, to replace DA and assuming its functions in the reward effects mediation. Moreover, this CPP for cocaine was blocked by the DA type 2 receptor agonist quinpirole, showing that DA neurons activity is still required for CPP induction even if these cells are unable to release DA. Regarding these arguments, we can easily hypothesize that DA-independent CPP evoked by morphine in DD mice could also be explained by similar developmental changes occurring in absence of DA. Taken together, these observations bring evidences for possible DA compensation instead of actual alternative mechanisms existing in physiological conditions.

3.3. Molecular targets of addictive drugs

As mentioned above, in the VTA the two neuronal populations are DA neurons and GABA neurons (Swanson, 1982; Margolis et al., 2006b). The DA neurons are projecting neurons, which are under the control of inhibitory GABA interneurons (Johnson and North, 1992); (Margolis et al., 2006a). Recently, a third population has been identified based on their immunoreactivity for V-Glut, a marker for glutamate, has been identified in the VTA (Yamaguchi et al., 2007). However, the function of these neurons remains elusive. Addictive drugs act on DA and/or GABA neurons of the VTA (Figure 2) but on different molecular targets (Luscher and Ungless, 2006).

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Morphine, cannabis and g-hydroxybutyric acid (GHB) can

be arranged in the same class of addictive drugs (Class I) due to their ability to increase the DA release in an indirect manner after removing the inhibitory control of GABA cells (Johnson and North, 1992; Cruz et al., 2004). Indeed, acting on their respective metabotropic Gi/o-protein coupled receptor (GPCR), they suppress the GABA cells activity resulting in a DA neurons disinhibition. In a similar way, addictive drugs belonging to the second class (Class II) target directly DA or GABA neurons, eventually increasing DA release in the NAcc. Drugs such as benzodiazepines, nicotine and ethanol are mediating their effects by acting on ionotropic receptors or ions channels located in GABA and DA neurons (Maskos et al., 2005). Finally, a third category of addictive drugs (Class III), composed of cocaine, amphetamine and ecstasy, directly acts on DA release at the level of the NAcc. Instead of increasing the cellular activity of DA neurons to boost the DA release, these drugs target directly the DA transporters on axons terminal (Figure 5). Cocaine blocks the reuptake of DA by disrupting the function of its transporters increasing the concentrations of DA targeted regions including NAcc and VTA itself (Chen et al., 2006). With amphetamine and ecstasy, the DA transporters are not blocked but reversed, a phenomenon that contributes to a similar DA accumulation in the NAcc (Seiden et al., 1993). This classification is helpful to understand how a large diversity of drugs of abuse can induce similar addictive effects (Figure 2). The specific knowledge on each addictive drug molecular target allows the design of specific and innovative therapy, which is one of the main aims of the addiction research. This strategy would prevent actions directly against DA release, since it is an important signal implicated in many molecular processes.

Figure 2. The molecular targets of main addictive drugs on DA neurons. G, Gi/o-coupled receptors; i, ionotropic receptors/ion channels; T, monoamine transporters. Modified from Luscher and Ungless, 2006.

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4. The Dopamine hypothesis: a cellular explanation for addiction

As mentioned above, a large body of work established that the rewarding properties of addictive drugs (as well as natural rewards) are due to their ability to increase DA at synapses that VTA DA neurons make with MSN of the shell region of the NAcc in the ventral striatum (Koob and Bloom, 1988; Pontieri et al., 1995). As previously described, addictive drugs are classified in multiple families that can stimulate or block differential targets, inducing several unrelated actions outside the reward system but that ultimately lead to DA release from the VTA to target regions (Luscher and Ungless, 2006).

Recent studies at the behavioral, physiological, computational and cellular level has begun to elucidate the mechanisms by which DA elevates the incentive value for drug taking to the point that control over the drug is lost (compulsion). It is clear that the most obvious difference between natural rewards and addictive drugs is that the latter have no intrinsic ability to serve as a biological need. However, having the same ultimate result – the increase in DA levels in target regions – addictive drugs mimic the effects of natural rewards and can thus shape behaviors. Indeed, it has been postulated that addictive drugs have competitive advantages over natural stimuli since they can finally produce much higher levels of DA release in a longer timescale (Schultz et al., 1997; Kelley and Berridge, 2002).

A still debated question is what DA encodes for in a physiological condition.

Initially, DA function was mainly associated with hedonic signals (pleasure), however it has been demonstrated that animals continued to prefer rewards (such a sucrose) even in condition of DA depletion, ruling out this role of DA (Berridge and Robinson, 1998;

Cannon and Palmiter, 2003). Indeed, Berridge and Robinson (1998) described that an animal can still “like” (hedonic signal) something in absence of DA, however the animal cannot use this information to motivate a behavior to obtain the reward (Berridge and Robinson, 1998). Massive injection of 6-OHDA (i.e. disruption of DA neurons) does not alter the ability of rats to make hedonic evaluations but it does prevent the incentive value of food, water and other rewards. In conclusion the authors suggest that three processes

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are coding for a normal reward: i) the hedonic activation, ii) the associative learning of the relationship between an event and its hedonic consequence and iii) the attribution of incentive salience to the event or their representation. Finally, DA projections and DA release in target regions code only for the attribution of incentive salience to motivational stimuli and their representation. Rats that lack DA still “like” the reward and still know the reward they “like”, however they fail to “want” the reward they “like” (Berridge and Robinson, 1998).

4.1. Anticipation of reward

In other studies, Schultz and colleagues made recordings from DA neurons while monkeys anticipated or consumed a reward, sweet juice in this case. Monkeys were trained to expect the juice after a fixed time following a visual or an auditory cue (Schultz et al., 1993; Hollerman et al., 1998). The DA neuron firing pattern was changing as the monkey learned the circumstances under which the reward occurred. DA neurons in awake monkeys present a tonic firing and superimposed brief phasic bursts of spike activity; the burst timing is determined by the prior experience of the animal with a reward. Indeed, an unexpected reward (i.e. delivery of juice) produces a transient increase in the firing rate of the neuron (Figure 3 top panel), but as the monkey learns that a cue (visual or auditory) predicts the reward, the timing of this phasic activity changes (Figure 3, middle panel). DA neurons now exhibit the phasic burst not in response to the delivery of juice, but earlier when the cue is presented. However if the cue is presented, but the reward is not given, there is a pause in the tonic DA firing at the time the reward was expected (Figure 3 bottom panel). When the reward comes in an unexpected moment or exceeds expectation, a phasic burst in firing is observed. The hypothesis beyond these studies is that phasic bursts and pauses encode for a prediction error signal. The bursts are the read out of the positive reward prediction, which results in better than expected; on the other hand the shut down of the firing is a negative prediction error, which results in an experience worse than expected (Montague et al., 2004).

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In the context of addictive drugs there are so far no data related to the control of the drugs over the prediction error signal. However, we could predict additional advantages of addictive drugs versus natural rewards; drugs have the ability to increase DA levels for a longer timescale and it would not decay over time. Thus, the brain would, in the case of the drug, repeatedly get the signal that drugs are always better than expected, strengthening this memory. Given the likelihood that addictive drugs exceed natural stimuli in reliability, quantity and persistence of increased DA levels, a consequence would be a profound overlearning of the cues that predict for the drug. However, much remains to be explained; the effects of the drug or the reward on DA kinetics has not yet being correlated and reward-related behaviors are only beginning to be studied (Hyman, 2005).

5. Addictive drugs hijack synaptic plasticity

Numerous evidences accumulated in the last decade demonstrated that addictive drugs might interfere with synaptic plasticity mechanism in brain circuits involved in reinforcement and reward processing. A very recent hypothesis is that addiction represents a pathological form of learning and memory (Hyman and Malenka, 2001).

Given that the mesolimbic DA system is a critical substrate for the neural adaptations that

Figure 3. Changes in the firing of DA neurons code for an error in the prediction of rewads. In vivo recordings of mesolimbic DA neurons in a non-anestetized monkey receiving or not (R or No R) a drop of juice with or without anticipation (CS or No CS).

Modified from Schultz et al., 1997.

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underlie addiction, interfering with synaptic functions in these areas can be a cellular mechanism correlated with behavioral modifications associated with compulsion.

5.1. Synaptic plasticity

A property of all synapses is their ability to undergo activity-dependent changes in their synaptic strength, defined as synaptic plasticity. The discovery in 1973 of the long- term potentiation (LTP) at excitatory synapses of the hippocampus (Bliss and Lomo, 1973) is the starting point of an exciting journey into the cellular basis and behavioral correlates of memory processes. It is clear that LTP and its counterpart long-term depression (LTD) are a basic molecular events at most excitatory synapses in the brain that are not only involved in learning and memory but also in synaptogenesis and synaptic stabilization or developmental fine-tuning of neural circuits (Malenka and Bear, 2004).

Many form of synaptic plasticity (LTP and LTD) have been described in the central nervous system. All of them are characterized by specific mechanisms of induction – necessary for the plasticity to be initiated (i.e. release of neurotransmitter or activation of specific receptors) – and expression – necessary for the plasticity to be maintained (i.e.

trafficking of receptors). Among the different forms of synaptic plasticity identified in literature so far (Kauer and Malenka, 2007), these are the main examples:

- N-methyl-D-aspartate (NMDA) receptors-dependent LTP. It requires a strong activation of synaptic NMDA receptors, after a depolarization that relieves the magnesium (Mg²⁺) block from the receptors itself. This allows a high calcium (Ca²⁺) entry in the postsynaptic terminal, which triggers an intracellular cascade of events, eventually leading to the trafficking of a-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptors at the synapse, strengthening the glutamate signal (Bliss and Lomo, 1973).

- Presynaptic LTP. It requires an activity-dependent Ca²⁺ rise in the presynaptic terminal that in turn activates adenyl cyclase and protein kinase A (Nicoll and Schmitz, 2005). This leads to a persistent increase in glutamate release from the presynaptic terminal.

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- NMDA receptors-dependent LTD. It requires a weak activation of NMDA receptors resulting in a small rise in Ca²⁺ in the postsynaptic terminal. Ca²⁺-dependent intracellular signals, including activation of phosphatases, lead to the dephosphorilation of AMPA receptors and the subsequent removal of these receptors from the syanspes via a clathrin- and dynamin-dependent process (Selig et al., 1995; Morishita et al., 2005).

- Metabotropic glutamate receptors (mGluR)-dependent-LTD. It requires activation of mGluRs, a weak Ca²⁺ rise and an intracellular cascade of events that ultimately leads to the withdrawal of AMPA receptors from the synapse. However other mechanisms can be implicated in the expression of this form of plasticity depending at which synapse or region of the brain the plasticity occurs (Bellone et al., 2008).

5.2. AMPA receptors

5.2.1 General features of AMPA receptors

In the mammalian central nervous system, AMPA-type glutamate receptors mediate the vast majority of fast excitatory synaptic transmission. AMPA receptors are tetramers made up of combinations of four subunits: GluR1, GluR2, GluR3, and GluR4 (named also “GluRA–D”) (Borges and Dingledine, 1998; Dingledine et al., 1999). All AMPA receptors subunit proteins have an extracellular N terminus, an intracellular C terminus, and four membrane-associated hydrophobic domains (M1–4, Figure 4).

Stargazin or other transmembrane AMPA receptors regulatory proteins (TARPs) also coassemble stoichiometrically with native AMPA receptors. The TARPs act as auxiliary subunits required for the receptor maturation, trafficking, and channel function (Chen et al., 2000; Rouach et al., 2005; Tomita et al., 2005; Nicoll et al., 2006; Ziff, 2007). AMPA receptors are widely expressed in neurons in all brain regions (Wisden and Seeburg, 1993; Belachew and Gallo, 2004). The great majority of AMPA receptors in the central nervous system exist as heteromers containing the subunit GluR2 (Wenthold et al., 1996;

Greger et al., 2002). GluR2 is a critical subunit in determining the AMPA receptors function, and many of the major biophysical properties of the native receptor, including, receptor kinetics, single-channel conductance, Ca²⁺ permeability, and block by endogenous polyamines. In addition, it is the most tightly regulated of the glutamate receptor subunits, with a number of specific regulatory processes at the level of gene expression, RNA editing, receptor assembly, and trafficking. Moreover, genetic

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manipulations of this subunit cause the most profound phenotype of all the AMPA subunits, demonstrating the critical importance of GluR2 for normal brain function (Brusa et al., 1995; Gerlai et al., 1998; Feldmeyer et al., 1999; Hartmann et al., 2004a).

Most of GluR2 protein contains an arginine residue (R) at position 607 in place of the genomically encoded glutamine (Q) (Sommer et al., 1991). This change is the result of hydrolytic editing of a single adenosine base in the pre-mRNA to an inosine by the adenosine deaminase enzyme ADAR2 (Higuchi et al., 1993). This Q/R editing is specific to the GluR2 subunit; more than 95% of GluR2 mRNA transcripts are edited in postnatal brain.

The additional positive charge introduced into the pore by the presence of R607 prevents both the passage of divalent cations (including Ca²⁺) and block by endogenous intracellular polyamines, and reduces single-channel conductance (Verdoorn et al., 1991;

Jonas and Burnashev, 1995). Thus, channels containing edited GluR2 subunits have a linear current-voltage relationship, are impermeable to Ca²⁺, and exhibit a relatively low single-channel conductance, while those lacking edited GluR2 are Ca²⁺ permeable, of a higher conductance, and are inwardly rectifying due to a voltage-dependent block by endogenous intracellular polyamines (Hestrin, 1993; Jonas et al., 1994; Bowie and Mayer, 1995; Geiger et al., 1995). However, although GluR2-lacking AMPA receptors exhibit significant Ca²⁺ permeability, they are less permeable to Ca²⁺ than NMDA receptors (Dingledine et al., 1999). The GluR2-dependent biophysical parameters of inward rectification, block by external polyamine, and Ca²⁺ permeability show a dose- dependence for the number of GluR2 subunits in the AMPA receptors complex (Washburn et al., 1997).

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5.2.2. GluR2 and AMPA receptors assembly

GluR2 plays a critical role in AMPA receptors assembly and trafficking. Receptors are tetramers (Rosenmund et al., 1998) formed in the endoplasmic reticulum (ER) as a dimer of dimers (Mayer, 2006). The initial stage of formation is the dimerization of two subunits that is dependent on the interactions in the N-terminal domain (NTD) (Ayalon and Stern-Bach, 2001). This is followed by a second dimerization step mediated by associations at the ligand binding and membrane domains, and this latter process is dependent on Q/R editing of GluR2 (Greger et al., 2003). The formation and stabilization of the tetramer is further promoted by NTD interactions.

In cells where GluR2 is highly expressed, the great majority of the AMPA receptors contain this subunit (Wenthold et al., 1996), and the preferred organization of receptor complexes containing GluR2 is a symmetrical heteromer (Mansour et al., 2001).

The assembly of AMPA receptors in the ER and subsequent ER exit is influenced by

Figure 4. GluR2 subunit and structure of the AMPA receptor complex. A. (A) Schematic of an AMPA receptor subunit. N-terminal domain (NTD), S1 and S2 ligand binding domains, membrane spanning domains (M1–4), Q/R and R/G RNA editing sites, flip/flop alternatively spliced region, glycosylation, and palmitoylation sites are indicated. The associated TARP/stargazin is also shown. (B) Schematic of predicted 3D structure of the tetrameric AMPA receptor complex, with NTD, S1 and S2, M2, and C terminus regions indicated. (C) GluR2 subunit domain structure with C terminus sequence detailed for the GluR2-short splice isoform (predominant form in the brain) and the GluR2-long isoform. Transmembrane domains indicated in yellow; flip/flop alternatively spliced region is shaded; editing, palmitoylation, phosphorylation, and protein interaction sites are as indicated. Modified from Isaac et al., 2007.

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subunit-specific interactions and editing of GluR2 at the Q/R site (Greger et al., 2002;

Greger et al., 2003; Greger et al., 2006). This regulated ER exit results in a large GluR2 pool in the ER of cell types that highly express this subunit (such as cortical pyramidal neurons), and this may serve to ensure that the great majority of receptors include GluR2.

In certain other cell types exhibiting relatively low levels of GluR2 expression, such as cortical GABAergic interneurons (Jonas and Spruston, 1994; Lambolez et al., 1996), the limited availability of GluR2 results in a significant fraction of receptors lacking GluR2.

Importantly, some subpopulations of inhibitory interneurons assemble and express both GluR2-containing and GluR2-lacking AMPA receptors in a single cell. Of particular interest is that these two types of receptors can be differentially targeted to synapses receiving distinct afferent input (Toth and McBain, 1998). This would suggest that stringent mechanisms are in place to regulate the availability of the GluR2 subunit during synthesis and targeting of GluRs in these cell types. Finally, there is also evidence that even in cells expressing high levels of GluR2, a functionally relevant population of GluR2-lacking AMPA receptors can be surface expressed under certain conditions (e.g., (Ju et al., 2004; Thiagarajan et al., 2005; Clem and Barth, 2006; Plant et al., 2006). This may specifically relate to GluR1 homomers produced by local dendritic synthesis of this subunit, potentially suggesting a differential assembly and trafficking for local dendritically synthesized AMPA receptors subunits (Ju et al., 2004; Sutton et al., 2006).

5.2.3. AMPA receptors trafficking

It is now well established that final insertion of AMPA receptors at the synaptic membrane involves tightly regulated trafficking events (Malinow and Malenka, 2002;

Bredt and Nicoll, 2003). This synaptic trafficking depends on the subunit composition of the receptor and on specific signals contained within the C termini. In the hippocampus, heterotetramers of GluR1–GluR2 and GluR2–GluR3 subunits, together with a smaller contribution from GluR1 homomers, represent the most common combinations at excitatory synapses (Wenthold et al., 1996). Based on experiments in which recombinant AMPA receptor subunits were expressed in hippocampal neurons, GluR2–GluR3 heterotetramers were found to cycle into and out of synapses continuously (Passafaro et al., 2001; Shi et al., 2001), whereas GluR1-containing receptors are added into synapses in an activity-dependent manner during LTP (Hayashi et al., 2000). The synaptic delivery

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of GluR1 is also regulated by physiological stimulation in living animals, as it has been reported for neocortical neurons upon sensory stimulation and in the lateral amygdala after fear conditioning (Sah and Lopez De Armentia, 2003; Takahashi et al., 2003). The subunit composition of the endogenous AMPA receptors that participate in regulated synaptic delivery has been more difficult to establish. Thus, both GluR2-lacking receptors (presumably GluR1 homomers) and GluR2-containing receptors (presumably GluR1–GluR2 heteromers) have been proposed to be rapidly inserted into synapses upon NMDA receptor activation in hippocampal slices (Bagal et al., 2005; Plant et al., 2006).

Although the details remain to be clarified, the importance of subunit composition for the regulation of synaptic delivery is well established. This has been recently corroborated by in vivo studies in which sensory stimulation, sensory deprivation and cocaine administration altered the prevalence of different AMPA receptor subunit assemblies at synapses (Bellone and Luscher, 2006; Clem and Barth, 2006; Goel et al., 2006).

Interestingly, GluR2-containing receptors enter spines constitutively, whereas the translocation of GluR2-lacking receptors into spines is triggered by LTP-inducing stimuli (Kopec et al., 2006). Therefore, it is tempting to speculate that the intracellular membrane transport machinery acts as a subunit-specific gate at the base of the spine, enabling the constitutive entry of GluR2–GluR3 receptors but restricting the access of GluR2-lacking receptors until the appropriate signaling events are triggered by synaptic activity.

The final step in the synaptic trafficking of AMPA receptors involves their functional insertion and stabilization at the postsynaptic membrane. Several members of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins are crucial for the synaptic targeting of AMPA receptors (Elias et al., 2006). Moreover, it has recently been shown that the exocyst acts within the dendritic spine to mediate the insertion of AMPA receptors into the postsynaptic membrane (Gerges et al., 2006). In fact, interference with the Exo70 subunit of the exocyst leads to the accumulation of AMPA receptors within the postsynaptic density, before fusion with the synaptic membrane. This observation suggests that AMPA receptor membrane insertion can occurs directly at the level of the postsynaptic density, and not laterally, as it had been previously hypothesized (Gerges et al., 2006).

In addition to this endosomal trafficking at synaptic terminals, there is considerable evidence for the presence of AMPA receptors at extrasynaptic plasma membrane

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locations. These surface receptors are highly mobile and can reach the synaptic membrane through lateral diffusion (Groc et al., 2004; Adesnik et al., 2005). The trapping and anchoring of these wandering AMPA receptors at the postsynaptic membrane relies on PDZ-dependent interactions between PSD95 (post-synaptic density 95) and TARPs (Groc et al., 2004; Adesnik et al., 2005). It is difficult to evaluate the relative contribution of surface and intracellular trafficking to the synaptic delivery of AMPA receptors.

Nevertheless, it is intriguing that lateral diffusion into synapses has been observed almost exclusively in primary neuronal cultures, whereas evidence for intracellular trafficking has been obtained from both brain slices and dissociated neurons. Therefore, it is possible that AMPA receptors follow the surface delivery route mainly in developing neurons. At later developmental stages, dendritic spines might acquire physical barriers that restrict lateral diffusion of extrasynaptic proteins into the synaptic membrane (Ashby et al., 2006).

5.2.4. Internalization, sorting and recycling of AMPA receptors

Synaptic AMPA receptors are internalized in an activity-dependent manner, leading to LTD. This process requires clathrin-mediated endocytosis (Carroll et al., 2001). In contrast to the subunit-specific rules for AMPA receptor delivery, the contribution of different receptor populations to activity-dependent removal remains controversial.

Hippocampal neurons that lack both GluR2 and GluR3 subunits display normal LTD, suggesting that GluR1 removal contributes to synaptic depression (Meng et al., 2003).

However, GluR2 subunits are removed during LTD in hippocampal neurons, and cerebellar LTD requires phosphorylation of GluR2 by PKC (Chung et al., 2003;

Seidenman et al., 2003). Therefore, both GluR1-containing and GluR2-containing receptors seem to participate in the synaptic trafficking associated with LTD. In fact, most experimental evidence is compatible with an initial indiscriminate internalization of all AMPA receptor populations upon LTD induction. However, it is increasingly appreciated that AMPA receptors undergo complicated intracellular sorting and recycling events after synaptic removal, and that these events might involve significant subunit specificity (Lee et al., 2004).

The molecular mechanisms that organize post-endocytic sorting of AMPA receptors and potential reinsertion into synaptic and/or extrasynaptic membranes are still

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unclear. Nevertheless, the balance between interactions of GluR2 with GRIP (glutamate receptor interacting protein) and ABP (AMPA binding protein) and with PICK1 (protein interacting C kinase) seems to be a crucial factor (Kim and Lisman, 2001; Perez et al., 2001; Hanley, 2006). In the case of hippocampal synapses, PICK1 is required for the removal of phosphorylated GluR2 and this role is facilitated by the Ca²⁺-dependent interactions between GluR2 and PICK1 (Hanley and Henley, 2005). Subsequently, a fraction of these internalized GluR2 subunits recycles back into synaptic sites, in a process probably mediated by direct interactions of PICK1with GRIP and ABP (Lu and Ziff, 2005), and NSF (N-Ethylmaleimide-sensitive fusion protein)-mediated dissociation of the GluR2–PICK1 complex (Hanley et al., 2002). The connection between these AMPA-receptor-binding proteins and the intracellular membrane-trafficking machinery is still being elucidated, but it has been recently proposed that the return of AMPA receptors to synaptic sites is mediated by phosphorylation-regulated interactions between GRIP and the neuron-enriched endosomal protein 21 (NEEP21) (Kulangara et al., 2007).

5.2.5. Ongoing dynamics of AMPA receptors

Several different techniques have demonstrated that glutamate receptors exchange between synaptic and extrasynaptic compartments (Newpher and Ehlers, 2008). It is possible to specifically track the surface population of glutamate receptors by using the pH-sensitive green fluorescence protein (GFP) viariant superecliptic pHluorin (SEP), which emits fluorescence at neutral pH of the extracellular space but is quenched within acidic internal compartments (Ashby et al., 2004; Ashby et al., 2006). Quantification of the bulk movements of surface SEP-GluR2 AMPA receptors using fluorescence recovery after photobleach (FRAP) revealed that 50% of surface GluR2 in spines is exchangeable, with a recovery phase that plateaus within 10–15 min (Ashby et al., 2006). This fluorescence recovery of SEP-GluR2 in spines is likely a result of surface lateral diffusion rather than plasma membrane insertion, as incubation of neurons with anti-GFP antibodies, a treatment that alters lateral diffusion of SEP-labeled surface receptors, slowed the fluorescence recovery of SEP-GluR2 in spines (Ashby et al., 2006). A similar fluorescence recovery rate was obtained following photobleaching of spine-localized with a fluorescence tag for GluR1 (Sharma et al., 2006). In addition, a recent FRAP study in intact hippocampal brain slices found that 30% of spine- and 60% of shaft-localized

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SEP-GluR2 is exchangeable (Heine et al., 2008), indicating that AMPA receptors are mobile in both dissociated culture and brain slices. Therefore, a major fraction of surface AMPA receptors in spines exist in a mobile population that undergoes exchange within minutes. Such rapid exchange of glutamate receptors at spines could provide the basis for acute changes in synaptic strength. Yet, the finding that approximately half the GluR2 AMPA receptors in spines do not exchange on this timescale indicates that a population of receptors is confined or immobile within synapses (Heine et al., 2008).

Both the exchange of mobile glutamate receptors and stabilization within synapses has been demonstrated by single particle tracking (SPT) experiments. Latex bead tracking of GluR2 revealed a wide range of diffusion coefficients for extrasynaptic AMPA receptors in hippocampal neurons, with a tendency for slower diffusion rates as neurons mature (Borgdorff and Choquet, 2002). Organic-dye-labeled antibodies subsequently allowed for the tracking of GluR2 in both synaptic and extrasynaptic compartments and for visualizing receptor exchange at synapses (Tardin et al., 2003). Similar to latex bead tracking, a wide range of diffusion coefficients exists for extrasynaptic GluR2. Within synaptic regions, two populations were detected, one apparently immobile pool and a second mobile population, consistent with the 50% mobile pool detected by FRAP methods (Ashby et al., 2006; Sharma et al., 2006). Using the mean square displacement to analyze results with SPT, GluR2 trajectories in synaptic and extrasynaptic domains revealed confined movements in synapses compared to primarily free Brownian diffusion in extrasynaptic domains. Importantly, extrasynaptic GluR2 AMPA receptors can enter into synaptic regions, providing evidence that extrasynaptic receptors can act as a readily available pool to supply synapses.

5.3. Addiction and synaptic plasticity

Since synaptic plasticity is required for neuroadaptations that result from a wide range of environmental stimuli, a potential scenario is that addictive drugs can cause long-term changes on behavior by initially altering the synaptic function and plasticity in drug-seeking-related brain circuits. Indeed, many evidences link various behavioral models of addiction with synaptic plasticity in brain areas involved in reinforcement and reward in line with the idea that addiction may develop following synaptic maladaptations.

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Studies demonstrating that blockade of NMDA receptors impairs the development of drug-induced behavioral adaptations in certain addiction animal models were the first demonstration that addictive drugs override processes implicated in storing informations (Kauer and Malenka, 2007). NMDA receptors blockade, which effectively blocks LTP and LTD in many brain regions (Malenka and Bear, 2004), also prevents a large numbers of behavioral task associated with drug reinforcement (i.e. CPP, behavioral sensitization and self-administration) (Kalivas and Alesdatter, 1993; Schenk et al., 1993; Kim et al., 1996). Importantly, NMDA receptors blockade specifically in the VTA prevents both behavioral sensitization and CPP (Kalivas and Alesdatter, 1993; Harris and Aston-Jones, 2003). However, these manipulations altered NMDA receptors function in all neuronal population in the VTA. We have recently circumvented this problem by using a mouse line where the NR1 subunit of NMDA receptors was deleted in adulthood (Engblom et al., 2008). In these mice, CPP and behavioral sensitization were normal, however they failed to reinstate drug-seeking behavior suggesting that the VTA might be implicated in late-stages drug-seeking behavior rather than short-term ones (Engblom et al., 2008) but see (Zweifel et al., 2008). Given the extremely important role of NMDA receptors in mediating synaptic plasticity, these findings suggest that learning processes are also essential in developing and maintenance of addictive behavior.

5.4. Drug-evoked plasticity in the VTA

Forms of synaptic plasticity have been described in DA neurons of the VTA.

Indeed excitatory synapses onto DA neurons of the VTA undergo a form of NMDA receptors-dependent LTP (Bonci and Malenka, 1999; Liu et al., 2005), a voltage-gated calcium channels-dependent LTD (VGCC), a protein kinase A (PKA)-dependent LTD (Jones et al., 2000; Thomas et al., 2000; Gutlerner et al., 2002) and an mGluR-dependent LTD (Bellone and Luscher, 2005). These findings set the stage for studies that directly tested whether in vivo administration of addictive drugs produced long-lasting synaptic adaptations at excitatory synapses onto DA neurons of the VTA.

Controlling the persistence of drug-evoked plasticity in the mesolimbic dopamine system

Thesis

Reference