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The role of the cannabinoid system in the neurochemistry underlying reward processes

Proefschrift

ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen

op gezag van de Rector Magnificus, dr. E. Sterken, in het openbaar te verdedigen op

vrijdag 4 januari 2013 om 16:15 uur

door

Jelle Kleijn

geboren op 5 november 1982 te Lelystad

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Promotor: Prof. dr. ir. B.H.C. Westerink Copromotor: Dr. T.I.F.H. Cremers Beoordelingscommissie: Prof. dr. J.M. Koolhaas

Prof. dr. P.G.M. Luiten Prof. dr. A.G.J. van der Zee

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Aan mijn ouders, die voor mij dit hebben mogelijk gemaakt. Jullie zijn mijn voorbeeld in het verschaffen van

mogelijkheden voor mijn kleijntjes.

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Table of contents

Chapter 1 Endocannabinoids and the CB-1 receptor

from involvement in drug addiction to sampling

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Chapter 2 Direct effect of nicotine on mesolimbic

dopamine release in rat nucleus accumbens shell

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Neuroscience letters 2011 Apr: 493 (1-2) 55-8

Chapter 3 Effects of amphetamine on dopamine release in the rat nucleus accumbens shell region depend on cannabinoid CB1 receptor activation

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Neurochemistry International 2012 Jun: 60 (8) 791-8

Chapter 4 CB-1 receptors modulate the effect of the Selective Serotonin Reuptake Inhibitor, citalopram on extracellular serotonin levels in the rat prefrontal cortex

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Neuroscience research 2011. Jul: 70(3) 334-7

Chapter 5 Optimizing the in vitro recovery of endocannabinoids sampled by microdialysis

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Chapter 6 The nicotine induced-dopamine release in

the reward pathway: role of extracellular endocannabinoids

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Chapter 7 Summary of the Thesis 149 Chapter 8 Nederlandse samenvatting proefschrift 155 Chapter 9 Acknowledgements / Dankwoord 161

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Chapter 1 Endocannabinoids and the CB-1 receptor from involvement in drug addiction to sampling J. Kleijn, B.H.C. Westerink University of Groningen, dept. Biomonitoring & Sensoring, Groningen, The Netherlands

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Drug addiction “Drug dependence” and the more traditional term “drug addiction” describes the human condition in which drug use becomes compulsive, often with serious adverse consequences. It concerns a state of psychological, as well as physical, dependence. Drug dependence can be viewed as a reversible pharmacological situation, whereas addiction describes a chronic, reoccurring disorder in which obsessive drug seeking and taking behavior persists despite negative consequences (Cami and Farre, 2003). In the year 2002, it was estimated that 2 billion people use alcohol, 1.3 billion people smoke tobacco cigarettes (WHO, 2002), and 185 million people use illicit drugs (UNDCP, 2002). There are many different types of illicit drugs of abuse, and regional differences exists in the use of the most popular ones. Addictive substrates are able to relief distress and induce euphoria. The brain is able to adapt to the frequent use of these compounds and the induced changes in central nervous system will lead to tolerance, physical dependence, sensitization, craving, and relapse (Cami and Farre, 2003). On the basis of learning and memory studies, theories of addiction have been developed. These theories overlap in some parts but are not all in consensus with each other. Generally, addictive drugs can induce two actions, either they act as positive reinforcers, resulting in euphoria, or they act as negative reinforcers, alleviating withdrawal symptoms of or dysphoria (see figure 1). When addicted, an environmental stimuli associated with previous drug use, can induce rewardive feelings or feelings of withdrawal or craving even in the absence of the drug (Spanagel and Weiss, 1999; Stolerman, 1992). Where reward is immediate, brief (hours) and decreases with repetition, the state of withdrawal is delayed, long-lasting (days), and increases with repetition.

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Figure 1: the psychological factors involved in development and maintenance of addiction (adjusted version of Rang and Dale’s Pharmacology 6th edition).

Pharmacological characteristics of drugs are important factors determining the way drugs are consumed. The lipophilicity of a drug determines the speed at which the drug passes the blood-brain barrier, whereas hydrophilic properties facilitate the administration of the drug (Farre and Cami, 1991). Furthermore, volatile drugs are prone to be inhaled, and heat resistant drugs are often smoked, like cannabis and tobacco. Other important characteristics of a drug making it highly addictive are the rapid onset and the intensity of the effect (Roset et al., 2001). Therefore even between different formulations of the same drug, there are differences in the addictiveness determined by the speed by which the compound reaches the brain (e.g., smoking “crack” cocaine is preferred to intranasal administration) (Hatsukami and Fischman, 1996). Compounds having a short half-life, like heroine, also produce more abrupt and intense syndromes of withdrawal, compared to methadone that has a long half-life (Farre and Cami, 1991).

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The likelihood of developing addiction is not only depending on the consumption of the drug, but personality and psychiatric state together with genetic and environmental factors play an equal role. Personality traits, like risk-taking and novelty seeking, and mental disorders are major conditioning factors in developing drug addiction (Cami and Farre, 2003; Helmus et al., 2001). Mental disorders, like schizophrenia, attention-deficit-hyperactivity-disorder, bipolar disorder, and depression are associated with a higher risk of developing addiction. When diagnosed for both a mental disorder and substance abuse, the success rate of treatment is usually low (Kavanagh et al., 2002). Genetic factors may also contribute to the risk of addiction by influencing metabolism and the pharmacological effect of drugs (Crabbe, 2001). When both parents are alcoholic the child has a higher chance of developing alcoholism at a later age (Schuckit and Smith, 2001). With regard to nicotine addiction, people who have a mutation in the allele coding for the nicotine degradation enzyme, are less likely to develop dependency when compared to people who are homozygous for this alleles (Rao et al., 2000). Furthermore, when the endocannabinoid system (likely involved in drug reward processes) is affected by a single-nucleotide polymorphism in the gene coding for fatty-acid-amide-hydrolase (FAAH: endocannabinoid inactivating enzyme) there is an increased likelihood of using illicit drugs (Sipe et al., 2002).

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Neurobiological basis of reward Despite the large differences in the working mechanisms of drugs of abuse, each binding to a distinct target protein in the brain which elicits a unique combination of behavioral and psychological effects, they share certain effects after both acute and chronical administration (Nestler, 2005). Virtually all drugs of abuse, including nicotine, cannabinoids, and amphetamines, activate the mesocorticolimbic dopaminergic pathway, also known as the reward pathway (Carboni et al., 1989; Di Chiara and Imperato, 1988; Imperato and Di Chiara, 1986; Imperato et al., 1986; Wise and Bozarth, 1987). The reward pathway plays a major role in the motivational system regulating the responses to natural rewardive processes, such as eating, drinking, having sex and social interaction. Drugs of abuse probably affect the reward pathway more intensely and with more persistence compared to the natural reinforcers. The interaction of drugs of abuse and the reward pathway might therefore induce alterations in the reinforcing mechanisms. The most important alterations in reinforcing mechanisms are tolerance, dependence, and sensitization. Tolerance might be a contributor for the intensification of drug intake seen during the development of an addiction. Where dependence contributes to the high rates of relapse in the early phases of withdrawal, sensitization plays an important role in an increased risk of relapse after longer withdrawal periods. The cell bodies of the reward pathway originate in the ventral tegmental area (VTA) of the midbrain (A10 cell group in rats), and run via the medial forebrain bundle to the nucleus accumbens (NAc), prefrontal cortex (PFC), amygdala (AMG) and limbic region (Koob and Nestler, 1997) (figure 2). Figure 2 gives a schematic view of the reward pathway with its most important connections and involved neurotransmitter systems. Although the VTA-NAc connection (especially the NAc shell region) is regarded as the most important and is therefore the subject of most studies investigating reward processes, other brain-structures like the ventral pallidum (VP), medial dorsal thalamus (MDT), hippocampus, PFC, and AMG also seem to play a significant role in reward processes (Everitt et al., 1991; Ikemoto, 2010).

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Figure 2: Schematic view of the reward circuitry and the neurotransmitter systems involved. DA neurons cell bodies are localized in the VTA project to the NAc and mPFC via the medial forebrain bundle. In addition connection to neurons in the hippocampus, AMG, VP are described. An inhibitory input via GABA neurons is present from the NAc to the VP and to the VTA (possibly as a negative feedback). The NAc is receiving stimulatory input via glutamate neurons from the mPFC, hippocampus and AMG. The MDT is connected via glutamate neurons to the mPFC. The VTA is known to have glutamatergic interneurons that stimulate the DA signals to the NAc.

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The VTA-NAC connection and reward It is well recognized that drugs of abuse activate a common circuitry in the brain’s limbic system (Di Chiara et al., 2004; Nestler, 2001). Most attention has been given to the reward circuitry and within this to the dopaminergic projection from the VTA to the NAc. This VTA-NAc connection is regarded as being the primary substrate for the acute rewarding effects of all drugs of abuse. The NAc can be divided into two different functional parts: the NAc core and NAc shell region. Both these regions of the NAc are implicated in processes related to reward and addiction (Bassareo et al., 2002; Di Chiara, 2002). The NAc shell and core seem to differ in the amount of sensitization to drugs (Cadoni and Di Chiara, 1999; Cadoni et al., 2000), their response to natural rewardive stimuli like food (Bassareo and Di Chiara, 1999) and their reaction in DA release to systemic drug administrations (Di Chiara, 2002). Overall the NAc shell seems to be involved in the initial reward processes (e.g. the “tasting” part of eating), while the NAc core is involved in the reward following the intake of the food (e.g. the feeling of satiety). While the DA increase in the NAc is the hallmark of reward, the DA cell bodies in the VTA seems to be fundamental in regulating the reward processes, and this nucleus is recognized as being the brain area initiating most rewardive processes. As a testament to the essential role of the VTA in reward it has been shown that the self-administration of nicotine by rats is inhibited by local administration of a nicotine-antagonist (dihydro-β-erythroidine) into the VTA. Furthermore, studies using nicotine acetylcholine receptor blockers or knock-outs in the VTA have shown the essential role of these receptors within this brain area for nicotine induced reward processes both neurochemically and behaviorally (Laviolette and van der Kooy, 2003; Maskos et al., 2005; Panagis et al., 2000; Picciotto et al., 1998; Pons et al., 2008; Walters et al., 2006). Additionally, it was shown that intra VTA infusions of heroin induces conditioned place preference (CPP) (Walker and Ettenberg, 2005), and that CPP induced by cocaine was completely inhibited by pre-treating the animals with local VTA microinjections of a glutamate antagonist (Harris and Aston-Jones, 2003). Taken together, these data suggest a crucial role of the VTA in regulating drug induced effects and hence mediating addiction. Apart from the DA neurons, within the VTA are at least two more neuronal pathways involved

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in reward (Cameron et al., 1997). One of these are GABAergic neurons, which project to the NAc (Van Bockstaele and Pickel, 1995) and PFC (Carr and Sesack, 2000), and locally synapse on the VTA DA neurons (Johnson and North, 1992; Steffensen et al., 1998). The other neuronal type projecting to the VTA is glutamatergic, which are originating from a wide range of neuronal substrates (mPFC, AMG, pedunculopontine nucleus and the subthalamic nucleus). Other brain areas involved in reward Recent studies have shown that other brain areas are also involved in reward, like the AMG, MDT, VP, hippocampus, and (m)PFC (Kalivas, 2004; Koob and Le Moal, 2001; Nestler, 2001; Robinson and Berridge, 2003; Volkow et al., 2004). Some of these areas are essential parts of the brain’s memory systems, leading to the now confirmed hypothesis that important reward processes engage emotional memories (Hyman and Malenka, 2001; Kalivas, 2004; Nestler, 2001, 2005; Wise, 2004). In CPP environmental stimuli are coupled to a primary reward (e.g. sucrose of a drug). Since the stimulus then predicts reward for the animal, the animal will therefore prefer the environment that is coupled to this rewardive stimulus. When bilateral lesions are induced in the basolateral AMG, using excitotoxic amino acids, the expression of CPP is abolished (Everitt et al., 1991). The same study showed that when the DA connection between the AMG and the VTA was lesioned CPP was again abolished, suggesting an important connection between the AMG and VTA for this behaviour. Additionally, in primates by applying aspiration lesions (in which the whole AMG is removed) severe impairments in stimulus-reward learning were observed (Murray and Mishkin, 1984, 1985; Spiegler and Mishkin, 1981). These studies indicate the importance of the AMG in remembering the features of rewardive stimuli. The role of the MDT in stimulus reward was studied in primates, where lesions of the MDT resulted in comparable effects to those observed after AMG lesioning (Gaffan and Murray, 1990). Although the AMG is known to have interactions with the NAc (Groenewegen, 1988; Kelley et al., 1982), no functional connections were identified between the AMG and MDT (Gaffan and Murray, 1990). Furthermore, lesions in the MDT completely prevented acquisition of place preference, while lesions in the VP only attenuated this behavior (McAlonan et al., 1993). While lesions, in both the

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MDT and VP, did affect the CPP they did not affect the consumption rate of the sucrose (the rewardive stimulus). This suggests that both the MDT and VP are more involved in the cognitive processes regulating reward and addiction, and have no influence on the direct reward (“liking” (Berridge, 1996)). By contrast, inducing a lesion in the VP pre-conditioning the animal in the CPP paradigm, did show a complete absence of place preference, while lesioning post-conditioning was ineffective (Hiroi and White, 1993). This finding does suggest that the VP is involved in the cognitive processes that result in addictive behavior. Another brain area that, like the AMG, is implicated in the cognitive reward processes is the hippocampus. There are indications that both the amygdalar- and the hippocampal-pathways are essential for cue-induced drug-seeking/taking (See, 2005). More specifically, the hippocampus is thought to be implicated in drug seeking and taking that is elicited by contextual (environmental) stimuli, which is confirmed by lesions in the hippocampus have shown to prevent the contextual reinstatement (relapse) of cocaine administration (Fuchs et al., 2005). The mPFC is another brain area believed to be important for the cognitive aspects of reward processes ultimately leading to the development or reinstatement of addiction. Furthermore, the mPFC is an important brain area in regulating impulsive behavior (Pattij and Vanderschuren, 2008), a form of behavior that plays an important role in the reinstatement of addiction (Fuchs et al., 2004). Neurochemically the mPFC receives dopaminergic input from the VTA, as does the NAc shell (Koob and Nestler, 1997), and shows a reduction in DA activity during amphetamine self-administration, controversially the NAc there is actually an increase in DA activity (Piazza et al., 1991). There are glutamatergic projections from the mPFC to the NAc which have shown to be involved in the reinstatement of cocaine-seeking (McFarland et al., 2003), which further emphasizes the significance of this brain area in addiction.

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Drugs of abuse Although there are a large number of drugs of abuse, like emphasized above, this thesis will focus on nicotine, amphetamine and cannabis, while comparisons with other drugs will be made when relevant. Nicotine Like other addictive substrates nicotine induces pleasure and reduces stress and anxiety. Nicotine addicts smoke tobacco to modulate levels of arousal and to control their mood (Benowitz, 2010). Even though nicotine improves concentration, and reaction time, and therefore is regarded as a stimulant, this is not the main reason that people keep on consuming cigarettes. Relief from withdrawal symptoms is also a strong motivator for smoking of cigarettes. Amongst the mentally ill, patients with schizophrenia show the highest smoking prevalence of about 70-80%, whereas it is 20-30% in the general population (Winterer, 2010). Patients with schizophrenia frequently suffer from negative symptoms (e.g. avolition, social withdrawal, anhedonia) and cognitive deficits, like impaired working memory and attention deficits (Gold and Harvey, 1993). Trying to explain the high smoking prevalence amongst these patients researchers came up with the self-medication hypothesis. This hypotheses states that patients with schizophrenia may consume nicotine to self medicate negative symptoms (reviewed by (Winterer, 2010). Whatever the reason is that these patients show such a high smoking prevalence, it may be concluded that these patients show a higher need for nicotinergic stimulation. Therefore, there are currently intensive efforts to develop a novel group of drugs targeting the nicotinergic system in order to treat the negative symptoms of schizophrenia (Winterer, 2010).

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Pharmacology of Nicotine As a testament for the addictive potential of nicotine it was shown that animals can be trained to push a lever to self-administer nicotine (Corrigall, 1999). Furthermore, cessation of regular nicotine administrations induces physical withdrawal signs and symptoms in this animal model of smoking addiction. These effects of nicotine are probably regulated mainly by nicotinic acetylcholine receptors, present in the brain. Nicotinic Acetylcholine Receptors Acetylcholine is an important excitatory neurotransmitter in the central nervous system (CNS). The effects of acetylcholine are mediated by two receptor types, the muscarinic and the nicotinic. There are five subtypes of the G-protein coupled muscarinic receptors (M1 to M5) of which the neural subtypes mediate processes like, for instance, memory. The nicotinic acetylcholine receptors (nAChR’s), known from the neuromuscular junction in ganglia are involved in fast excitatory transmission in the brain. All nAChR’s are, like GABAA, glycine, and 5-HT3, members of the ligand-gated ion channel family (Akabas and Karlin, 1995; McGehee and Role, 1995). These nicotinic receptors consist of a pentameric cylindrical structure organized around a central pore which is permeable to Na+, K+, and Ca2+ (Cooper et al., 1991). Cloning the nAChR’s gave eight possible neuronal subunits of which the pentameric cylindrical structure is designed (α2 - α7 and β2 - β4) (Chini et al., 1994; Elgoyhen et al., 2001; Lukas et al., 1999). Neuronal nAChR’s are composed of a combination of α- and β-subunits. The numbers of available nAChR subunits facilitate a large amount of possible receptor subtypes. Moreover, a single neuron may express more than one nAChR subtype, with each subtype serving a different function. Therefore making a prediction of the distribution of nAChR subtypes, based on the gene expression of each subunit, is not an easy task. From in situ hybridization and immunohistochemical studies it has become clear that the subunits α4, β2, and α7 are the most abundantly expressed in the mammalian brain (Hill et al., 1993; Marks et al., 1992; Seguela et al., 1993; Wada et al., 1989). Although there is partly overlap of expression, each α-subunit has a unique set of neuronal structures (Wada et al., 1989). Of all α-subunits, α4 is the most abundant in the brain and together with the β2 subunit accounts for more than 90% of nicotine binding sites in the rat brain (Flores et al., 1992; Wada et al., 1989; Whiting and Lindstrom, 1986). Like the α4 the α7-

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subunit is distributed throughout the brain with dense populations in the cortical and limbic areas (Orr-Urtreger et al., 1997). In contrast, the α2, α3, and α5 are only found in limited areas of the mammalian brain (Lena and Changeux, 1997; Wada et al., 1990). From the β-subunits, β2 is the most abundant and its mRNA is widely expressed, together but not exclusively, with the α4 mRNA (Hill et al., 1993; Lena and Changeux, 1997). Their distribution parallels the location of high-affinity nicotine binding sites in the brain, like in the thalamus, striatum and cortex (Clarke et al., 1985).

Like all ligand-gated ion channels, opening of the nAChR receptor channel requires the binding of a ligand, like nicotine. When the ligand is bound to the receptor-site all subunits will undergo a conformational change opening a pore and allowing ions (Na+, K+) to flow in or out of the cell eventually leading to depolarization of the neuron. Nicotine and dopamine When nicotine reaches the brain it binds and activates nAChRs throughout the whole brain (Clarke et al., 1985). Within the reward circuitry nicotine is believed to bind both the nAChRs in the VTA (Maskos et al., 2005; Picciotto, 1998; Pons et al., 2008; Walters et al., 2006) and NAc shell region (Clarke et al., 1985; Harfstrand et al., 1988; Kamiya et al., 1982; Mifsud et al., 1989). Upon stimulation of the nAChRs the DA neuron in the VTA is stimulated directly or indirectly by activating nAChRs located on either glutamate or GABA neurons within the VTA. The result of this stimulatory effect in an increase in NAc shell DA release. However, within the NAc shell itself there are also nAChRs located that, upon activation by binding with nicotine, could directly stimulate local DA release from DA terminals. The fact that nicotine is binding at both sides of the reward circuitry makes it difficult to understand the exact neurochemical processes that determines the rewardive effect of nicotine. Since there are different types of receptors (α7, α4β2) localized in the VTA and NAc the key of nicotine reward is probably activation of a unique combination of these receptor types.

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Amphetamine Amphetamine like drugs and stimulants such as amphetamine, dextroamphetamine, but also methylphenidate (Ritalin) are known to have both central and peripheral effects. The main central effects are an increase in locomotor activity, euphoria, excitement, and stereotyped behavior. Amphetamines are also known to have a strong anorexic effect and have been used for weight loss. In the periphery amphetamine increases blood pressure and inhibits gastrointestinal motility. Amphetamine is also used by students to improve performance during exams. The compound has indeed been shown to increase cognitive tasks in humans (Barch and Carter, 2005). During repeated use within a short time-frame, amphetamine might induced a state of psychosis (Shoptaw et al., 2009), closely resembling an acute schizophrenic attack (hallucinations, paranoia, and aggressive behavior). When the effect of amphetamine wears down, there is usually a period of deep sleep from exhaustion, followed by feelings of lethargy, depression, anxiety and hunger (Zorick et al., 2010). These effects might be the result of depletion of catecholamine stores. Dependence on amphetamine appears to be the result of these unpleasant withdrawal effects and the memory of euphoria, which will motivate the user to administer another dose. Repeated amphetamine use will rapidly develop tolerance, especially the peripheral sympathomimetic and anorexic effects, but more slowly to the other effects (locomotor and stereotypics) (Wolgin, 2000). Amphetamine-like drugs as medicine Amphetamine has the characteristics of a highly addictive drug of abuse. The effect of amphetamine is fast, pleasurable and when it wears down leaves withdrawal feelings which motivate the following administration. Indeed, amphetamine has shown to be self-administered by rats trained to push a lever to receive an administration of the drug (Lyness et al., 1979). Furthermore, because of its importance in a range of psychiatric disorders, including personality disorders, drug addiction, and attention-deficit/hyperactive-disorder (ADHD), impulsive behavior has received a great deal of attention in scientific studies. Amphetamine is a often used model for studying the impulsive decision making in animal models (Pattij and Vanderschuren, 2008; van Gaalen et al., 2006).

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In this regard it is of interest to note that amphetamine-like drugs are frequently prescribed as medicine for ADHD. Since a major physical effect of amphetamine is hyperactivity, it seems paradoxically that amphetamines are the medicine of choice in the treatment of ADHD. Methylphenidate (Ritalin) is commonly used to treat ADHD at doses lower than those causing euphoria and other side-effects. ADHD is a common condition in children in which the over-activity and lack of attention-span disturbs their social development and educational performance. In this group of subjects amphetamines are regarded as a safe and effective drug. Despite of the effectiveness and safety of these drugs, which is confirmed by controlled clinical trials (e.g. (Greenhill et al., 2006; Wigal et al., 2004), the clinical usefulness is limited by the risks of adverse effects (hypertension, insomnia, anorexia, tremors, and the risk of dependence). Amphetamine and dopamine Amphetamines are known to increase the extracellular levels of dopamine and noradrenaline (NE) in the brain. As dopamine is the crucial transmitter in the reward circuitry, the characteristic behavioural effects of amphetamine are very likely explained by this pharmacological effect. This hypothesis is supported by the observation that the stereotyped behavioral effects of amphetamines are absent after depleting the brain from DA with the specific neurotoxin 6-hydroxydopamine or by preventing the catecholamine syntheses by pretreatment with α-methyltyrosine (Gold et al., 1989; Jones et al., 1989). There are multiple ways by which amphetamine can increase catecholamine levels in the synaptic cleft, First, amphetamine binds to the pre-synaptic membrane of DA neurons and induces the release of DA from its terminals. Second, amphetamine can bind and inhibit the monoamine degradation enzyme monoamine oxidase in the DA neurons. Third, amphetamine can stimulate DA containing vesicles to release DA into the synaptic cleft, and fourth, amphetamine is able to prevent DA re-uptake by binding and blocking the DA re-uptake transporter.

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Cannabis Although cannabis/marijuana is prescribed for medication purposes (pain relief, treatment of nausea or premenstrual syndrome) for centuries, the use of marijuana for recreational purposes has always accompanied its medical benefits. Marijuana or hashish induces the feeling of relaxation and well-being while it impairs cognitive processes. However, an overdose of cannabis can induce panic attacks and even psychotic episodes (Hall and Solowij, 1998). Among patients with schizophrenia or other mental disorders there is a relative high incidence of cannabis consumption (Zammit et al., 2002). Compared to other drugs of abuse, like ethanol, the withdrawal symptoms of cannabis (restlessness, irritability, and insomnia), are subtle and are mainly restricted to the heavy users (Budney et al., 2001). The long-term effect of repetitive use of high cannabis doses is a subject of debate and concern. Although there is evidence for the negative effects of cannabis use on memory (Bolla et al., 2002; Solowij et al., 2002), the impact of other with cannabis use associated symptoms (like the marijuana amotivational syndrome) remain unclear (Hall and Solowij, 1998). Neurochemistry of Cannabis Although the addictive properties of cannabis have been debated during many years,, it is now accepted that cannabis is in fact addictive. Cannabis has shown to act upon reward processes in a manner which is consistent with other drugs of abuse (Lupica et al., 2004). Cannabis induces CPP, which is mediated by CB-1 receptors, since its antagonist SR141716A reverses the process (Gardner, 2002; Tanda and Goldberg, 2003). In initial experiments cannabis was not active in a self-administration paradigm (where animals are trained to push a lever, or do a nose poke to receive a dose of cannabis) (reviewed by (Tanda and Goldberg, 2003), However after careful control of dose and vehicle cannabis robustly induced self-administration in primates at doses comparable to those observed in marijuana smokers (Tanda and Goldberg, 2003; Tanda et al., 2000). Consistent with most drugs of abuse, cannabis also showed to lower the threshold for intra-cranial self stimulation (ICSS) in the medial forebrain bundle (Gardner and Lowinson, 1991), In the late 1980’s it was suggested that specific CB receptors existed, and that the effect of cannabinoids was not just the result of alterations of cellular membrane structures (Devane et al., 1988; Howlett et al., 1990).

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Soon after this suggestion the first CB receptor, the CB-1 receptor, was cloned and sequenced (Matsuda et al., 1990). The discovery of this receptor was followed shortly by the identification of a second CB receptor, called the CB-2 receptor (Munro et al., 1993). The CB-1 receptor is mainly found in the CNS whereas the CB-2 receptor is mainly present in lymphoid tissue and has an important function in the immune system (Nocerino et al., 2000). However, more recently the CB-2 receptor was also found in the brain (reviewed by (Atwood and Mackie, 2010) and although the central relevance of the CB-2 receptor remains under discussion, they have recently been implicated in cocaine induced reward (Xi et al., 2011). Some studies also suggested the existence of an additional non-CB-1 and non-CB-2 receptor (Begg et al., 2005; Breivogel et al., 2001; Freund and Hajos, 2003; Fride et al., 2003; Hajos et al., 2001; Jarai et al., 1999; Ryberg et al., 2007; Zimmer et al., 1999) of which the neurological significance needs more investigation. Because of its role in mediating neurotransmission in the CNS the CB-1 receptor will be the main focus of this thesis. CB-1 receptors are the most abundant G-protein coupled receptors present in the brain (Howlett, 2002). CB-1 receptors are of the metabotropic type and coupled to Gi/o proteins whose activation results in an inhibition of adenyl cyclase activity. A decrease in adenyl cyclase activity consequently results in a decrease in cyclic AMP content in the cytosol. This also closes Ca2+ channels, opens of K+ channels, and stimulation of the kinases that phosphorylate tyrosine, serine, and threonine (Howlett, 1998; McAllister and Glass, 2002). CB-1 receptors are located on the presynaptic nerve ending and are therefore believed to inhibit the release of glutamate, GABA, serotonine, and other neurotransmitters (Schlicker and Kathmann, 2001; Wilson and Nicoll, 2002). Stimulation of CB-1 receptors inhibits both the excitatory input of glutamate (Melis et al., 2004b; Pistis et al., 2002; Robbe et al., 2001) and the inhibitory input of GABAergic neurons (Szabo et al., 2002) within the VTA on DA neurons. Regulating the excitatory and the inhibitory input within a system projects the CB-1 receptor as an important modulator of (DA) nerve activity. Additionally, cannabinoids have shown to decrease acetylcholine (ACh) release and activity (Carta et al., 1998; Gessa et al., 1998; Gessa et al., 1997; Gifford et al., 2000; Narushima et al., 2007). Controversially, also

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stimulatory effects of cannabinoids on ACh release are shown (Acquas et al., 2000; Acquas et al., 2001; Degroot et al., 2006; Verrico et al., 2003). Norepinephrine nerve activity is also frequently linked the activity of CB-1 receptors (Mendiguren and Pineda, 2006; Oropeza et al., 2005; Schlicker et al., 1997; Tzavara et al., 2001), while other neurotransmitters are less often linked to CB-1 receptor activity, such as histamine and cholecystokinin (Beinfeld and Connolly, 2001; Cenni et al., 2006). The localization of CB-1 receptors in the brain is consistent with the known effects of cannabinoids. The highest concentrations of CB-1 receptors are found in areas involved in memory (e.g. hippocampus), motor coordination (e.g. cerebellum), and emotions (e.g. prefrontal cortex) (Herkenham et al., 1990; Tsou et al., 1998). These reports also show that in the terminal region of the brain reward circuitry average to high CB-1 receptors densities are found, while in the VTA only low concentrations of CB-1 receptors were reported. The relatively low levels of CB-1 receptors within the VTA do not implicate that there is no functional role of CB-1 receptors on processes within this region. In fact, several studies have shown that cannabinoids directly affect VTA neuron activity (Cheer et al., 2003; Melis et al., 2004a; Melis et al., 2004b; Riegel and Lupica, 2004; Szabo et al., 2002), suggesting an important role in reward processes (Zangen et al., 2006). Since the CB-1 receptors are so widely distributed, abundant in high numbers, and upon receptor stimulation have an inhibitory effect on multiple neurotransmitter systems it seems only logical that the activity of this receptor has major influences on both endogenous and exogenous (e.g. drug induced/medication) processes. A pronounced and clinically relevant interaction of the CB-1 receptor with an SSRI (selective serotonine reuptake inhibitor; the most prescribed class of antidepressants) will be described in Chapter 4.

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Endogenous Cannabis Although investigation of the rewarding effects of cannabis is interesting and highly relevant, the focus of this thesis will be the role of endocannabinoids (ECs) in the regulation of brain neurotransmitter release and activity within the reward circuitry. ECs, like anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are retrograde messengers which implicate that they are synthesized postsynaptically in an activity-dependent manner (on demand), travel the synaptic cleft and interact with presynaptic CB-1 receptors on both excitatory and inhibitory afferents (Harkany et al., 2008). After binding to the CB-1 receptor, their action is inhibition of nerve activity and thus decreasing neurotransmitter release. In the early 1990s, AEA (Devane et al., 1992; Di Marzo et al., 1994) and 2-AG (Mechoulam et al., 1995; Stella et al., 1997; Sugiura et al., 1995) were discovered as the first endogenous ligands to the CB-1 receptor. Following AEA and 2-AG other possible ECs have also been proposed, like virodhamine (Porter et al., 2002), noladin ether (Hanus et al., 2001), and arachidonoyldopamine (Porter et al., 2002), but their physiological role and natural occurrence remains a subject of debate. AEA and 2-AG have different structures, are synthesized via different pathways, and have different degradation pathways. AEA is degraded by fatty-acid-amide-hydrolase (FAAH) into arachidonic acid and ethanolamine, whereas 2-AG is mainly degraded by monoacylglycerol lipase (MAGL). Furthermore, these ECs appear to be formed under the different conditions and to be differently affected by several manipulations, including pharmacological stimulation [reviewed by: (Di Marzo et al., 2004; Freund et al., 2003; Piomelli, 2003)]. There are also endocannabinoids that have no affinity for the CB-1 receptor, like OEA and PEA. These ECs bind to the nuclear transcription factor peroxisome proliferator-activator (PPAR-α) receptor, on which they display agonistic actions (Fu et al., 2003). These receptors appear to be generally distributed in the CNS (Moreno et al., 2004), and present in the cortex, VTA, midbrain, medulla, hippocampus, substantia nigra, and olfactory tubercle (Cullingford et al., 1998; Deplanque et al., 2003; Kainu et al., 1994; Kreisler et al., 2007; Moreno et al., 2004). AEA and 2-AG are believed to be involved in a wide range of processes, symptoms, and diseases, like: pain, motor-functions, schizophrenia, cognitive functions, sleep, feeding and appetite [reviewed in (Fride, 2002)].

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While there is less known about the physiologic role of OEA and PEA, they have been implicated to have neuroprotective effects, to modulate DA neurons, to increase neurosteroïd production and to play a role in memory consolidation, seizure protection, and inhibition of tumor proliferation (Campolongo et al., 2009; Mazzola et al., 2009; Melis et al., 2008; Morales et al., 2007; Sasso et al., 2010; Sheerin et al., 2004; Sun et al., 2007). However, this thesis will mainly focus on the role of ECs in regulating reward processes, and their relevance in the development and reinstatement of drug addiction. AEA and 2-AG can serve as powerful reinforcers of self-administration in primates when injected intravenously (Justinova et al., 2005; Justinova et al., 2011). Furthermore, when given intravenously AEA increases extracellular DA levels in the NAc shell, a neurochemical feature regarded as the hallmark of reward (Solinas et al., 2006). Importantly these reinforcing effects of AEA are CB-1 mediated, and are potentiated by FAAH inhibition, which increases the levels of AEA, OEA and PEA in the synaptic cleft (Solinas et al., 2006). In addition, systemic AEA administrations was found to increase food intake (Hao et al., 2000), which was mimicked by local-infusion of AEA into the NAc (Mahler et al., 2007). Whereas AEA appears to stimulate food intake, 2-AG is suggested to be more important for appetitive aspects than the consummatory aspects of food reward (Kirkham et al., 2002). This individuality of each EC in regulating specific reward processes is further emphasized by the fact that the extracellular levels of each EC respond different to self-administration of drugs of abuse (Caille et al., 2007). ECs are thought to modulate the rewarding properties of nicotine through a CB-1 mechanism (Merritt et al., 2008). Administration of the CB-1 antagonist rimonabant, decreased nicotine self-administration and CPP in rats (Cohen et al., 2005a; Cohen et al., 2005b; Le Foll and Goldberg, 2004), confirming an involvement of endocannabinoid signaling in nicotine reinforcement. Supporting this notion, CB-1 knock-out mice did not display nicotine induced-CPP (Castane et al., 2005; Merritt et al., 2008). Furthermore, inhibition of FAAH, by administration of URB597, showed to dose-dependently increase the nicotine rewarding properties, measured by CPP (Merritt et al., 2008). These results indicate that ECs binding to the CB-1 receptor are crucial to nicotine’s rewardive value. However, OEA and PEA have also shown to be involved in nicotine induced reward processes. More specifically, there is one study that

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demonstrates their inhibitory role on nicotine induced DA release in mesolimbic areas (Melis et al., 2008). These findings introduce the PPAR-α receptor as a novel target for anti-addiction drug research.

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Analytical methods for ECs To understand the mechanism of action of drugs in vivo monitoring techniques of the neurochemistry of the CNS are essential. There are several methods for monitoring brain constituents, like neurotransmitters, during or after a pharmacological challenges. Tissue constituents can be analyzed post-mortem, implicating the possibility for studying multiple analytes (depending on the analytical method and sample preparation). However, a major disadvantage of this technique is the amount of animals required when studying the effects on different points in time. Furthermore, post-mortem degradation, release or even syntheses of the analyte, like ECs, hamper the physiological relevance of these findings (Bazinet et al., 2005; Hansen and Diep, 2009; McCue et al., 2009). Techniques that display an acceptable time-resolution and are able to monitor effects of a stimulus in a specific tissue in a freely moving animal are intracerebral electrophysiological recordings or the use of implanted biosensors and microdialysis probes. Electrophysiological recording is a method that relies on the electrical properties of tissues and cells. It implicates recording of the electrical activity of neurons, and particularly their action potential activity. While electrophysiology studies detect the activity of specific neurons, microdialysis and biosensors monitor the extracellular content of neurotransmitters and related compounds. There are different types of biosensors used to monitor CNS constituents. For routine use commercial available amperometric biosensor are of interest. There are two types of biosensors; those using cyclic voltametry [carbon fibers used by the group of Wightman (Kita and Wightman, 2008)] and those who are based on enzymatic catalysis of a reaction that produces or consumes electrons (by redox enzymes). The advantage of the latter is the specificity (enzyme dependent) and high time-resolution (sub-second measurements). However, disadvantages are the fact that only one analyte is monitored, possible interference signals of easily oxidizing compounds (like arachidonic-acid), and biofouling. Microdialysis represents an I-shaped semi-permeable membrane tube that is implanted stereotactically into the brain area of interests. A dialysis buffer, closely resembling the ionic composition of the extracellular fluid, perfuses the membrane via tubing attached to a syringe pump. Substances in the brain, that are small enough to pass through the membrane (e.g. neurotransmitters and peptides), will diffuse into the buffer and carried through the connective tubing to a collection vial (Parent et al., 2001;

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Torregrossa and Kalivas, 2008). Depending on the analyte or group of analytes, samples can then be analyzed by means of analytical methods such as HPLC, LC-MS(/MS), ELISA, etc. An extra dimension is added to the microdialysis method when a second probe is implanted (called the dual-probe method). This approach enables to study interactions between specific pathways such as the mesolimbic DA pathway (Westerink et al., 1996). Because of its robustness and the possibility of sampling multiple analytes, microdialysis is the preferred method in this thesis. Because ECs display a high lipophilicity current sample conditions for microdialysis result in an unacceptable low recovery. In order to improve the recovery of the ECs we have optimized the sampling conditions [see chapter 5 and (Buczynski and Parsons, 2010)].

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Objectives of this Thesis This thesis is based on five different studies, each with a unique experimental setup. The main objective of this thesis was to critically evaluate and further investigate the role of the cannabinoid CB-1 receptor and its ligands in the neurochemistry of reward processes that are relevant for addiction. In Chapter 2 we developed and evaluated the nicotine induced reward processes by monitoring extracellular DA in the VTA and the NAc shell). The microdialysis technique was used to monitor DA. The efficacy of the nicotine-induced release of DA in the two brain areas were compared. In Chapter 3 amphetamine was used to increase DA levels in the NAc and PFC, both areas are involved in the regulation of impulsivity, a behavioral parameter important for reinstatement of addiction. The goal of this study was to investigate whether the effect of amphetamine on monoamines such as DA, NE and serotonin – determined by the microdialysis method - was regulated by CB-1 receptors. In Chapter 5 we describe an elaborate the microdialysis method of the ECs: AEA, 2AG, OEA, and PEA. Because these compounds display a high lipophilicity current sample conditions for microdialysis cannot be used. In order to improve the recovery of the ECs we have optimized the sampling conditions. One of the critical parameters appeared the recovery of perfusion fluid itself. An altered fluid recovery might either drain or perfuse the extracellular fluid in the sampled brain region. In the final study the role of ECs in the VTA-DA reward circuitry was studied during nicotine administration (Chapter 6). In this chapter we used the optimal sampling conditions for the ECs (determined in Chapter 5), and applied the double probe microdialysis setup and pharmacological treatment that was developed in Chapter 2. Although these were the main focus points of the thesis, no unexpected interesting and probably clinically relevant finding would be left unevaluated. An example of such observation is presented in chapter 4, where we found an interaction between the CB-1 receptor and the efficacy of the SSRI citalopram to increase extracellular 5-HT. This interaction might have clinical implications for the use of anti-depressives.

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Chapter 2 Direct effect of nicotine on mesolimbic dopamine release in rat nucleus accumbens shell Published in Neuroscience letters 2011 Apr: 493 (1-2) 55-8 J. Kleijn 1, J.H.A. Folgering4, M.C.G. van der Hart 2, H. Rollema 3 , T.I.F.H. Cremers 1 , B.H.C. Westerink 1 1: University of Groningen, dept. Biomonitoring & Sensoring, Groningen, The Netherlands 2: Brains On-Line LLC, San Francisco, USA 3: Neurosci., Pfizer Global Res. & Dev, Groton, CT 4: Brains On-Line, Groningen, the Netherlands

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Abstract Nicotine stimulates dopamine (DA) cell firing via a local action at somatodendritic sites in the Ventral Tegmental Area (VTA), increasing DA release in the Nucleus Accumbens (NAcc). Additionally, nicotine may also modulate DA release via a direct effect in the NAcc. This study examined the contribution of the latter mechanism on NAcc DA release by applying nicotine systemically, as well as locally in the VTA and NAcc shell region in rats. Furthermore, the effect of i.v. nicotine on cell firing rate of dopaminergic neurons in the VTA was measured. Systemic administration of nicotine (0.32mg/kg s.c.) increased extracellular DA levels in the NAcc to ~1.5 fold of baseline, while DA levels in the VTA remained unaffected. A similar DA increase was observed after local NAcc infusion of nicotine (1µM and 10µM). However, 10 to 1000-fold higher nicotine concentrations were required in the VTA to produce a comparable 150% increase in extracellular DA levels in the ispilateral NAcc. Additionally, electrophysiological experiments showed that the dopaminergic firing rate in the VTA showed a trend towards an increase after a nicotine dose of 0.1mg/kg i.v. Taken together these data indicate that the effects of nicotine on DA release at the level of the NAcc might be more important for the rewarding effects than originally proposed.

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Introduction The addictive nature of smoking tobacco is thought to be mainly due to one compound: nicotine (Stolerman and Jarvis, 1995). The DA hypothesis of drug addiction states that the crucial common reinforcing property of drugs of abuse is an increased DA release (Di Chiara and Imperato, 1988; Wise and Bozarth, 1987), in the NAcc (Nestler, 2005; Nisell et al., 1994b). Nicotine fits this hypothesis as supported by studies where the toxin 6-hydroxydopamine injected into the rat NAcc blocked nicotine self-administration (Corrigall et al., 1994). Other research showed that nicotine stimulates DA cell firing and subsequently increases DA release in the NAcc via local action at somatodendritic sites in the VTA (Ikemoto, 2007). These sites are populated by nicotinic acetylcholine receptors (nAChRs) and are activated during nicotine induced reward (Corrigall et al., 1994; Wolske et al., 1993). Central nAChRs consist of variable numbers of α and β receptor subunits. The α7 and the α4β2 receptor are widely expressed on dopaminergic neurons in both the NAcc and VTA (Clarke et al., 1985; Kamiya et al., 1982). Convincing results from receptor blockades and nAChR knockout (KO) mice have shown that a range of nAChRs (β2, α4, α6, α7) in the VTA are crucial for nicotinic reward (Laviolette and van der Kooy, 2003; Panagis et al., 2000; Picciotto et al., 1998; Pons et al., 2008). When the nAChR β2-subunit was knocked-out mice no longer exhibited conditioned place preference (CPP) (Walters et al., 2006). After restoring the β2-subunit by a VTA lentiviral vector injection, the nicotine-induced DA release and electrophysiological response to nicotine were restored (Maskos et al., 2005). Additionally, the α4 and α6 subunit containing nAChRs within the VTA have shown to play a crucial role in the self-administration of nicotine (Pons et al., 2008), and the α7 is suggested to be specifically involved in the mediation of nicotine induced reward signals (Laviolette and van der Kooy, 2003). Taken together, these data suggest a crucial role of the nAChRs in the VTA in regulating nicotine effects and hence mediating addiction. However, the nAChRs on DA neurons in the VTA are pharmacologically different of those on the nerve terminals in the NAcc, as the sensitivity to nicotine, Acetylcholine, and epibatidine were shown to differ in both areas (Reuben et al., 2000). Furthermore, nicotine has apart from effects in the VTA, also shown to have its primary binding sites in the NAcc, as local

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infusions of nicotine in the NAcc cause an increase in NAcc DA release (Mifsud et al., 1989). It was shown that blocking the α7-nAChRs in the NAcc before systemic nicotine prevents the increase in NAcc terminal DA (Fu et al., 2000). These direct effects of nicotine in the NAcc suggest a role of NAcc nAChRs in mediating accumbal DA responses to nicotine. Therefore, we investigated the role of the nicotine on dopaminergic neurons in both the VTA and the NAcc shell in rats. Nicotine was administered systemically, or locally infused into the VTA or NAcc and effects on DA release were monitored by microdialysis. Additionally, the effect of i.v. nicotine administration on the firing rate of VTA DA neurons was measured using electrophysiological recordings.

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Materials and Methods Animals Male Sprague-Dawley rats (n=62; 275-320g; Harlan, The Netherlands) were used. After surgery, animals were individually housed in plastic cages (35x35x40cm) with ad libitum food and water. Experiments were approved by the IACUC of the University of Groningen. Drugs and drug treatment Nicotine-di-tartrate was purchased from Sigma-Aldrich (CAS:65-31-6) and apomorphine from Research Biochemicals International (CAS:314-19-2). Nicotine was dissolved in saline for systemic injections, or in Ringer’s solution for infusion by retrograde microdialysis (pH 7). Doses and concentrations of nicotine are given as free base. Apomorphine was dissolved in 0.5mg/ml ascorbic acid. Surgery and microdialysis I-shaped 9mm long microdialysis probes (1.5mm exposed membrane; polyacrylonitril, MWcut-off 40-50kDa; Brainlink, the Netherlands) were implanted simultaneously in the NAcc shell (coordinates from bregma and dura;Paxinos and Watson, 2007: A/P 2.0, L/M 1.2, V/D -7.9) and the VTA (coordinates: A/P -5.0, L/M 0.9, V/D -8.2). Probes were implanted under isoflurane anesthesia (2.5%, 0.6l/min (O2)) and local analgesia with bupivacaine and finadyne (2mg/kg, 1ml/kg s.c.) for postoperative analgesia. Microdialysis experiments were carried out after a 24-48h recovery period by perfusing the probes with Ringer’s solution (in mM: NaCl 140.0, KCl 4.0, CaCl2 1.2, MgCl2 1.0) at a flow rate of 1.5µl/min (CMA402 syringe-pump, CMA microdialysis). To study the effect of systemic nicotine, microdialysis samples were taken from both the NAcc and the VTA and analyzed for DA. To study the effect of local nicotine infusions, retrograde dialysis was used to administer nicotine directly into either the NAcc or VTA (in separate experiments). In these experiments DA was determined in the NAcc only. 20 minute samples were collected using a fraction collector (Univentor820 microsampler, TSE systems) and samples were stored at -80oC until analysis. After the experiment the animal was euthanized using pentobarbital and probe placement was histologically evaluated.

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DA analysis DA was quantified by HPLC with electrochemical detection. A Shimadzu LC-10-AD pump was used in combination with a C18 column (150x4.7mm2; Supelco LC18, USA), connected with an electrochemical detector (ESA 5011A, potential first cell: 300mV; potential second cell: -300mV). The mobile phase consisted of 0.1M sodium acetate adjusted to pH 4.2 with acetic acid, 1.8mM octane sulfonic acid, 0.3mM Na2-EDTA, and 120ml/L MeOH at a flow rate of 1.0ml/min. The detection limit of the assay was ~0.05nM DA. Electrophysiological experiments Electrophysiological experiments were carried out similar to Dremencov et al. (Dremencov et al., 2009). Additionally the cell type was verified by administration of, the DA agonist, apomorphine. Rats were anesthetized with chloral hydrate (400mg/kg i.p. additionally 100mg/kg when required). Although DA neurons are commonly studied using chloral hydrate, it cannot be excluded that the anesthesia influenced the results. Data analysis Microdialysis data were expressed as percentages of baseline DA levels. To determine nicotine sensitivity of the NAcc and VTA, the Area Under Curve (AUC), based on relative response, during treatment (t=0 to t=260 min) was calculated after baseline substraction. For effect of treatment statistical analysis was performed with one or two-way analysis of variance for repeated measures (ANOVA), followed by Student Newman Keuls post-hoc analysis. Electrophysiological data were analyzed by students T-test for independent variables. The overall level of significance was set at p<0.05.

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Results Systemic nicotine preferentially increases DA release in NAcc shell Injections with 0.32 mg/kg s.c. nicotine caused a significant increase in extracellular DA in the NAcc shell 20 min after administration to 163 ± 20% of basal at t = 40min (F(10,45)=6.642; p < 0.001; n=5; figure 1). From 80 minutes onward, there was no significant difference in DA levels compared to baseline. DA release in the VTA, from the same rat, was not significantly affected by nicotine administration (figure 1). Basal concentrations (not corrected for recovery) of DA in dialysates, from the VTA were (mean ± SEM in pMol / sample) 11.3±1.9 (n=5), and from the NAcc shell 53.4 ± 7.9 (n=49).

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Figure 1: The effect of systemic (s.c.) administration (t=0 min) of 0.32 mg/kg nicotine on DA release in the NAcc shell (solid circles) and VTA (open circles). Data are expressed as percentages of basal levels ± SEM (n=5). * p<0.05 vs. baseline.

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The NAcc shell is more sensitive to nicotine infusions, compared to the VTA When 1µM nicotine was locally infused into the NAcc shell by retrograde dialysis, DA release to the NAcc shell increased to 150% of basal levels (F(3,290)=4.575; p=0.013; figure 2). Increasing the concentration to 10µM produced an increase that was not significantly different from the response to 1µM nicotine. Infusing 0.1µM nicotine or vehicle into the NAcc did not affect accumbal DA release.

Figure 2: The effect of infusions in the NAcc shell (top panel) or VTA (bottom panel) with vehicle (open circles), 0.1µM nicotine (solid squares), 1µM nicotine (open squares), 10µM nicotine (solid circles), 100µM nicotine (open triangles), or 1000µM nicotine (solid triangles) on DA release in the NAcc shell. Infusions started at t=0 min and lasted for 260 minutes (bar). Data are expressed as percentages of basal levels ± SEM (NAcc: n=6; VTA: n=5).

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Nicotine infusions into the VTA via retrograde dialysis, only produced significant increases in DA release in the NAcc shell at concentrations over 10µM (F(3,285)=7.629; p=0.001; figure 2). Infusions with 10µM had no effect, while 100µM and 1000µM nicotine increased DA release compared to vehicle treatment. Both treatments elevated DA release to about 150% of baseline, with no significant difference between the 100µM and 1000µM treatment. The NAcc shell is 100-times more sensitive to nicotine than the VTA In figure 3 the mean AUC with baseline subtraction is given for each dose infused in NAcc or VTA, from 0 to 260 minutes. When the local infusions into the VTA and NAcc are compared in a dose-response curve it shows that the response in the NAcc is 100-times more sensitive than in the VTA. Both brain areas show a plateau of nicotine-induced DA release which is reached within one order of magnitude.

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Figure 3: Dose-response curve of nicotine infused in NAcc shell (solid circles) and VTA (open circles). Data represented are the AUC (%*min) plus SEM of each local infusion into NAcc or VTA (µM). Dotted line represents the NAcc basal DA release.

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Systemic nicotine increases VTA dopaminergic cell firing Administration of 0.1mg/kg i.v. nicotine showed a trend towards an increase in dopaminergic cell firing to 131% of pre-drug firing rates (figure 4). A lower nicotine dose (0.02mg/kg i.v.) was not effective. Administration of apomorphine (0.02mg/kg i.v.) decreased cell firing, this together with other criteria (position, waveform, firing rate) demonstrates that the cells monitored were indeed dopaminergic. The basal rate of DA neurons in the VTA was (mean ± SEM) 4.38 ± 0.84Hz (n=8).

base nic 0.02 nic 0.1 Apo 0.02

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Figure 4: The effect of systemic nicotine [0.02 and 0.1 mg/kg i.v. (grey)] on dopaminergic cell firing in the VTA. The effect of apomorphine [0.02 mg/kg i.v. (black)] demonstrates that dopaminergic neuronal firing was monitored. + p<0.1 two-sided vs. baseline.

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Discussion This study presents a sensitive response to nicotine in the NAcc shell, indicating that this region might play an important role in nicotine induced reward. Infusing 1µM nicotine into the NAcc shell resulted in an increase in accumbal DA to about 150% of basal. Infusing 100µM nicotine into the VTA increased accumbal DA to similar level as the 1µM NAcc shell infusion. These findings provide evidence that the current hypothesis of nicotine-induced reward might not be complete and the NAcc may play a more important role than previously thought. Therefore, the nicotinergic interaction with nAChRs, resulting in the all important rewarding accumbal DA release, might take place in the NAcc rather than in the VTA. Conversely, other microdialysis and electrophysiological studies in knockout mice demonstrated that the primary effect of nicotine is stimulation of DA cell firing by local action in the VTA (Hildebrand et al., 1999; Maskos et al., 2005; Nisell et al., 1994a, b). Selective activation of α4β2 nAChRs (the most abundant subtype in the VTA), induced rewardive effects shown with a systemic nicotine self-administration model (Pons et al., 2008), and in a CPP test (Tapper et al., 2004). However, in accordance with this and other studies the α7-nAChR in the NAcc is of high importance (Fu et al., 2000; Harfstrand et al., 1988; Kamiya et al., 1982). Blocking the nAChRs in the NAcc using the α7 specific antagonist α-bungarotoxin, the systemic nicotine-evoked DA release in the NAcc was reduced by 50% (Fu et al., 2000). These results indicate that at least part of the nicotine-induced increase in mesolimbic DA is the result of an interaction of nicotine with the NAcc nAChRs. Since diverse types of nAChRs are present in both the VTA and the NAcc, of which in reward the α4β2 and α7 are particularly implicated (Fu et al., 2000; Laviolette and van der Kooy, 2003; Maskos et al., 2005; Panagis et al., 2000; Picciotto et al., 1998; Walters et al., 2006). It is probably the ratio between receptor densities of these nAChR subtypes that dictates the effect of the VTA and NAcc in reward. This study shows a dose response for nicotine infusions in both the NAcc and VTA. The concentrations used in this study are previously reported although never in one single study (Mifsud et al., 1989; Nisell et al., 1994a, b). The 1µM and 10µM infusions into the NAcc shell increase accumbal DA, in line with previous studies (Mifsud et al., 1989). Misfud et al. have shown that infusion of 2.4µM nicotine into the NAcc area increased DA

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release to 490%. Although these authors report an increase in DA after 2.4µM nicotine infusion, the magnitude of their increase is far higher than ours. This discrepancy between responses may be explained by essential methodological differences between the studies (higher calcium concentration in Ringers solution, shorter recovery after surgery). VTA nicotine infusion concentrations of 100µM and 1000µM are required to evoke an increase in DA in the ipsilateral NAcc to about 150% of basal, as supported by previous reports (Nisell et al., 1994a, b). After infusing nicotine (1000µM) into the VTA or NAcc the increase in DA has been reported to be more pronounced when nicotine was infused in the VTA than in the NAcc (Nisell et al., 1994a). However in another study, NAcc nicotine (1000µM) infusions increase NAcc DA release to the same level (~150%) as the VTA nicotine (1000µM) infusions (Nisell et al., 1994b). Although the VTA infusions in the results of this study show a comparable DA response, which validate the model, the main difference between the study results are the effective doses in the NAcc. This discrepancy might be explained by the sub-region of the NAcc in which DA was monitored. The present study sampled the NAcc shell region specifically, where Nisell et al. placed the probe more lateral from midline and therefore positioned the probe more in the core region of the NAcc. As drugs of abuse preferentially increase DA in the NAcc shell, compared to the core region (Pontieri et al., 1995; Pontieri et al., 1996; Tanda et al., 1997). It seems crucial to study the dopaminergic response to nicotine in the NAcc shell region, an argument strengthened by a study where the NAcc shell was shown to be more responsive to systemic nicotine, in increasing DA release, compared with the NAcc core in acute and chronically pretreated rats (Nisell et al., 1997). In this study we found a clear difference between effective nicotine dose in the NAcc shell and VTA, on increasing accumbal DA release (see figure 3). Both the NAcc and VTA show a similar pattern reaching a maximum nicotine-induced DA release within one order of magnitude. It is expected that higher nicotine concentrations are needed to saturate the VTA, in a dual probe experiment, to elicit a maximal increase of extracellular DA in the NAcc (Westerink et al., 1992). However, the likelihood that this is the main explanation for the difference in responsiveness between NAcc and VTA is small, since the time to reach the maximal accumbal dopaminergic response is similar in both brain areas.

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Following a pharmacologically active dose of 0.32mg/kg s.c. nicotine, DA release was significantly increased in the NAcc shell region, but not in the VTA. DA release in the VTA is probably of dendritic origin and indicates, in freely moving animals, an increase in neuronal activity (Kalivas and Duffy, 1991). To further examine the role of nicotine on dopaminergic activity in the VTA, the effect of systemic nicotine on VTA DA cell firing was monitored. After a relatively high nicotine dose (0.1mg/kg i.v.) only a trend to an increased cell firing (to 131% of controls) was observed. While the lower dose of 0.02mg/kg i.v. had no effect. These results are in line with a study where similar and even higher doses of nicotine (up to 0.775mg/kg i.v.) were used to stimulate dopaminergic firing in rats (Pierucci et al., 2004). Although, these results are at odds with a study in β2 knock-out mice (Maskos et al., 2005), in which 0.03mg/kg i.v. nicotine significantly increased dopaminergic cell firing in the mouse VTA. These discrepancies might be explained by species differences. This would mean that VTA neurons in the rat are less responsive to nicotinic stimulation than those in mice, and could therefore differentially regulate DA release after i.v. nicotine administration. For the understanding of reward the distinction between the NAcc shell and core is important. Similarly it has been suggested that the VTA displays distinct functions in reward processes when the anterior or posterior VTA are compared. E.g., it was shown that applying the nAChR antagonist mecamylamine in the anterior VTA completely blocked the increase in accumbal DA release seen after systemic ethanol administration(Ericson et al., 2008; Tizabi et al., 2002), whereas mecamylamine in the posterior VTA was ineffective (Ericson et al., 2008). Therefore the location within the VTA, might be critical and could explain the moderate dopaminergic response on firing rate of DA neurons as observed in this study. The dose-response presented here for local nicotine infusion shows that the NAcc shell is more sensitive to nicotine than the VTA in increasing accumbal DA release. This provides evidence that the current hypothesis of nicotine-induced reward might not be complete and the NAcc could play a more important role than previously proposed. Therefore it is recommended that future studies implicate the NAcc as a possible key-player in regulating nicotine reward.

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Laviolette, S.R., van der Kooy, D., 2003. The motivational valence of nicotine in the rat ventral tegmental area is switched from rewarding to aversive following blockade of the alpha7-subunit-containing nicotinic acetylcholine receptor. Psychopharmacology (Berl) 166, 306-313.

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Chapter 3 Effects of amphetamine on dopamine release in the rat nucleus accumbens shell region depend on cannabinoid CB1 receptor activation Published in Neurochemistry International 2012 Jun: 60 (8) 791-8

J. Kleijn1*, J. Wiskerke2*, T.I.F.H. Cremers1, A.N.M. Schoffelmeer2, B.H.C. Westerink1, T. Pattij2† * Authors contributed equally to this manuscript,† Corresponding author: T. Pattij, Ph.D., E-mail: t.pattij@vumc.nl 1:Dept. Biomonitoring & Sensoring, University of Groningen, The Netherlands 2:Dept. Anatomy and Neurosciences, Neuroscience Campus Amsterdam, VU university medical center, The Netherlands

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Abstract The psychostimulant drug amphetamine is often prescribed to treat Attention-Deficit/Hyperactivity Disorder. The behavioral effects of the psychostimulant drug amphetamine depend on its ability to increase monoamine neurotransmission in brain regions such as the nucleus accumbens (NAC) and medial prefrontal cortex (mPFC). Recent behavioral data suggest that the endocannabinoid system also plays a role in this respect. Here we investigated the role of cannabinoid CB1 receptor activity in amphetamine-induced monoamine release in the NAC and/or mPFC of rats using in vivo microdialysis. Results show that systemic administration of a low, clinically relevant dose of amphetamine (0.5 mg/kg) robustly increased dopamine and norepinephrine release (to ~175-350% of baseline values) in the NAC shell and core subregions as well as the ventral and dorsal parts of the mPFC, while moderately enhancing extracellular serotonin levels (to ~135% of baseline value) in the NAC core only. Although systemic administration of the CB1 receptor antagonist SR141716A (0-3 mg/kg) alone did not affect monoamine release, it dose-dependently abolished amphetamine-induced dopamine release specifically in the NAC shell. SR141716A did not affect amphetamine-induced norepinephrine or serotonin release in any of the brain regions investigated. Thus, the effects of acute CB1 receptor blockade on amphetamine-induced monoamine transmission were restricted to dopamine, and more specifically to mesolimbic dopamine projections into the NAC shell. This brain region- and monoamine-selective role of CB1 receptors is suggested to subserve the behavioral effects of amphetamine.

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Introduction The psychostimulant drug amphetamine is not only one of the leading prescription drugs to treat Attention-Deficit/Hyperactivity Disorder (ADHD) (Elia et al., 1999; Kutcher et al., 2004; Swanson et al., 2011), but also a widely abused addictive substance (Costa e Silva, 2002; Miller et al., 1989). Amphetamine’s behavioral effects are known to depend on its ability to robustly enhance monoamine transmission throughout the brain (Fleckenstein et al., 2007; Robertson et al., 2009). Other neurotransmitter systems have, however, been implicated as well, including the endogenous cannabinoid system. Endocannabinoids (eCBs), which via the cannabinoid CB1, and to a lesser extent CB2, receptors function as a negative feedback mechanism in the brain (Iversen, 2003; Kano et al., 2009), have particularly been implicated in mediating amphetamine-induced locomotor activity (Pryor et al., 1978; Tzavara et al., 2003; 2009), psychomotor sensitization (Corbille et al., 2007; Thiemann et al., 2008a; 2008b), and the persistence of psychostimulant addiction (Wiskerke et al., 2008). Moreover, we recently showed that the effects of a low dose of amphetamine on impulsive behaviors in rats can be prevented by pharmacological blockade of CB1 receptors (Wiskerke et al., 2011b). Together, these findings underscore that activation of the eCB system may represent a mechanism subserving amphetamine-induced behaviors. Indeed, acute challenges with amphetamine have been shown to modulate eCB levels in several brain regions including the nucleus accumbens (NAC) (Thiemann et al., 2008a). Putative involvement of eCBs in amphetamine-induced behaviors is presumably related to the close interactions that exist between the eCB and monoamine neurotransmitter systems in the brain. Thus, there is ample evidence for functional interactions of the eCB system with dopamine (DA), norepinephrine (NE), and serotonin (5-HT) systems within brain areas such as the medial prefrontal cortex (mPFC) and NAC (Egerton et al., 2006; Lopez-Moreno et al., 2008; Schlicker and Kathmann, 2001). In addition, a large literature already exists on the role of CB1 receptors in the effects of various other abused substances on specifically NAC DA release, effects that in contrast to those of amphetamine depend on DA neural activity (Cheer et al., 2007; Serrano and Parsons, 2011). In general, although the results from studies employing CB1 receptor knockout mice

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have sometimes yielded mixed results, pharmacological studies have shown that acute CB1 receptor blockade reduces alcohol- and nicotine-induced, but not opiate- or cocaine-induced, tonic DA release in the NAC (for review, see Serrano and Parsons, 2011). In contrast, the CB1 receptor antagonist SR141716A has recently been shown to abolish transient DA release induced by alcohol, nicotine, and cocaine in rats (Cheer et al., 2007). Given the vast amount of studies indicating CB1 receptor involvement in amphetamine-induced behaviors and the occurrence of eCB-monoamine interactions in the brain, surprisingly little is known about the role of CB1 receptors in amphetamine-induced monoamine release that is independent of DA neural activity (Sulzer, 2011). In order to examine such a modulatory role of the eCB system and its possible monoamine- and brain region-specificity, we investigated the effects of behaviorally relevant doses of SR141716A (De Vries et al., 2001; Pattij et al., 2007; Wiskerke et al., 2011b) on amphetamine-induced increases in monoamine release in the NAC and mPFC. To that end, we used in vivo microdialysis to monitor the release of DA, NE, and 5-HT in the NAC shell and core subregions and the ventral and dorsal parts of the mPFC of freely moving rats following administration of SR141716A (0-3 mg/kg) alone and in combination with a low dose of amphetamine (0.5 mg/kg). A low dose of the latter drug was chosen given its clinical relevance (Elia et al., 1999; Kutcher et al., 2004) and because this dose is devoid of stereotypy effects (Kuczenski et al., 1991). Moreover, we recently showed that impulsive behavior in rats induced by this dose of amphetamine is CB1 receptor-dependent (Wiskerke et al., 2011b).

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Material and Methods Animals Male Wistar rats (275-320g; Harlan, The Netherlands) were used. After surgery, animals were individually housed in plastic cages (35x35x40cm). Animals were maintained at approximately 90% of their free-feeding weight starting 1 week prior to the beginning of the experiments by restricting the amount of standard rodent food pellets (Harlan Teklad Global Diet, Blackthorn, UK). Water was available ad libitum throughout the entire experiment. The experiments were conducted during the dark phase (19:00h – 07:00h) in a temperature (22 ± 2˚C) and humidity (55 ± 10 %) controlled room. Experiments were approved by the ethical committee of the University of Groningen (the Netherlands), and all efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, if available. Drugs and drug treatment SR141716A was kindly donated by Abbott (Weesp, the Netherlands), and dissolved as previously described (De Vries et al., 2001) in a mixture of ethanol, TWEEN80, and sterile saline in a ratio 1:1:18. (+)-Amphetamine-sulphate (O.P.G., Utrecht, the Netherlands) was dissolved in sterile saline. Amphetamine and SR141716A were freshly prepared on each testday and injected intra-peritoneally (i.p.) in a volume of 1 ml/kg bodyweight. Surgery and in vivo microdialysis Each rat was equipped with two microdialysis probes: one probe in the NAC (either shell or core subregion) and one in the mPFC (either ventral or dorsal part). Both microdialysis probes were implanted simultaneously under isoflurane anesthesia (2.5%, 0.6l/min (O2)) and local analgesia with bupivacaine and finadyne (2mg/kg, 1ml/kg s.c.) for postoperative analgesia. For the ventral and dorsal mPFC I-shaped 6mm long microdialysis probes (2mm exposed membrane; polyacrylonitril, MW cut-off 40-50kDa; Brainlink, the Netherlands) were implanted (coordinates from bregma and dura;Paxinos and Watson, 2007: A/P 3.0, L/M 1.0 (angle: 5˚), V/D -4.1 and A/P 3.0, L/M 0.8 (angle: 5˚), V/D -2.4, respectively). For the NAC shell and core (A/P 2.0, L/M 0.9, V/D -7.9 and A/P 1.6, L/M 1.8, V/D -7.7, respectively) 9mm long I-shaped microdialysis probes were implanted (1.5 mm exposed membrane; polyacrylonitril, MW cut-off 40-50kDa; Brainlink,

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the Netherlands). After surgery (performed on Fridays) animals were allowed to recover for 48h. Subsequently, all animals were tested in two microdialysis experiments, conducted on the following Monday and Friday to ensure that drugs administered on the first test-day would not interfere with the second test-day. Each animal was tested once using the probe in the nucleus accumbens (shell or core) and once using the probe in the mPFC (dorsal or ventral part), in a semi-random order, to reduce the number of rats required for this study. The drug dosages administered on each test-day were also chosen semi-randomly so no animal would receive the same treatment twice and to prevent any treatment-order effects. Microdialysis experiments were carried out by perfusing the probes with Ringer’s solution (in mM: NaCl 140.0, KCl 4.0, CaCl2 1.2, MgCl2 1.0) at a flow rate of 1.5µl/min (CMA402 syringe-pump, CMA microdialysis). 15 min samples were collected using a fraction collector (Univentor820 microsampler, TSE systems) and samples were stored at -80oC until analysis. The experiment started with the collection of four baseline samples followed by administration of SR141716A (1 or 3 mg/kg i.p., doses based on our previous work (De Vries et al., 2001; Pattij et al., 2007; Wiskerke et al., 2011b)) or vehicle. Another four samples were taken to determine the effect of SR141716A on monoamine release, after which amphetamine (0.5 mg/kg i.p., dose based on our previous work (Schoffelmeer et al., 2002; Wiskerke et al., 2011a; 2011b)) was administered and the effect of the drug interaction on monoamine release was monitored for 3 hours. At the end of the experiment, the animals were euthanized using an overdose of pentobarbital (20%) and probe placement was histologically evaluated. Monoamine analysis Complete sample dialysate monoamine content was determined as previously described (Rollema et al., 2011) using a Shimadzu LC-10-ADvp setup connected with Sciex API4000 MS/MS units (Applied Biosystems, the Netherlands). Chromatographic separation was performed using a reversed phase Synergi MAX-RP 100 x 3 (2.5µm particle size) column. Through soft ionization components were analyzed in the MS/MS with high sensitivity (LOQ: 0.05 nM) and selectivity. Data acquisition was performed using Analyst® 1.4.2.

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Data analysis For each analyte baseline samples (t=-60 to 0 min) of each drug treatment group were submitted to a one-way analysis of variance (ANOVA) for repeated measures per brain region with drug dose as between subjects factor, to exclude possible group differences. Subsequently, microdialysis data were expressed as percentages of baseline. To evaluate the effect of amphetamine on monoamine release in subregions of the NAC and mPFC, per brain region a repeated measures ANOVA was executed for each analyte with time as within subjects variable, followed by Student Newman Keuls post-hoc analyses to determine individual time point differences whereby t=60 min was used as a control value. For the effect of drug (amphetamine + SR141716A) treatment, statistical analysis was performed per analyte per brain region with a two-way repeated measures ANOVA, testing for the effect of drug dose and time, followed by Student Newman Keuls post-hoc analyses. In these analyses, to determine the effects of SR141716A alone, samples collected between t=0-60 min were analyzed, whereas for the effects of SR141716A on amphetamine-induced monoamine release samples collected between t=60-240 min were analyzed. The overall level of significance was set at p<0.05.

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Results The effects of administration of SR141716A (1 or 3 mg/kg) on monoamine release induced by acute amphetamine administration (0.5 mg/kg) were assessed by in vivo microdialysis in the NAC shell and core sub regions, as well as the ventral and dorsal parts of the mPFC. There were no significant differences between baseline values of any of the monoamines (DA, NE, or 5-HT) among different doses within one brain area. The pooled baseline monoamine levels per brain region recovered in this study are presented in table 1. Table 1: Mean basal monoamine levels in brain dialysates (not corrected for recovery)

Monoamine Brain area fmol/sample (± SEM)

n = (total)

F-value (treatment-effect)

Dopamine NAC shell 61.0 ± 8.6 19 F(2,10)= 0.58; N.S.

NAC core 47.4 ± 4.4 19 F(2,10)= 0.23; N.S.

ventral mPFC 8.4 ± 1.7 20 F(2,10)= 0.72; N.S.

dorsal mPFC 8.3 ± 1.6 20 F(2,10)= 3.21; N.S.

Norepinephrine NAC shell 7.0 ± 0.8 16 F(2,8)= 1.08; N.S.

NAC core 7.4 ± 1.4 16 F(2,10)= 1.02; N.S.

ventral mPFC 10.7 ± 0.9 16 F(2,8)= 0.25; N.S.

dorsal mPFC 11.8 ± 1.4 17 F(2,9)= 3.21; N.S.

5-HT NAC shell 4.0 ± 0.3 19 F(2,10)= 0.48; N.S.

NAC core 4.4 ± 0.6 19 F(2,10)= 0.48; N.S.

ventral mPFC 3.5 ± 0.3 19 F(2,10)= 1.24; N.S.

dorsal mPFC 6.3 ± 0.9 20 F(2,11)= 0.44; N.S.

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SR141716A dose-dependently blocks amphetamine-induced DA release in the NAC shell Results from separate analyses over the pre-amphetamine period (t=0-60 min) showed that neither dose of SR141716A alone altered DA levels in any of the brain areas monitored (figure 1; Treatment effects: NAC shell: F(2,59)=2.71, N.S.; NAC core: F(2,64)=3.56, N.S.; ventral mPFC: F(2,64)=0.47, N.S.; dorsal mPFC: F(2,66)=1.28, N.S.). In contrast, separate analyses of the amphetamine-vehicle treated animals showed that amphetamine increased extracellular DA levels in the NAC shell and core to approximately 200% of baseline (shell: F(12,60)=14.78, p<0.001; core: F(12,60)=18.51, p<0.001), with post-hoc testing revealing a significant amphetamine effect between t=90-165 min in both subregions of the NAC. In the ventral and dorsal parts of the mPFC amphetamine caused increases of ~270% and ~190%, respectively (ventral mPFC: F(12,43)=6.92, p<0.001; dorsal mPFC: F(12,59)=3.65, p<0.001). Post-hoc testing revealed that amphetamine increased mPFC dopamine release between t=90-105 min.

Figure 1: Effects of the CB1 receptor antagonist SR141716A (1 mg/kg (open circle), 3 mg/kg (closed triangle); i.p.) or vehicle (closed circle), administered at t=0 min, on DA release induced by amphetamine (0.5 mg/kg; i.p.; administered at t=60 min) in the NAC

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shell (upper left), NAC core (upper right), ventral mPFC (lower left) and dorsal mPFC (lower right). Data are expressed as mean percentage DA release of baseline ± SEM (n=5-7 per treatment per brain region). Asterisks indicate p<0.05 as compared to the vehicle. Only in the NAC shell, prior administration of SR141716A dose-dependently affected amphetamine-induced DA release with statistical analyses showing significant treatment (F(2,184)=5.51, p=0.015), time (F(12,184)=14.94, p<0.001), and treatment × time effects (F(24,184)=1.67, p=0.033). Further comparisons revealed that particularly from 30 to 90 minutes following amphetamine administration (t=90-150 min) there was a significant reduction in amphetamine-induced DA release after administration of 3 mg/kg SR141716A versus vehicle administration. In fact, prior administration of 3 mg/kg SR141716A completely prevented amphetamine-induced increments in NAC shell DA release (F(12,55)=1.94, N.S.). For all other brain regions, SR141716A did not affect amphetamine induced DA release (Treatment: NAC core: F(2,191)=2.60, N.S.; ventral mPFC: F(2,181)=0.28, N.S.; dorsal mPFC: F(2,190)=0.22, N.S.). SR141716A does not affect amphetamine-induced NE release in the NAC or mPFC In the amphetamine-vehicle treated animals, acute administration of amphetamine increased extracellular NE levels in all brain areas monitored (figure 2). The relative increase in extracellular NE was most pronounced in the mPFC, with an increase of ~345% (F(12,48)=7.34, p<0.001; significant post hoc tests between t=90-135 min) and ~220% (F(12,48)=8.07, p<0.001; significant post hoc tests between t=90-120 min) as compared to baseline in the ventral and dorsal mPFC, respectively. In the NAC shell and core, amphetamine increased NE levels to ~205% (F(12,48)=7.69, p<0.001; significant post-hoc tests between t=90-135 min), and 175% (F(12,48)=4.00, p<0.001; significant post-hoc tests between t=75-240 min) of baseline values, respectively. Prior administration of SR141716A did not affect amphetamine-induced NE release (Treatment: NAC shell: F(2,139)=0.19, N.S.; NAC core: F(2,155)=0.21, N.S.; ventral mPFC: F(2,140)=0.39, N.S.; dorsal mPFC: F(2,157)=0.21, N.S.). Furthermore, SR141716A alone did not affect NE levels in any brain area monitored (Treatment: NAC shell: F(2,45)=2.47, N.S.; NAC core: F(2,52)=1.19, N.S.; ventral mPFC: F(2,48)=0.52, N.S.; dorsal mPFC: F(2,55)=0.70, N.S.).

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Figure 2: Effects of the CB1 receptor antagonist SR141716A (1 mg/kg (open circle), 3 mg/kg (closed triangle) i.p.) or vehicle (closed circle) administered at t=0 min, on NE release induced by amphetamine (0.5 mg/kg i.p.; administered at t=60 min) in the NAC shell (upper left), NAC core (upper right), ventral mPFC (lower left) and dorsal mPFC (lower right). Data are expressed as mean percentage NE release of baseline ± SEM (n=5-6 per treatment per brain region). Amphetamine moderately increases 5-HT levels in the NAC core via a CB1 receptor-independent mechanism During the 300 min of sampling the levels of 5-HT did not deviate more than 50% from baseline values in any brain region monitored. Nonetheless, administration of amphetamine (0.5 mg/kg) in the amphetamine-vehicle group resulted in a significant increase in 5-HT release in the NAC core (figure 3; F(12,54)=3.47, p<0.001). Post-hoc analysis revealed that only at t=105 min the level of extracellular 5-HT was significantly increased to ~135% of baseline. In the NAC shell, a one-way ANOVA for repeated measures revealed a time effect in the amphetamine-vehicle group (F(12,57)=3.40, p<0.001). However, further analyses did not identify any time point to be significantly different from baseline (t=60 min) in this brain

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region, thereby questioning the biological relevance of this finding. In the mPFC, administration of amphetamine had no effect on 5-HT levels (ventral mPFC: F(12,57)=1.34, N.S.; dorsal mPFC: F(12,57)=0.48, N.S.). Furthermore, there was no effect of administration of SR141716A alone on extracellular 5-HT levels in any brain region (Treatment: NAC shell: F(2,57)=1.29, N.S.; NAC core: F(2,59)=1.80, N.S.; ventral mPFC: F(2,64)=0.65, N.S.; dorsal mPFC: F(2,63)=0.83, N.S.). Likewise, no significant effects were detected of SR141716A followed by amphetamine on 5-HT release in the NAC or mPFC (Treatment: NAC shell: F(2,166)=0.49, N.S.; NAC core: F(2,176)=0.65, N.S.; ventral mPFC: F(2,182)=0.77, N.S.; dorsal mPFC: F(2,184)=0.42, N.S.).

Figure 3: Effects of the CB1 receptor antagonist SR141716A (1 mg/kg (open circle), 3 mg/kg (closed triangle) i.p.) or vehicle (closed circle) administered at t=0 min, on 5-HT release induced by amphetamine (0.5 mg/kg i.p.; administered at t=60 min) in the NAC shell (upper left), NAC core (upper right), ventral mPFC (lower left) and dorsal mPFC (lower right). Data are expressed as mean percentage 5-HT release of baseline ± SEM (n=5-7 per treatment per brain region).

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Discussion In the present study, we examined the in vivo effects of the cannabinoid CB1 receptor antagonist SR141716A on amphetamine-induced monoamine release in the NAC and mPFC by challenging rats systemically with a low dose (0.5 mg/kg) of amphetamine. Such a dose of amphetamine is clinically relevant (Elia et al., 1999; Kutcher et al., 2004) and has previously been shown in rats to robustly enhance locomotor activity (Schoffelmeer et al., 2002) and to affect impulsivity (van Gaalen et al., 2006a; 2006b; Wiskerke et al., 2011a; 2011b) in rats without causing stereotyped behaviors (Kuczenski et al., 1991) under our experimental conditions. The locomotor activating and addiction-related behavioral effects of psychostimulants, such as amphetamine, are known to be mediated by the mesocorticolimbic monoamine systems (Fleckenstein et al., 2007; Robertson et al., 2009) in concordance with other neurotransmitter systems, including the eCB system (Corbille et al., 2007; Pryor et al., 1978; Thiemann et al., 2008a; 2008b; Tzavara et al., 2003; 2009; Wiskerke et al., 2008). Moreover, we recently showed that the effects of a low dose of amphetamine (0.5 mg/kg) on measures of impulsive behavior in rats could be reversed by pre-treatment with the CB1 receptor antagonists SR141716A and O-2050 (Wiskerke et al., 2011b). Despite this large body of behavioral evidence supporting a role for CB1 receptor activity in amphetamine’s effects, data on the underlying mechanisms are scarce. Several brain regions of the mesocorticolimbic system including the mPFC and particularly the NAC have been implicated in amphetamine’s actions on the eCB system (Hiranita et al., 2008; Rodriguez et al., 2011; Thiemann et al., 2008a). Furthermore, to our knowledge, only a single study has reported on the role of the eCB system in amphetamine-induced monoamine release, showing that genetic deletion of the CB1 receptor did not influence DA release in the NAC of mice following an acute amphetamine challenge (Tzavara et al., 2009). In contrast, we observed that pretreatment with SR141716A dose-dependently abolished the amphetamine-induced increase in DA release specifically in the NAC shell. This apparent discrepancy between our data and those of Tzavara and co-workers (2009) may well be due to the fact that these researchers used constitutive CB1 receptor knockout mice, whereas in the current study the

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effects of acute pharmacological blockade of CB1 receptors on amphetamine-induced monoamine release were studied. Importantly, our findings were not resulting from homergic effects of SR141716A and amphetamine on DA transmission, since SR141716A by itself did not significantly affect DA release in the NAC shell. In fact, administration of SR141716A alone did not significantly affect monoamine release in the NAC or mPFC, suggesting absence of an eCB tone regulating monoamine transmission in these brain regions under our experimental conditions as previously reported by others (e.g. Kleijn et al., 2011; Nakazi et al., 2000; Oropeza et al., 2005; Pistis et al., 2002; Solinas et al., 2006). Two earlier studies, however, did show stimulatory effects of SR141716A on monoamine release in both the mPFC and NAC (Need et al., 2006; Tzavara et al., 2003). It is possible that differences in experimental design, including route, dose, and volume of drug administration and the microdialysis technique employed explain this discrepancy in findings. Indeed, in both aforementioned studies, effects of SR141716A on monoamine release were mainly observed at a dose of 10 mg/kg. However, behavioral effects of SR14716A in rodents are usually already observed within a dose range of 1-3 mg/kg (De Vries et al., 2001; Pattij et al., 2007; Thiemann et al., 2008b; Tzavara et al., 2003; 2009; Wiskerke et al., 2011b). Hence, we here did not test higher doses, even though we acknowledge that different results may have been obtained with higher doses of SR141716A. It should also be mentioned that SR141716A is thought to have inverse agonistic properties under certain experimental conditions, i.e. that in the absence of an endogenous tone at the CB1 receptor it may have effects opposite to those of a CB1 receptor agonist, probably due to a reduction of constitutive receptor activity (Pertwee, 2005). Although a contribution of such effects to the current results cannot be ruled out at present, they are unlikely to fully explain our results given that we recently excluded a role for the inverse agonistic properties of SR141716A in dopamine-dependent behavior. Thus, under similar experimental conditions employed here, we fully replicated the effects of SR141716A on impulsive behaviors in rats with the neutral CB1 receptor antagonist O-2050 (Wiskerke et al., 2011b). The effects of SR141716A on amphetamine-induced monoamine release were restricted to DA transmission, as amphetamine-induced NE and 5-HT release were not affected by SR141716A pretreatment in the brain regions monitored. It should be noted here that, in contrast to the effects on DA and

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NE and in line with previous studies (Baumann et al., 2011; Dalley et al., 2002; Pum et al., 2007), the effects of amphetamine on 5-HT release were moderate, which may be related to the lower affinity of amphetamine for the 5-HT transporter versus the DA and NE transporter (Rothman and Baumann, 2003). Therefore, although we cannot exclude the possibility that SR141716A might reduce amphetamine-induced 5-HT release when higher doses of the psychostimulant, that do increase 5-HT release (Baumann et al., 2011; Kuroki et al., 1996), are administered, it seems safe to conclude that an interaction between the eCB and 5-HT systems does not play a major role in the robust locomotor and neurocognitive effects of low doses of amphetamine. Altogether, our data suggest that under our experimental conditions, which were specifically designed to match the experimental conditions in our recent study on the role of the CB1 receptor in amphetamine-induced impulsivity (Wiskerke et al., 2011b), CB1 receptor activation mediates the behavioral effects of amphetamine primarily by enhancing DA release in the NAC shell. Our study is not the first to show that CB1 receptors can control tonic DA release in the NAC shell induced by a drug of abuse (for review, see Serrano and Parsons, 2011). In addition, acute SR141716A-treatment was previously found to reduce alcohol-, nicotine-, and cocaine-induced DA transients into the NAC shell of rats (Cheer et al., 2007). The current results confirm and extend those findings by showing that CB1 receptors also modulate amphetamine-induced tonic DA release. In contrast to those of other drugs of abuse, the DA-releasing effects of amphetamines are independent of vesicle exocytosis at DA terminals. Thus, amphetamines induce robust DA release via reversed transport of cytosolic DA through the DA transporter in combination with a complex series of intracellular actions including effects on DA-related enzymes and molecules such as tyrosine hydroxylase, monoamine oxidase, and vesicular monoamine transporter-2 (for excellent reviews on this subject, see e.g. Fleckenstein et al., 2007; Robertson et al., 2009; Sulzer, 2011). Our findings therefore suggest that CB1 receptor modulation of drug-induced alterations in DA release is independent of the propagation of action-potentials in DA neurons. Instead, the current findings together with the abovementioned previous findings suggest that eCB involvement in drug-induced DA transmission is heavily dependent on the release pattern and the anatomical locus of DA release. In this respect it is of interest that the current effects

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of SR141716A were found to be restricted to mesolimbic, but not mesocortical DA projections. The observed lack of effect of prior SR141716A administration on amphetamine-induced mPFC DA release was somewhat surprising given that both endogenous and exogenous CB1 receptor agonists have consistently be shown to enhance basal DA release in the NAC as well as the mPFC (Chen et al., 1990a; 1990b; Oropeza et al., 2005; Solinas et al., 2006; Tanda et al., 1997). On the other hand, our finding that CB1 receptor control over mesolimbic DA release is selective for the NAC shell is consistent with a previous study showing that CB1 receptor agonists induced DA release in the NAC shell but not the NAC core (Tanda et al., 1997). It is as yet unclear how CB1 receptors modulate amphetamine-induced, action potential-independent DA release. This might include G-protein-mediated inhibition of one or more of the presynaptic processes in DA nerve terminals acted upon by amphetamine to induce non-vesicular DA release. CB1 receptors are densely expressed in the NAC shell (Pickel et al., 2006; Wenger et al., 2003). However, DA neurons are generally believed to be devoid of CB1 receptors (Herkenham et al., 1991; Mailleux and Vanderhaeghen, 1992; Matsuda et al., 1993), albeit there is some inconsistency in the literature in this respect (Pickel et al., 2006; Wenger et al., 2003). Assuming that DA terminals are devoid of CB1 receptors, one must assume that activation of CB1 receptors reduces the negative feedback regulation of DA neural activity. Since drugs were administered systemically in the present study, the anatomical locus of the CB1 receptors involved in amphetamine-induced enhancement of DA levels in the NAC shell also remains elusive. Therefore, SR141716A may inhibit amphetamine-induced DA transmission indirectly by modulating glutamatergic and particularly GABAergic neurotransmission within the NAC shell or one of its afferent structures (Lopez-Moreno et al., 2008; Sperlagh et al., 2009). Perhaps noteworthy in this regard is the observation that infusion of SR141716A or its structural analogue AM251 into the NAC core, a brain region with GABAergic efferents to the NAC shell (van Dongen et al., 2005), suppressed methamphetamine-induced stereotypy (Morra et al., 2010). It is also conceivable that CB1 receptor blockade may have affected GABAergic and/or glutamatergic input onto the DA cell bodies in the ventral tegmental area (VTA) that innervate the NAC (Lupica and Riegel, 2005; Melis et al., 2004; Szabo et al., 2002). To resolve this

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issue, future studies should combine in vivo microdialysis in the NAC shell with local infusion of CB1 receptor (ant)agonists in various mesocorticolimbic brain regions. In conclusion, we showed that systemic administration of the cannabinoid CB1 receptor antagonist SR141716A blocked the increase in DA release in the NAC shell but not in the NAC core, or the dorsal and ventral parts of the mPFC upon an acute challenge with a low dose of amphetamine. Moreover, the effects of this psychostimulant on NE and 5-HT release in the NAC and mPFC were unaffected by pharmacological blockade of CB1 receptors. This monoamine- and brain site-specific role of CB1 receptors may subserve the psychomotor and neurocognitive effects of amphetamine. Acknowledgements: This research was supported by a grant from the Dutch Top Institute Pharma (T5-107-1). None of the authors has any disclosure or conflict of interest to report.

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Chapter 4 CB-1 receptors modulate the effect of the Selective Serotonin Reuptake Inhibitor, citalopram on extracellular serotonin levels in the rat prefrontal cortex Published in Neuroscience research 2011. Jul: 70(3) 334-7 Jelle Kleijn1, Thomas I.F.H. Cremers2, Corry M. Hofland2, Ben H.C. Westerink1 1:Department of Biomonitoring & Sensoring, University of Groningen, The Netherlands 2:Brains On-line BV, Groningen, the Netherlands

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Abstract A large percentage of depressed individuals uses drugs of abuse, like cannabis. This study investigates the impact of cannabis on the pharmacological effects of the antidepressant citalopram. Using microdialysis in the prefrontal cortex of rats we monitored serotonin levels before and after cannabinoid (WIN55,212-2 or rimonabant) and citalopram administration. Stimulating CB-1 decreased the effect of citalopram on increasing serotonin levels in the prefrontal cortex. Blocking CB-1 augmented this effect of citalopram. Although repeating these experiments in a chronical setting is recommended the present results might have implication for the clinical effects of citalopram.

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Introduction Predominantly amongst adolescents, there is a strong link between the use of drugs of abuse, especially cannabis, and mood disorders (Bovasso, 2001; Rey et al., 2002; Rey and Tennant, 2002). There are three hypotheses explaining this link. First, the self-medication hypothesis which states that the use of drugs, like cannabis, is a way to manage the depressive symptoms (Khantzian, 1985; Miller-Johnson et al., 1998). The second hypothesis suggests that comorbidity between cannabis dependence and depression likely arises from both genetic and environmental factors (Fu et al., 2002; Lynskey et al., 2004). The third hypothesis, states that high levels of cannabis use, especially during adolescence, trigger the onset of depression (Bovasso, 2001; Rey and Tennant, 2002). Whichever hypothesis is true, the facts remains that there is a comorbidity between cannabis use and depression (Kaminer et al., 2007; Rowe et al., 1995; Troisi et al., 1998). Furthermore, there are studies suggesting that depression has a negative effect on treatment outcomes of substance use disorder (Cornelius et al., 2004; Riggs et al., 1995; Subramaniam et al., 2007). However, little is known about the influence of the use of “drugs of abuse”, like cannabis, on the effectiveness and outcome of treatment of depression using SSRI’s. Although various clinical studies have reported depressed patients using cannabis display poor illness treatment success rates (Raphael et al., 2005). In this rat study we asked whether stimulation or blocking the CB-1 receptor acutely modulates the effect of an acutely administered SSRI on extracellular serotonin (5-HT) levels in the brain. To answer this question we used microdialysis to sample the rat medial prefrontal cortex (mPFC) and LC-MS/MS to asses the 5-HT levels. Since the CB-1 receptors in the PFC are up-regulated in the PFC of rats subjected to different models of depression (Bortolato et al., 2007; Hill et al., 2008), we have chosen to focus on the PFC. As a typical SSRI the antidepressant citalopram was chosen.

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Material and Methods All animal experiments were conducted according to the guidelines of the animal ethical committee of the University of Groningen. Male Wistar rats (225-325 g, Harlan; the Netherlands) were maintained on a 12 hour light/dark cycle. The CB-1 antagonist rimonabant (Kemprotec limited, Middlesbrough UK) and the CB-1 agonist WIN55,212-2 (Tocris Bioscience, UK) were suspended in 1% Tween80 in saline. Citalopram (provided by Lundbeck) was dissolved in saline. Rimonabant and citalopram were injected subcutaneous (s.c.1 ml/kg) and WIN55,212-2 was injected intraperitoneally (i.p.1 ml/kg). Rimonabant (1mg/kg) and WIN55,212-2 (1 mg/kg) were injected 30 minutes before citalopram was given on t=0 minutes. Based on earlier studies (Rea et al., 2010) a dose close to the EC 50% effect (3 mg/kg) of citalopram was chosen. No vehicle-vehicle group was included since similar experiments have shown no effect of vehicle treatment on baseline 5-HT levels in the mPFC (Allers et al., 2010). Using isoflurane anesthesia, a microdialysis probe (Brainlink, The Netherlands) was positioned in the medial prefrontal cortex (mPFC; 0.34AP; -0.08ML; -0.50DV mm relative to bregma and dura) according to the stereotaxic brain atlas (Praxinos and Watson, 2007). Ringers solution (in mM: NaCl 140.0, KCl 4.0, CaCl2 1.2, MgCl2 1.0) was used to perfuse the microdialysis probe (4 mm polyacrylonitrile membrane (cut-off 40-50 kDa) at a flow rate of 1.5 µl/min. After 2.5 hours of stabilization, collection of 20 minute fractions was started. Analyses were performed on a Shimadzu LC-10-ADvp setup connected with Sciex API4000 MS/MS units. 5-HT and its internal standard, 5-HT-d4 are derivatized with SymDAQ (Symmetrical DiAldehydes Quaternary ions) and through soft ionization analyzed with high sensitivity (LOQ: 0.05 nM) and selectivity. Data acquisition was performed using Analyst®1.4.2. All data were expressed as mean ± SEM and submitted to parametric tests (two-way analysis for repeated measures (ANOVA)), followed by Student Newman-Keuls method for post hoc comparisons. Statistical values reaching p ≤ 0.05 were considered significant.

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Results To study the role of the CB-1 receptor on the effectiveness of citalopram to enhance mPFC extracellular 5-HT, we injected rats either the CB-1 receptor agonist WIN55,212-2 or the CB-1 receptor antagonist rimonabant followed by citalopram. There was no significant difference between groups in baseline serotonin levels (F(4,58)= 1,582; P<0.218). The overall baseline PFC 5-HT levels were (mean ± SEM in fMol/sample): 6.32 ± 0.71 (n = 25). CB-1 receptor agonist decreases the effect of citalopram on increasing mPFC 5-HT levels Co-administration of WIN55,212-2 ( 1 mg/kg i.p.) and citalopram ( 3 mg/kg s.c.) (n = 5) largely suppressed the citalopram-induced increase in 5-HT (figure 1), whereas WIN55,212-2 (n = 5) itself had no effect on extracellular 5-HT levels in the mPFC (F(2,116) = 7,820; P<0.007). Citalopram (3 mg/kg, i.p.) increased mPFC 5-HT levels to about 420% of baseline, which started directly after administration, and was significantly higher compared to the other treatments during the first 80 minutes.

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Figure 1: Effect of WIN55,212-2 (1mg/kg i.p.), alone, or in combination with citalopram (3mg/kg s.c.) on 5-HT levels in the mPFC. Filled circle: WIN55,212-2; open circle: citalopram; filled triangle: WIN55,212-2 + citalopram. Baseline 5-HT levels within groups were: WIN-VEH 5.79 ±

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0.75 (n = 5); WIN-CIT 8.63 ± 1.31 (n = 5); and VEH-CIT 7.90 ± 2.59 (n = 5) (mean ± SEM in fMol/sample). Data are expressed as percentage of baseline ± SEM (n=5). * p<0.05 vs. other treatments. 5-HT levels were (mean ± SEM in fMol/sample): 6.32 ± 0.71 (n = 25).

CB-1 receptor antagonist augments the effect of citalopram on increasing mPFC 5-HT levels Combining the CB-1 receptor antagonist rimonabant (1 mg/kg s.c.) with citalopram (3 mg/kg s.c.) resulted in the opposite effect on extracellular mPFC 5-HT levels (figure 2). This combination seemed to prolong and augment the effect of citalopram on 5-HT levels alone (F(20,110) = 4,711; P<0.001). While there was no overall difference between treatments administering citalopram alone and in combination with rimonabant, dosing the animals with both rimonabant and citalopram resulted in an significant increase in mPFC 5-HT levels compared to other treatments at t =100, 120, and 160 minutes. Rimonabant itself had no effect on 5-HT release in the mPFC.

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Figure 2: Effect of rimonabant alone, or in combination with citalopram on prefrontal cortex 5-HT levels. Filled circles: rimonabant (1mg/kg s.c.), open circles: citalopram (3mg/kg s.c.); filled triangles: rimonabant + citalopram. Baseline 5-HT levels within groups were: RIM-VEH 5.52 ± 1.24 (n = 5); RIM-CIT 3.77 ± 0.74 (n = 5); and VEH-CIT 7.90 ± 2.59 (n = 5) (mean ± SEM in fMol/sample). Data are expressed as percentage of baseline ± SEM (n=5). * p<0.05 vs. other treatments.

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Discussion In our experiments, acute stimulation of the CB-1 receptors with WIN55,212-2 suppressed the effect of citalopram on increasing mPFC 5-HT levels. In contrast, blocking the CB-1 receptors with rimonabant augmented this effect of citalopram. The citalopram induced increase in extracellular 5-HT in the mPFC, to about 400 % of baseline, was clearly suppressed to only 200% of baseline by WIN55,212-2. In contrast, rimonabant enhanced the citalopram-induced increase in 5-HT levels, to about 550 % of baseline 120 minutes after citalopram administration. WIN55,212-2 or rimonabant alone did not affect extracellular 5-HT. This effect of rimonabant is in line with a study which showed that rimonabant in a dose of 1 mg/kg had no effect on 5-HT levels, although 3 mg/kg and 10 mg/kg both increased 5-HT efflux in the mPFC (Tzavara et al., 2003). That only high doses of rimonabant induce an effect on 5-HT release is supported by Darmani et al. who reported that an increase in 5-HT release after 20-40 mg/kg rimonabant (Darmani et al., 2003). The present data suggest an interaction between the pharmacological effects of citalopram on extracellular 5-HT levels and the CB-1 receptor. The most likely explanation might be found in the assumption that a CB-1 agonist, even in the rather low dose of 1mg/kg i.p., is inhibiting the activity of 5-HT neurons in the mPFC resulting in less 5-HT available for uptake inhibition with citalopram. This explanation is supported by studies that show lower levels of serotonergic activity (Sagredo et al., 2006), release (Nakazi et al., 2000), or the serotonin precursor 5-HTP (Moranta et al., 2009) in the prefrontal cortex after CB-1 agonist administration. A second, somewhat less likely explanation for the effect of WIN might be found in the assumption that CB-1 activation positively effects the transport of 5-HT and therefore directly counteracts the action of citalopram. This hypothesis is supported by a study that revealed a co-distribution of protein markers for monoamine release and re-uptake as well as CB-1 receptors on serotonergic neurons (Lau and Schloss, 2008). Lau et al. stated that, because of this co-expression, CB-1 receptors are able to inhibit serotonergic transport. Therefore, endogenous cannabinoids (ECs) could inhibit both release and reuptake of 5-HT by binding the CB-1 receptors. WIN55,212-2

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might therefore regulate serotonergic release and reuptake, preventing the effect of citalopram, explaining our results. The inhibition by WIN55,212-2 of the citalopram-induced increase in extracellular 5-HT was immediate, whereas the augmentive effect of rimonabant was only detectable 100 minutes after the administration of citalopram. An explanation for this effect might be more pharmacological than just a difference in route of administration. The later onset of rimonabant might be caused by the fact that citalopram-induced 5-HT levels had reached plateau values, implicating that the effect of rimonabant became apparent when the levels of 5-HT started to decline. A similar additive effect of the combination of citalopram and rimonabant as shown in this study was reported by Takahashi et al. who showed that cannabinoid CB-1 antagonists have an additive effect on certain behavioral parameters when administered concomitantly with citalopram or sertraline (Takahashi et al., 2008). In that study the combination of sub threshold dose of rimonabant and citalopram both decreased immobility time in the forced swim test and tail suspension test. The present results raise the question whether the observed acute interaction between the CB-1 receptor and 5-HT uptake inhibition has any clinical implications. As stated in the introduction there is comorbidity between cannabis use and depression (Kaminer et al., 2007; Rowe et al., 1995; Troisi et al., 1998). Therefore, do depressed patients treated with an SSRI limit the effect of their treatment by using cannabis? Indeed depressed patients using cannabis display poor illness treatment success rates (Raphael et al., 2005). Prospective studies of patients with a comorbidity of depression and substance abuse, demonstrate that treatment outcomes, such as symptoms prevalence, hospitalizations rates, and life-quality factors are worse when compared to single disorder patients (Chouljian et al., 1995; Linszen et al., 1994; Osher et al., 1994; Swofford et al., 1996). Finally it is emphasized that this study is based on acute administrations of both cannabinoids and citalopram. Chronic treatment would be more relevant when comparing to the clinical situation in depression. It cannot be excluded that the observed acute effect of SSRI on 5-HT level is relevant to the reported anxiety-like responses caused by acute SSRI (Bagdy et al., 2001), and that cannabinoids may counteract this effect.

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Summarizing the results of this study, it was shown that acute stimulation and blocking of the CB-1 receptor modulates the effect of citalopram on extracellular 5-HT levels in the mPFC. Extrapolating this observation to clinical practice suggests that cannabis use might lower the anti-depressive effect of an SSRI. This interaction of CB-1 (de)activation and SSRI effectiveness might be relevant, since a large percentage of depressed patient’s uses cannabis. It is of great importance to study these observations in more detail since the clinical impact of these findings might be considerable.

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Chapter 5 Optimizing the in vitro recovery of endocannabinoids sampled by microdialysis Manuscript in preparation J. Kleijn1, T. Klein2, C. M. Hofland2, K.D. Huinink4, M. Stienstra1, T.I.F.H. Cremers3, B.H.C. Westerink1

1: University of Groningen, dept. Biomonitoring & Sensoring, Groningen, the Netherlands 2: Brains On-Line, Groningen, the Netherlands 3: Brains On-Line LLC, San Francisco, USA 4: Brainlink, Groningen, the Netherlands

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Abstract

Endocannabinoids (ECs) are lipophilic compounds and are transported through the polar environment with the help of chaperone molecules. These characteristics affect the recovery rates of microdialysis probes and complicate the sampling and quantitation of ECs. To optimize the microdialysis setup we have studied the effect of four different microdialysis probes combined with three different perfusate additives on the in vitro recovery rates of ECs. The results show that adding β-cyclodextrines (β-CDs) to the perfusate is the most effective way of to increase the EC in vitro recovery rate. Other additives were not effective in increasing EC recovery rates. The Brainlink-NO probe appeared to be the best choice since it realizes the highest recovery rates combined with only a minor effect on fluid recovery. To minimize tissue damage caused by ultra-filtration or excessive drainage due to increased fluid recovery we optimized the (β-CDs) concentration. We emphasize to use 2% of β-CDs as this concentration had no effect on fluid recovery. In this study we identified the Brainlink-NO probe in combination with 2% of β-CDs as the most effective and save microdialysis approach.

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Introduction Since its development in the early 1980s in vivo microdialysis is extensively used as a in vivo sampling technique of brain tissue. Since microdialysis uses a virtually closed system, it results in minimal tissue disturbance during sample collection. Samples can be continuously taken and since they originate from the extracellular space they resemble relevant physiological concentrations much better than those provided after post-mortem brain tissue analysis. Samples collected during microdialysis have a low need for pre-analytical sample preparation since the membrane acts as a filtering barrier against large molecules like enzymes that could degrade the constituents. Microdialysis is most often used for sampling polar neurotransmitters (e.g. monoamines, acetylcholine and amino acids) that easily diffuse though the membrane into the lumen of the probe. However, more recently the focus for sampling larger and more hydrophobic like ECs, has increased. However for proper detection of ECs modifications of the microdialysis circumstances are needed The cannabinoid system has been the focus of a large number of studies and the role of ECs has shown to be extremely diverse. Surprisingly, to the best of the authors knowledge there are only 9 experimental reports and one review (Buczynski and Parsons, 2010) of in vivo rat microdialysis of ECs, derived from four different research groups. Some studies focused on fundamental principles of EC signaling (Bequet et al., 2007; Giuffrida et al., 1999), others studied the role of ECs in pain (Walker et al., 1999) or other disease-states (Villanueva et al., 2009). Several EC microdialysis studies were directed to the role of ECs on effects induced by drugs of abuse (Alvarez-Jaimes et al., 2009; Caille et al., 2007; Ferrer et al., 2007; Orio et al., 2009). Despite small variations in methodology such as sampling conditions, brain areas and analytical procedures, baseline EC concentration across these studies were consistent for both AEA and 2-AG (low nanomolars), with 2-AG showing higher (2-8x higher) concentrations. The efficiency by which an analyte is sampled through the membrane is described by the term recovery. Recovery is influenced by factors like the flow rate and composition of the perfusate, type of membrane (material and pore-size), and the tissues response to probe placement (biofouling). The term recovery can be divided into “absolute recovery” (e.g. the mass of

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analyte collected per unit of time) and “relative recovery” (concentration in % related to the concentration of the sampled extracellular fluid). The absolute recovery of an analyte is directly proportional to the perfusate flow rate (Wages et al., 1986). The first report of EC sampling that studied the recovery used a flow rate of 10µl/min (Giuffrida et al., 1999). This study lower flow rates resulted in EC levels below the limit of detection (LOD). However high flow rates, such as 10µl/min, create a lot of internal probe pressure and might affect the extracellular equilibrium (Dykstra et al., 1992). Most authors agree that flow rates ranging from 0.6-2.0µl/min are preferred. The disadvantage of slower flow rates is that they result in smaller sample sizes which might put the analytical method under significant strain. Another factor influencing the microdialysis recovery of an analyte is the membrane type. There is a wide variety in membrane materials, such as polyacrylonitrile, regenerated cellulose, polycarbonate, and polysulfone. For endocannabinoids the most often used membrane is made of polyethylsulfone (PES), which typically has a large pore size of 6-40 kDa, and a low EC adsorption (Bequet et al., 2007). ECs are smaller than 1 kDa and should be easily sampled using the low molecular weight cut-off membranes. However, the fact that ECs might bind to carrier proteins might seriously hamper the recovery. To sample carried-bound EC, membranes with a molecular cut-off >67kDa (size of albumin)] might be helpful. The major factor that determines the EC recovery is probably the perfusate composition. ECs are lipophilic and as a consequence will show poor solubility when a polar perfusion fluid is used. This poor solubility will affect the passage of ECs through the membrane. Therefore, the majority of microdialysis studies that sample ECs used an additive in the perfusion fluid, namely β-CDs. Because β-CDs have a hydrophilic outer surface and a hydrophobic central cavity, this additive can form inclusion complexes with non-polar molecules (Schneiderman and Stalcup, 2000) like ECs. Including relative high concentrations of β-CDs (up to 30%) in the perfusate, has shown to result in a 10-fold increase in recovery (Walker et al., 1999). Additionally, β-CDs might reduce the adsorption of ECs to the inner wall of the probe and tubing (Bequet et al., 2007), and lower the concentration of free ECs in the lumen of the probe which creates a concentration gradient increasing EC recovery (Khramov and Stenken, 1999). When using an additive there is a risk that the compound diffuses to the tissue directly surrounding the probe. Cyclodextrines can seriously damage cell functioning by extracting

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cholesterol from plasma membranes (Bari et al., 2005; Ohvo and Slotte, 1996). To reduce tissue damage or toxical effects induced by cyclodextrines, alternative additives should be considered. Other additives that are often used to increase recovery of lipophilic compounds are bovine serum albumin (BSA) (Jensen et al., 2007; Trickler and Miller, 2003) or dextran (60-1250kDa) (Dahlin et al., 2010; Rosenbloom et al., 2006). The advantage of these additives, when compared to β-CDs, is that they are already effective in relatively low concentrations (0.02-2%). Using lower concentrations of additives will result in a smaller effect on the osmolality of the perfusate, which creates less disturbance of the extracellular equilibrium. In addition, lower additive concentrations create less ion suppression during MS detection, creating less strain on the analytical method. In the literature there is a wide range of methods used to analyze ECs from biological matrixes. Most of them use chromatographic separation. Although there are also studies that show chemical derivatization of ECs followed by UV spectroscopy (Arai 2000, Yagen 2000, Wang 2001), this mechanism suffers from unspecific binding of the derivatization agent. Most methods use a combination of either mass-spectrometry coupled to gas chromatography (GC-MS) or liquid chromatography (LC-MS). Both of these methods produce limits of detection in the low nanomolar range (Hardison 2006, Alvarez-Jaimes L. 2009). Using LC coupled to mass spectrometry set on single ion monitoring (SIM) has proven to be a reliable method for EC quantitation. However, using tandem mass spectrometers creates a significant increase in selectivity since both the ion and one of its fragmented products can be monitored (multiple reaction monitoring: MRM). As previously stated, high concentrations of cyclodextrines could induce ion-suppression and therefore negatively affect the quantitation of ECs. As a consequent, it is recommended to first determine its effects on the analytical method, before using this or an alternative additive to enhance recovery. The goal of this study was to determine the optimal membrane and perfusate composition to sample endogenous cannabinoids (anandamide (AEA), 2-arachidonoylglycerol (2-AG), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA)). In order to achieve this we have chosen four

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types of microdialysis probes that differ in both membrane and probe design. Furthermore, we have compared three different types of additives in the perfusion fluid in order to determine which combination results in the most efficient EC sampling. The primary focus of this study was to identify the optimal microdialysis conditions and therefore the experimental setup consisted of relatively small group sizes (n≈5).

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Material and Methods In vitro microdialysis In this study we have used four different types of probes, which were equipped with three different membrane types (table 1). For all experiments the (total) flow-rate was set at 0.6 µl/min (CMA402 syringe-pump, CMA microdialysis). For the MetaQuant (MQ)- probe (Brainlink, The Netherlands) we used 0.5 µl/min dilution-flow and 0.1 µl/min ultraslow-flow. To limit ultrafiltration and therefore loss of perfusion fluid with the OM-probe (Brainlink, The Netherlands), the collectors were placed lower (40-60cm) than the probe to create a gravity driven pulling force (as described by (Rosenbloom et al., 2006)). All probes had an active membrane length of 4 mm. Ringers solution (in mM: NaCl 140.0, KCl 4.0, CaCl2 1.2, MgCl2 1.0) was used as standard pefusate. We have chosen three different additives in multiple concentrations to enhance EC recovery: β-CD (1-2-5-10-15-20-30%), BSA (0.2%), and Dextran 60kDa (1-2-5-10-15%).

Table 1: Overview of the different probe characteristics used in this study

Probe type Membrane Material Cut-off (kDa) MAB 6 Polyethersulfon 15 NO Polyacrylonitril 40-50 MQ Polyacrylonitril 40-50 OM Polyethylene 3000

During in vitro experiment the probes were connected with PEEK tubing and first stabilized for two-hours in Ringers solution, after which they were placed in EC bath solution and four 30 minute samples were taken using a fraction collector (Univentor820 microsampler, TSE systems). The EC bath solution consisted of 10-8 M AEA, PEA, OEA, and 10-7 M 2-AG in Ringers solution with 0.2% BSA (to prevent non-specific binding of ECs to the inner wall of the vial) and 0.02M formic acid (FA) (preventing degradation). The stock solution was stirred and kept on 37˚C during the experiment. The 100% reference sample (bath concentration) was taken at the end of the experiment to correct for possible degradation. Analysis EC in vitro recoveries were analyzed using LC-MS/MS. AEA, 2-AG, PEA, and OEA (deuterated versions as internal standards) were injected in

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samples of 18µl into the HPLC-MS/MS system by an automated, and cooled samples injector (SIL-20 AC, Shimadzu, Japan). Components were separated using a phenyl column (Inertsil Ph-3 (2.1x100 mm, 3 µm particles) GC sciences, Japan) perfused with a linear gradient of acetonitril (ACN) in UP (table 2). The gradient consisted of solvent A (UP/ACN 0.2 /99.8 with 0.1% FA) and solvent B (UP/ACN 10/90 with 0.1% FA), and the runtime/sample was approximately 7 minutes. All dilutions were prepared in 0.2% BSA in Ringers solution to prevent non-specific adsorption. Calibrations were conducted daily (linear range: 20pM to 20nM).

Table 2: LC scheme ACN

Gradient Time (min) %B Valve 0.0 30 3.0 MS 4.0 100 5.0 100 6.0 30 7.0 Waste 7.01 Stop

MS analysis was performed using an API 5000 MS/MS system consisting of an API 5000 MS/MS detector and a Turbo Ion Spray interface, probe temperature set at 500 ˚C. The acquisitions were performed in positive mode. The instrument was operated in multiple-reaction-monitoring (MRM) mode (see table 3 for MRM settings). Data were calibrated and quantified using Analyst data system (Applied Biosystem, version 1.4.2.).

Table 3: MS/MS MRM settings

Analyte Q1 Q3 2-AG 379.2 287.3 AEA 348.2 62.2 PEA 300.3 62.2 OEA 326.2 62.2

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Data analysis Data are presented as percentage of bath concentration. All data are expressed as mean ± SEM. Because of the small group sizes (n=3-5) there was no statistical analysis performed for the comparison of additives within probe types. For effect of additive on fluid recovery the statistical analysis between groups was performed with a one-way analysis of variance for repeated measures (ANOVA), followed by Student Newman Keuls post-hoc analysis. The overall level of significance was set at p < 0.05.

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Results & Discussion To determine the optimal conditions for sampling endocannabinoids we have studied the role of the composition of the perfusion fluid and the membrane material on in vitro recovery rates.

Figure 1: The effect of four different pefusate compositions (blank Ringer, Ringer-BSA, Ringer-Dextran 60kDa, and Ringer-β-CD) on the recovery rate of endocannabinoids using 4 different probe types: Microbiotech 6 (MAB6), Brainlink-NO (NO), Brainlink-MetaQuant (MQ), and the Brainlink-openmembrane probe (OM) (n=3-5). Direct comparison of the perfusate compositions and probe types (figure 1) shows that adding β-CDs to the perfusion fluid had the highest effect on EC recovery. For all four probe types β-CDs increased EC recovery to rates ranging from 40% ± 22.4 SEM to 112% ± 41.7 SEM for the NO-probe to a maximum recovery ranging from 299% ± 28.4 SEM to 611% ± 105.2 SEM for the MQ-probe. Using 0.2% BSA had no effect on EC recovery rates and

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was as ineffective as the control group of probes perfused with blank Ringer. Adding 2% Dextran 60kDa was only effective when used in combination with the NO-probe. In this approach 2% Dextran 60kDa increased EC recovery to rates ranging from 17% ± 8.6 SEM for AEA to 73% ± 37.4 SEM for PEA. Overall AEA showed the lowest recovery and PEA showed the highest recovery rates especially when sampled through a MQ-probe, which resulted in 30.9% ± 4.7 compared with blank Ringer and to 611% ± 105.2 SEM using 30% β-CD. These results clearly indicate that cyclodextrines are the most advantageous addition to increase the in vitro recovery of ECs. Where using blank Ringer and 0.2% BSA resulted in (average) recovery rates lower than 5%, but including 30% β-CDs often resulted in >100% recovery. Although recovery rates higher than 100% are suggesting either active carriage over the membrane or an increased fluid recovery, both of which could result in adverse effects in vivo, these results clearly confirm the earlier postulated notion that β-CDs are an effective tool in increasing EC microdialysis recovery (Buczynski and Parsons, 2010). The ineffectiveness of BSA as a perfusate additive in increasing EC levels was unexpected. BSA has proven to prevent fluid loss (ultra filtration), when used as a perfusion additive in microdialysis, and is therefore expected to increase recovery rates (Trickler and Miller, 2003). Another mechanism by which BSA could increase recovery of lipophilic compounds like ECs is that it will bind to the lipophilic binding sites in the lumen of the probe, both at the membrane site and tubing, so that ECs will stay in solution when transported to the collection vial (Jensen et al., 2007). A Third hypothesis by which BSA could aid recovery of large or lipophilic compounds is by binding to the lipophilic compound and thereby facilitating transport of these compounds to the probe and through the tubing. The ineffectiveness of BSA in the present study might be explained by the relatively low concentration that was used. Another possible explanation is that BSA bound too strong ECs keeping the compounds in solution both during the experiment and analysis. The analytical method was designed to prevent entry of BSA into the MS/MS, which could cause contamination, damage, and ion suppression, and therefore the eluens was switched to waste the first 3

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minutes. ECs bound to BSA might consequently not have reached the MS/MS. Dextran has proven to be a useful additive in microdialysis studies by increasing the recovery of large lipophilic molecules, by preventing fluid loss like BSA (Rosenbloom et al., 2006). In that study, Dextran 70kDa 6% increased the absolute recovery while actually lowering the relative recovery of proteins. In the present study Dextran only showed to be effective in combination with the NO-probe, where it increased recovery to 17.1% for AEA to 73.1% for PEA. These results were unexpected since the beneficial effect of adding Dextran should be mainly present in combination with a large pore membrane, where Dextran could increase recovery by preventing fluid-loss. In all four probe and membranes types adding β-CDs to the perfusate resulted in the highest recovery rates, some even higher than the optimal 100%. Since including a relatively high concentration of β-CDs will affect the osmotic balance, which will result in an inward movement of fluid into the perfusion fluid in order to correct for the difference in osmolality on both sides of the membrane. Although the scale of the excessive fluid recovery will determine the effect it has in vivo, tissue drainage of extra cellular fluid and constituents might induce pathological events. Therefore in the present study the amount of fluid recovered was carefully monitored (figure 2). The MAB6 probes recovered the highest amount of fluid with a fluid recovery of 164% ± 5.4 SEM (n=4), while the MQ-probes recovered the lowest amount of extra fluid with 110% ± 1.4 SEM (n=4). The NO- and OM-probe in combination with 30% β-CDs recovered fluid amounts of 125% ± 4.7 SEM (n=5) and 137% ± 3.9 SEM (n=4).

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Figure 2: The effect of 30% β-CDs on the fluid recovery of four different probe types. The area in red resembles the excess of fluid recovered.

There seems to be a relation between flow-speed at the membrane and the amount of fluid recovery when using 30% β-CDs. The probes with the highest internal diameter (the MAB6: ID ± 0.48mm and OM-probe: ID 0.33mm) that consequently have the lowest flow-speed at the membrane, displayed the highest fluid recovery. Although the MQ-probe has an even lower speed at the membrane with only one sixth of the total flow passing at the membrane, the absolute fluid recovery is lower than the other probes. When correcting for the lower membrane flow in the MQ-probe the fluid recovery increases from 110% to 160%, which is similar to the MAB6 probe. With the settings applied in the present study, the NO-probe suffers the lowest amount of β-CD induced ultra-filtration. Surprisingly, none of the studies previously reported and discussed the increase in fluid recovery when using β-CDs. Cyclodextrines are able to extract cholesterol from plasma membranes (Ohvo and Slotte, 1996) which results in a disruption of the lipid raft integrity (Bari et al., 2005; Ohvo and Slotte, 1996). Since this lipid membrane controls the CB1 receptor binding and signaling (Bari et al., 2005) any direct tissue contact of cyclodextrines might affect EC levels. However, Rosenbloom et al showed that an increase

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in vitro fluid recovery not directly implies that in vivo the fluid recovery is also affected (Rosenbloom et al., 2006). The latter study showed that adding Dextran 70 kDa 6% to the perfusion fluid resulted in an increase in in vitro fluid recovery to more than 180%. Under the same conditions in vivo only a minor increase in fluid recovery to 110% was observed. This difference between in vitro and in vivo effects could mean that monitoring in vitro fluid recovery is not a relevant measure controlling in vivo ultra-filtration. To establish the optimal cyclodextrine concentration six lower concentrations of β-CDs were used in combination with a NO-probe (figure 3). Without adding β-CDs the recovery is virtually absent (EC recovery < 5%), while adding 30% β-CDs resulted in recoveries higher than 100%. Figure 3 shows a dose-response of β-CDs on the recovery of ECs. Adding 2% or 5% was as effective as 10% and resulted in recovery rates ranging from 15.2% for AEA to 52.5% for PEA.

Figure 3: The effect of β-CDs (0-30%) on the recovery rate of endocannabinoids using a NO-probe.

In the present study addition of β-CDs 30% resulted in recovery rates ranging from 40 to more than 600% (figure 1). These recovery rates are higher than the 5.09-5.90% range initially reported in the first study conducting EC microdialysis (Walker et al., 1999). In that study a low

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recovery of about 0.34% using blank perfusate was reported, which is similar to the average recovery rate of 1.19% found in the present study. Reviewing the difficulties of sampling ECs Buczynski et al, showed that 10, 20 and 30% β-CDs are similar in effectiveness and realized a recovery rate of around 4% for AEA and 8% for 2-AG (Buczynski and Parsons, 2010). To the author’s opinion, the difference in recovery rate between the results of the present study and those reported in literature is difficult to explain by the methodological differences (perfusion fluid: Ringer vs. aCSF; connective tubing: PEEK vs. FEB) between our and their experimental setup. The most feasible factor responsible for the differences between the current results and those of Walker et al and Buczunski et al is the type of dialysis membrane used. The results of the fluid recoveries of different concentrations of β-CDs show a dose-response relation and are presented in figure 4. Increasing the concentration of β-CDs above 5% resulted in fluid recoveries higher than 100%, which were significantly different from 15% and higher concentrations (F(7,27)=9.021, p<0.001).

Figure 4: The effect of different concentrations of β-CDs (0-30%) on the fluid recovery of during collection using a Brainlink-NO probe. * p<0.01 relative to 0% β-CDs.

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Considering the pathological effects we observed during in vivo experiments (incidentally inducing paralysis; unpublished observations) using high concentrations of β-CDs in the perfusion fluid, all measures should be taken to prevent ultrafiltration and fluid drainage from the extracellular space. Therefore the optimal sampling conditions are not primarily those based on the highest possible relative recovery rates. The optimal sampling conditions are rather a balance between relative EC recovery and fluid recovery. Based on our results it is concluded that a β-CD concentration of 2% is conjunction with a NO-probe is the optimal sampling condition. Using these conditions we were able to sample and quantify ECs in in vivo experiments without inducing unwanted behavioral effects (Chapter 6 of this thesis).

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Conclusion The fact that only four laboratories were able to sample ECs from freely moving animals, using microdialysis, might be related to difficulties in controlling the recovery rate of these compounds. In the present study the optimal conditions for sampling ECs were determined. It was found that that a β-CD concentration of 2% is conjuction with a NO-probe is the optimal sampling condition. By applying these conditions an excess of fluid recovery was prevented and an acceptable recovery of the ECs for in vivo analysis (manuscript in preparation) was ensured.

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Chapter 6 The nicotine induced-dopamine release in the reward pathway: role of extracellular endocannabinoids Manuscript in preparation J. Kleijn1, T. Klein2, K.D. Huinink4, T.I.F.H. Cremers3, J.H.A. Folgering2, B.H.C. Westerink1

1: University of Groningen, dept. Biomonitoring & Sensoring, Groningen, the Netherlands 2: Brains On-Line, Groningen, the Netherlands 3: Brains On-Line LLC, San Francisco, USA 4: Brainlink, Groningen, the Netherlands

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Abstract The role of endocannabinoids (ECs) in the reward circuit has recently been the subject of various studies. It is generally assumed that ECs play a regulatory role regarding the reward-induced dopamine (DA) release in the NAc shell. Since extracellular ECs are difficult to sample, because of their lipophilic properties, microdialysis studies aimed at the pharmacology of the ECs are scarce. Here we studied the effects of nicotine on extracellular ECs and dopamine (DA) in the nucleus accumbens (NAc). Two microdialysis probes were implanted in the ventral tegmental area (VTA) and the ipsilateral NAc respectively. Nicotine was administered systemically or by local infusion through the microdialysis probes, whereas the extracellular levels of ECs (AEA, 2-AG, OEA, PEA) as well as DA were monitored in the VTA and NAc. To further evaluate the role of the CB-1 receptor in nicotine induced reward processes the CB-1 antagonist rimonabant was co-administered with nicotine. A LC-MS/MS method was used to analyze the ECs and DA in the dialysis samples. The results of this study showed that administration of nicotine (systemically as well as locally) enhanced DA release in the NAc shell. The increase of DA release in the NAc shell after local infusion of nicotine was dose-dependently inhibited by rimonabant, confirming the modulating role of the cannabinoid system in reward. Furthermore, while systemic nicotine increased the levels of all four ECs, the increase in AEA and OEA were inhibited by rimonabant. This indicates that AEA and OEA are implicated in the modulation of nicotine reward processes. Further evaluation of the endocannabinoid system is needed to confirm and explain the results presented here.

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Introduction Tobacco use by cigarette smoking is known to lead to serious health issues, such as various forms of cancer, pulmonary and cardiac diseases. Nicotine has been recognized to be the main addictive component of tobacco smoke, (Stolerman and Jarvis, 1995). Although clinical and pre-clinical studies indicate that several neurotransmitter systems mediate the neurobiological effects underlying nicotine addiction (George and O'Malley, 2004; Tanda and Goldberg, 2000), dopamine (DA) in the mesolimbic system is regarded as a crucial factor mediating the rewarding effects of drugs of abuse, including nicotine (Di Chiara and Imperato, 1988). While DA release in the nucleus accumbens (NAc) shell received much attention, focus of research has recently shifted towards systems that modulate the release of DA, like the endocannabinoid and opioid systems (reviewed by (Maldonado and Berrendero, 2010)). The distribution of CB-1 receptors in the CNS has been studied extensively (Herkenham, 1992; Herkenham et al., 1990; Moldrich and Wenger, 2000; Tsou et al., 1998). CB-1 receptors are found all over the brain and in significant densities in both the NAc and the VTA (Wenger et al., 2003). Furthermore, CB-1 receptors are known to co-localize with other receptor types, like opioid receptors (Salio et al., 2001). A large body of evidence demonstrates that the endocannabinoid system is involved in nicotine-induced reward processes (Maldonado et al., 2006; Scherma et al., 2008a). Three different cannabinoid CB-1 receptor antagonists (rimonabant, AM251, and SLV330) have been shown to effectively decrease nicotine self-administration in rats (Cohen et al., 2002; de Bruin et al., 2011; Shoaib, 2008). Furthermore, nicotine has been shown to induce conditioned place preference (Le Foll and Goldberg, 2005), indicating the addictive potential of nicotine, which both in rat and mice, has shown to partially depend on CB-1 receptors (Biala et al., 2009; Budzynska et al., 2009; De Vries et al., 2005; Le Foll and Goldberg, 2004; Merritt et al., 2008). The possibility of nicotine receptors and CB-1 receptors co-localizing within the NAc and/or VTA might suggest a role (direct or indirect) of CB-1 receptors in nicotine induced reward processes. Monitoring extracellular DA levels with microdialysis or voltammetry showed that the nicotine-induced effects on DA in the NAc shell are blocked by rimonabant pretreatment (Cheer et al., 2007; Cohen et al., 2002). In addition, rimonabant was also found to block the nicotine-induced effects

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on DA in the NAc shell and in the bed nucleus of the stria terminalis (Cohen et al., 2002), a structure connected with both the NAc and ventral tegmental area (VTA) and critical for the acquisition and maintenance of drug addiction (Epping-Jordan et al., 1998; Erb and Stewart, 1999). In humans, rimonabant has shown potential as an antirelapse agent in nicotine-addiction and obesitas. Nicotine addicts displayed significantly higher quitting rates when using rimonabant compared to placebo-treated smokers (Cahill and Ussher, 2007; Fernandez and Allison, 2004). Rats chronically treated with nicotine showed higher anandamide (AEA) tissue content levels in the limbic forebrain, a region important in reward processes (Gonzalez et al., 2002). By contrast, in the hippocampus, striatum, and cerebral cortex chronic nicotine treatment actually decreased both AEA and 2-arachidonoylglycerol (2-AG) levels, whereas CB-1 receptor mRNA levels remained unaffected. Furthermore, blocking the enzymatic degradation of AEA by administration of URB597 prevented the development of nicotine induced conditioned place preference (CPP), and acquisition of nicotine self-administration (Scherma et al., 2008b). It is assumed that increased synaptic AEA following administration of URB597 reduced the nicotine-induced enhanced DA release in the NAc shell, probably through inhibition of glutamate release in the VTA. Similar inhibitory effects on nicotine-induced DA release in the NAc shell were observed by two other ECs: oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) (Melis et al., 2008). Using electrophysiological methods Melis et al showed that OEA and PEA blocked the effects of nicotine by activation of the peroxisome proliferator-activated receptor-α (PPAR-α), a nuclear receptor transcription factor involved in aspects of energy management. Ultimately the activation of PPAR-α was hypothesized to lead to a negative regulation of neuronal nicotinic acetylcholine receptors. While nicotine affects DA release both in the VTA and NAc shell (Kleijn et al., 2011b), the role of ECs within these regions during systemic or local nicotine administration remains to be determined. In an earlier study we hypothesized that the nicotine-induced DA release is modulated in the NAc shell rather than in the VTA. In the present microdialysis study we investigated the role of extracellular ECs in the nicotine-induced DA release in the VTA-NAc shell pathway. An LC-MS-MS method was developed to

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determine the ECs in dialysates. Microdialysis probes were implanted in both the NAc shell and VTA and samples were collected during nicotine administration (both systemic and local through the microdialysis probe). In addition the effect of rimonabant on the neurochemical effects of nicotine were determined. Microdialysis samples were analyzed for DA and the ECs: AEA, 2-AG, PEA, and OEA.

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Material and Methods Animals Male Sprague-Dawley rats (275-320g; Harlan, The Netherlands) were used. After surgery, animals were individually housed in plastic cages (35x35x40cm). Food (Harlan Teklad Global Diet, Blackthorn, UK) and water was available ad libitum throughout the entire experiment. The experiments were conducted during the light phase (07:00h – 19:00h) in a temperature (22 ± 2 ˚C) and humidity (55 ± 10 %) controlled room. Experiments were approved by the committee for animal experimentation of the University of Groningen. Drugs and drug treatment The CB-1 antagonist rimonabant (Kemprotec limited, Middlesbrough UK) was dissolved as previously described (Kleijn et al., 2011a) in a mixture of 1% TWEEN80 and sterile saline. Nicotine-di-tartrate purchased from Sigma-Aldrich, was dissolved in either saline for systemic injections, or in Ringer’s solution for infusion via retrograde microdialysis into the VTA or NAcc shell. Solutions were brought to pH 7 using sodium hydroxide. After preparation on the day of experiment, solutions of nicotine and rimonabant were injected sub-cutaneously (s.c.) in a volume of 1 ml/kg bodyweight or infused locally in concentrations ranging from 1 to 100 µM. Surgery and in vivo microdialysis Two microdialysis probes were implanted simultaneously in each rat: one probe in the NAc and one in the VTA. Both microdialysis probes were implanted under isoflurane anesthesia (2.5%, 0.6l/min O2). Local analgesia was applied with bupivacaine, and finadyne (2mg/kg, 1ml/kg s.c.) and was given for general peri- and post-operative analgesia. For the NAc shell (coordinates: A/P 2.0, L/M 1.2, V/D -7.9) and VTA (coordinates: A/P -5.0, L/M 0.9, V/D -8.2) 9 mm long I-shaped microdialysis probes were implanted (1.5 mm exposed membrane; polyacrylonitril, MW cut-off 40-50 kDa; Brainlink, the Netherlands). After surgery animals were allowed to recover for 24 h. Animals were randomly distributed between dose groups. Microdialysis experiments were carried out by perfusing the probes with Ringer’s solution (in mM: NaCl 142.0, KCl 3.0, CaCl2 1.2, MgCl2 1.2) at a flow rate of 1.5 µl/min (CMA402 syringe-pump, CMA microdialysis). For

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sampling ECs β-cyclodextrin 2% was added to the Ringers solution and the flow rate was adjusted to 0.6µl/min, both changes incorporated to increase analyte recovery (see chapter 5). Twenty minute samples were collected using a fraction collector (Univentor 820 microsampler, TSE systems) and samples were stored at -80 oC until analysis. The experiment started with the collection of four base-line samples during which at t = -30 min an administration of rimonabant (1 or 3 mg/kg) was given. At t = 0 min nicotine or vehicle was administrated, either systemically (0.5 mg/kg s.c. (nicotine salt)) or locally [NAc: 1 µM, and VTA: 100 µM (both concentrations determined to be the lowest effective dose in earlier studies (Kleijn et al., 2011b))] and the effect of the drug interaction on DA and EC release was monitored for 3 hours. At the end of the experiment, the animals were euthanized using an overdose of pentobarbital (20 %) and probe placement was histologically evaluated. DA analysis Dialysate DA content was determined by an LC-MS/MS method as previously described (Rollema et al., 2011) using a Shimadzu LC-10-ADvp system connected to a Sciex API 4000 MS/MS unit (Applied Biosystems, the Netherlands). Chromatographic separation was performed using a reversed phase Synergi MAX-RP 100 x 3 (2.5 µm particle size) column. After a soft ionization moiety derivatization, DA levels were analyzed in the MS/MS with high sensitivity (LOQ: 0.05 nM) and selectivity. Data acquisition and analysis was performed using Analyst® 1.4.2. EC analysis AEA, 2-AG, PEA, and OEA (deuterated versions as internal standards) were injected in samples of 20µl into the HPLC-MS/MS system by an automated, and cooled samples injector (SIL-20 AC, Shimadzu, Japan). Components were separated using a phenyl column (Inertsil Ph-3 (2.1x100 mm, 3 µm particles) GC sciences, Japan) perfused with a linear gradient of acetonitril (ACN) in UP (Table 1). The gradient consisted of solvent A (UP/ACN 0.2 /99.7 with 0.1% formic acid (FA)) and solvent B (UP/ACN 10/90 with 0.1% FA). All dilutions were made with 0.2% BSA in Ringers solution to prevent non-specific adsorption.

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Table 1: LC scheme ACN

MS analysis was performed using an API 5000 MS/MS system consisting of an API 5000 MS/MS detector and a Turbo Ion Spray interface, probe temperature set at 500 ˚C. The acquisitions were performed in positive ionization mode. The instrument was operated in multiple-reaction-monitoring (MRM) mode (see Table 2 for MRM settings). Data acquisition and analysis was performed using Analyst® 1.4.2.

Table 2: MS/MS MRM settings Analyte Q1 Q3 2-AG 379.2 287.3 AEA 348.2 62.2 PEA 300.3 62.2 OEA 326.2 62.2

Data analysis For baseline samples (t = -60 to 0 min) of each drug treatment group were per brain region submitted to a one-way analysis of variance (ANOVA) for repeated measures with drug dose as between subjects factor, to exclude possible group differences. Subsequently, microdialysis data were expressed as percentages of mean baseline within each subject. To evaluate the effect of drug (nicotine, rimonabant) treatment on DA and EC levels, statistical analysis was performed per analyte per brain region with a two-way repeated measure ANOVA, testing for the effect of treatment and time, followed by Student Newman Keuls post-hoc analyses. The overall the level of significance was set at p<0.05.

Gradient Time (min) %B Valve

0.0 30 3.0 MS 4.0 100 5.0 100 6.0 30 7.0 Waste 7.01 Stop

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Results The effects of administration of rimonabant (1 or 3 mg/kg s.c) on DA and EC release enhanced by acute nicotine administration by systemic application (0.5 mg/kg s.c.) or local infusion (100 µM infused into the VTA or and 1µM infused into the NAc shell) were assessed by in vivo microdialysis of the VTA and the NAc shell. Basal values There were no significant differences between baseline values of the various experimental groups for extracellular DA or any of the extracellular ECs (2-AG, AEA, OEA or PEA) both in the VTA and NAc shell. The baseline neurotransmitter levels per brain region are presented in Table 3. When EC levels in the VTA and the NAc shell were compared, only PEA was present in significantly higher levels in the VTA (F(1,58)= 9.885; p=0.003) (*). Table 3: Mean basal neurotransmitter levels in brain dialysates (not corrected for recovery)

Monoamine Brain area Concentration (nM)

(± SEM) n = (total study)

F-value (treatment-effect)

Dopamine NAC shell 2.09 ± 0.31 93 F(18,74)= 0.919; N.S.

2-AG VTA 1.64 ± 0.40 59 F(9,43)= 1.239; N.S.

NAC shell 0.99 ± 0.19 61 F(9,45)= 1.370; N.S.

AEA VTA 0.10 ± 0.02 59 F(9,43)= 1.538; N.S.

NAC shell 0.08 ± 0.01 61 F(9,45)= 1.050; N.S.

OEA VTA 3.27 ± 0.30 59 F(9,43)= 1.88; N.S.

NAC shell 1.97 ± 0.23 61 F(9,45)= 1.393; N.S.

PEA VTA 17.38 ± 2.34 * 59 F(9,43)= 1.832; N.S.

NAC shell 11.17 ± 0.97 61 F(9,45)= 1.935; N.S.

*significantly higher basal level compared to the NAc shell

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Systemically administered nicotine enhanced DA release in the NAc shell: effects of rimonabant Systemic administration of 0.5 mg/kg (s.c.) nicotine resulted in a significant increased in DA release in the NAc shell, to about 160% of baseline (Figure 1). This increase lasted from 20 to and including 160 minutes after administration. Although this nicotine induced DA release was found to be significant for both treatment (F(5,267)=6.544; p<0.001), time (F(10,267)=11.010; p<0.001) and the interaction of treatment and time (F(50,267)=3.506; p<0.001), there was no effect of co-administrating rimonabant (1 and 3 mg/kg s.c.) with nicotine. However, there is a tendency towards a decrease in nicotine induced DA release following a co-administration of rimonabant 1 mg/kg s.c. (p=0.067).

Figure 1: Effects of rimonabant (1 mg/kg (open circle), 3 mg/kg (closed triangle); s.c.) or vehicle (closed circle), administered at t = -30 min, on DA release induced by nicotine (0.5 mg/kg; s.c.; administered at t = 0 min) in the NAc shell. As control administrations vehicle-vehicle (open triangle), rimonabant (1mg/kg s.c.) / vehicle (closed square), and rimonabant (3 mg/kg s.c.) / vehicle (open square) were included. Data are expressed as mean percentages of baseline ± SEM (n = 5-7 per treatment).

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Local administration of nicotine into the VTA enhanced DA release in the NAc: effect of rimonabant Local infusions of 100 µM nicotine into the VTA induced a significant increase in DA levels within the ipsilateral NAc shell, compared to vehicle treatment (F(3,190)=8.957; p=<0.001; Figure 2). Although the increase in DA release reached a maximum of about 150 % of baseline 60 min after injection, there was no significant effect over time (F(10,190)=0.730; p=0.695). Co-administration of rimonabant reduced the effect of VTA nicotine infusion significantly, while rimonabant itself had no effect on NAc shell DA levels. There was no difference in effectiveness between the two doses of rimonabant in reducing nicotine’s effects in DA levels.

Figure 2: Effects of s.c. rimonabant, 1 mg/kg (open circles) or 3 mg/kg (closed triangles) on DA release in the NAc shell induced by local nicotine (100 µM, closed circle) into the VTA. As control administrations vehicle-vehicle (open triangle), rimonabant (1mg/kg s.c.) and vehicle (closed square), and rimonabant (3 mg/kg s.c.) and vehicle (open square) were included. Rimonabant was administered at t = -30 min Data are expressed as mean percentages of baseline ± SEM (n = 5-7 per treatment).

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Local administration of nicotine into the NAc enhanced DA release in the NAc: effect of rimonabant Infusing of nicotine in low doses (1µM) into the NAc shell, while simultaneously sampling this region, induced an increase in DA levels to about 150% of baseline (Figure 3). The increase in DA levels persisted duration the nicotine infusion and was significant from t= 40 to t= 200 minutes (F(10,177)=2.289; p=0.015). The increase in NAc shell DA levels was fully suppressed by co-administration of both doses of rimonabant (1 and 3 mg/kg s.c.; F(3,177)=6.281; p=0.004). Administration of vehicle or rimonabant alone did not affect DA levels within the NAc shell.

Figure 3: Effects of rimonabant 1 mg/kg (open circle), 3 mg/kg (closed triangle); s.c.), on DA release in the NAc shell induced by local nicotine infusion (1µM; starting at t=0 min, closed circles). As control administrations vehicle / vehicle (open triangle), rimonabant (1mg/kg s.c.) / vehicle (closed square), and rimonabant (3 mg/kg s.c.) / vehicle (open square) were included. Rimonabant was administered at t = -30 min Data are expressed as mean percentages of baseline ± SEM (n = 5-7 per treatment).

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Effect of systemically administrated nicotine on extracellular EC levels in the VTA: effects of rimonabant Nicotine (0.5 mg/kg) had a pronounced effect on the extracellular levels of 3 of the 4 studied ECs (Figure 4) in the VTA. Nicotine increased extracellular AEA levels to about 450% compared to baseline (F(11, 316)=10.497; p<0.001; post hoc test between t= 60-220 min). Nicotine induced an increase in extracellular OEA levels in the VTA. The maximum increase was about 550% of baseline, 220 minutes after systemic nicotine administration (F(11,318)=35.010; p<0.001; post hoc test between t= 60-220 min). Nicotine also increased PEA levels within the VTA to a maximum of about 470% of baseline 220 minutes after systemic nicotine administration (F(11,310)=55.773; p<0.001; post hoc test between t= 40-220 min). Although, systemic administration of nicotine did not significantly affect VTA 2-AG levels compared to baseline, there was an effect of treatment and in time within this group of animals (treatment: F(5,305)=7.545; p<0.001); time: (F(11, 305)=3.907; p<0.001). There was no significant effect of nicotine on extracellular 2-AG in the VTA when compared to vehicle treatment, although there is a trend to increase its levels (p=0.06). Figure 4 also shows the effect of rimonabant (1 and 3 mg/kg s.c.) on systemic nicotine induced EC release within the VTA. Rimonabant alone did not affect EC levels in the VTA compared to the vehicle-vehicle treatment. Furthermore, no time dependent change was observed for any of the ECs in the vehicle / vehicle group. Rimonabant dose-dependently inhibited the nicotine induced increase in AEA release (F(5,316)=15.296; p<0.001). While rimonabant 1 mg/kg already decreased the effect of nicotine on AEA levels (p=0.042), it was not as effective as the higher dose of rimonabant (3 mg/kg), which fully suppressed the nicotine-induced AEA increase. The nicotine induced increase in OEA was also dose dependently inhibited by co-administration of rimonabant (1 and 3 mg/kg s.c.; F(5,318)=12.530; p<0.001). Co-administration with rimonabant did not affect the nicotine-induced PEA release within the VTA.

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Figure 4: Effects of rimonabant (1 mg/kg (open circle), 3 mg/kg (closed triangle); s.c.) or vehicle (closed circle), administered at t = -30 min, on nicotine (0.5 mg/kg; s.c.; administered at t = 0 min) induced VTA EC release (2-AG: upper left; AEA: upper right; OEA: lower left; PEA: lower right). As control administrations vehicle-vehicle (open triangle), rimonabant (1mg/kg s.c.) / vehicle (closed square), and rimonabant (3 mg/kg s.c.) / vehicle (open square) were included. Data are expressed as mean percentages of baseline ± SEM (n = 5-7 per treatment). Effect of systemically administered nicotine on extracellular levels of EC in the NAc shell: effect of rimonabant Nicotine (0.5 mg/kg) increased the extracellular levels of 2-AG, AEA, OEA and PEA in the NAc shell (Figure 5). Systemic administration of nicotine significantly increased 2-AG levels in the NAc shell to about 250% of controls (F(11, 305)=3.907; p<0.001; post hoc test between t= 140-220 min) and vehicle treatment (F(5,288)=3.588; p=0.011). Nicotine (s.c.) induced a significant increase of AEA levels to 330% of baseline (F(11, 320)=25.710; p<0.001; post hoc test for t= 120,160-220 min).

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OEA levels rose to about 450% of controls after nicotine treatment (F(11,

320)=25.710; p<0.001; post hoc test between t= 60-220 min), PEA levels within the NAc shell region increased to a maximum of about 450% of baseline 200 minutes after systemic nicotine administration (F(11,319)=64.171; p<0.001; significant post hoc test between t= 40-220 min). The effect of rimonabant (1 and 3 mg/kg s.c.) on systemic nicotine induced EC release within the NAc is also shown in Figure 5. Rimonabant inhibited the nicotine induced increase of OEA levels in the NAc shell, while leaving the increase in 2-AG, AEA, and PEA levels unaffected. The increase in extracellular OEA levels in the NAc shell after nicotine (s.c.) administration was dose-dependently inhibited by co-administration of rimonabant (F(5,

320)=7.524; p<0.001). From t = 100 minutes after nicotine administration the effect of rimonabant 3 mg/kg s.c. resulted in a significantly lower NAc shell OEA level compared to nicotine treatment alone. Rimonabant alone did not affect EC levels in the NAc shell compared to the vehicle-vehicle treatment. For 2-AG, AEA, and PEA there was no effect in time of vehicle-vehicle treatment. However, vehicle-vehicle treatment did affect PEA levels within the NAc shell (F(11,319)=64.171; p<0.001; post hoc test between t = 60-200 min).

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Figure 5: Effects of rimonabant (1 mg/kg (open circle), 3 mg/kg (closed triangle); s.c.) or vehicle (closed circle), administered at t = -30 min, on nicotine (0.5 mg/kg; s.c.; administered at t = 0 min) induced NAc shell EC release (2-AG: upper left; AEA: upper right; OEA: lower left; PEA: lower right). As control administrations vehicle-vehicle (open triangle), rimonabant (1mg/kg s.c.) / vehicle (closed square), and rimonabant (3 mg/kg s.c.) / vehicle (open square) were included. Data are expressed as mean percentages of baseline ± SEM (n = 5-7 per treatment). Effects of local infusion of nicotine into the VTA on extracellular levels of EC in the VTA and ipsilateral NAc shell: effects of rimonabant Infusion of 100 µM of nicotine into the VTA did not affect 2-AG (F(3,186)=2.026; N.S) and AEA (F(3,200)=0.286; N.S) levels (Figure 6). In addition, within the NAc shell the levels of 2-AG and AEA were not affected by the infusion of nicotine or the vehicle in the VTA. No effects were seen after systemic administration of rimonabant or vehicle (2-AG: F(3,200)=0.907; N.S; for AEA: F(3,197)=2.657; N.S). Although there were effects over time on the OEA levels and for treatment and time on PEA levels, none of them were nicotine dependent. For OEA

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there was an effect in time in both the VTA (F(11,202)=1.932; p=0.037) and NAc shell (F(11,212)=3.384; p<0.001), post-hoc analyses revealed that these changes did not occur compared to baseline. Although nicotine infusion into the VTA had no effect on EC levels, including PEA, administration of vehicle or rimonabant 3 mg/kg did, however, affect PEA levels in the VTA (treatment x time: F(33,200)=2.561; p<0.001) and the NAc shell (F(3,215)=4.687; p=0.012). Within the VTA, PEA levels were increased from t= 80 after rimonabant or t=100 minutes after vehicle / vehicle administration and lasted for the duration of the experiment. In the NAc shell, PEA levels were increased from t=200 minutes after rimonabant or t=60 minutes after vehicle / vehicle administration and lasted for the duration of the experiment (F(11,215)=21.627; p<0.001).

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Figure 6: Effects of nicotine infusion (100µM) into the VTA on the release of extracellular 2-AG, AEA, OAE and PEA (closed circles) in the VTA and NAc shell. In addition the effect of co-administration with rimonabant 3 mg/kg (open circle) administered at t = -30 min is shown. As control administrations vehicle / vehicle (closed triangle) and rimonabant (3mg/kg s.c.) / vehicle (open triangle) are included. Data are expressed as mean percentages of baseline ± SEM (n = 5-7 per treatment).

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Effect of local infusion of nicotine into the NAc shell on extracellular levels of EC in the NAc shell and VTA: effects of rimonabant Infusion of 1 µM nicotine into the NAc shell did not affect extracellular levels of 2-AG in the NAc or VTA (NAc shell: F(3,186)=0.919; N.S; and VTA: F(3,194)=0.489; N.S) Even so no effects were seen on AEA levels (NAc shell: F(3,186)=1.771; N.S; and VTA: F(3,199)=2.872; N.S) (Figure 7). Although infusing nicotine into the NAc shell did not affect OEA and PEA levels within the NAc shell or VTA, these ECs were affected by administration of vehicle or rimonabant 3 mg/kg. Within the VTA OEA increased in animals receiving systemic rimonabant 3 mg/kg (s.c.) compared to the other treatment groups (vehicle / vehicle, NAc nicotine infusion without and with rimonabant) (F(3,200)= 6,579; p=0.003). The levels of PEA, within the NAC shell and the VTA, were increased by the administration of both vehicle / vehicle and rimonabant 3 mg/kg (s.c.) alone. In the VTA, there was an effect for treatment, time and the interaction of treatment and time (F(33,197)=3,729; p<0.001) which lasted from t=100 minutes and t=40 minutes till the end of experiment for vehicle / vehicle and rimonabant 3mg/kg, respectively. The levels of PEA increased within the NAc shell after rimonabant and vehicle administration, compared to other treatments (F(3,206)=6,878; p=0.002), which started at t=60 (vehicle) and t=40 (rimonabant) and lasted till the end of the experiment (F(11,206)=17,166; p<0.001).

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Figure 7: Effects of nicotine infusion (1µM) into the NAc on the release of extracellular 2-AG, AEA, OAE and PEA (closed circles) in the VTA and NAc shell. In addition the effect of co-administration with rimonabant 3 mg/kg (open circle), administered at t = -30 min, is shown. As control administrations vehicle / vehicle (closed triangle) and rimonabant (3mg/kg s.c.) / vehicle (open triangle) are included. Data are expressed as mean percentages of baseline ± SEM (n = 5-7 per treatment).

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Discussion Similar to most other drugs of abuse nicotine has been shown to increase the activity of DA neurons in the VTA and consequently enhance DA release in the NAc (Cheer et al., 2007; Kleijn et al., 2011b; Nisell et al., 1994a). In the present study we have applied the microdialysis technique to investigate whether the endocannabinoid system is involved in this nicotine-dopamine interaction. To that end the CB-1 antagonist rimonabant was co-administered with nicotine whereas extracellular DA was recorded both in the VTA and NAc shell. In the second part of this study we have investigated whether the sampling of extracellular ECs by microdialysis could contribute to substantiate a possible nicotine-DA-CB-1 interaction. The nicotine-induced DA release in the VTA and NAc shell: effect of rimonabant The present results reproduced the literature findings, as systemic and local nicotine administrations induced a rapid increase in extracellular DA in the VTA and NAc shell. It is generally accepted that the CB-1 receptor is implicated in regulating nicotine’s rewarding effects, as co-administration of nicotine with a CB-1 receptor antagonist led to a decrease in nicotine self administration, prevents reinstatement to nicotine seeking (de Bruin et al., 2011; Forget et al., 2009; Shoaib, 2008), and blocked nicotine induced DA transients in the NAc shell (Cheer et al., 2007). However in the present study the effect of 3 mg/kg nicotine s.c. on NAc shell DA was not blocked by co-administration with the CB-1 antagonist rimonabant. Although we have no clear explanation for this finding at the present time, it should be noted that the dose of 1 mg/kg s.c. tended to inhibit the nicotine-induced DA release in the NAc shell (p<0.1). More convincing results regarding the nicotine-DA-CB-1 interaction were obtained when nicotine was administered locally through the microdialysis probe. Local infusions of nicotine rapidly increased NAc shell DA levels to about 150% of baseline, which is similar to previous reports (Mifsud et al., 1989; Nisell et al., 1994a, b). The fact that infusing nicotine directly into the VTA and the NAc increased NAc shell DA release implies that both regions play a direct role in the regulation of nicotine-induced reward. Infusing

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nicotine into the NAc shell as well as into the VTA increased DA release in the NAc shell, again indicating that both regions play a direct role in the regulation of nicotine-induced reward (Kleijn et al., 2011b). Interestingly the increase in DA release in the VTA as well as in the NAc was dose-dependently inhibited by systemic administrations of rimonabant. This finding suggests that the effects of local nicotine infusions on DA release are CB-1 receptor mediated. Although rimonabant was administered systemically, and therefore able to bind CB-1 receptors all over the CNS, the implicated CB-1 receptors are not necessarily restricted to the VTA or NAc shell. This hypothesis is in line with the findings in an experiment in which self-administration of ethanol was inhibited by intra-NAc infusion of rimonabant (Caille et al., 2007). The effect of nicotine and rimonabant on endogenous EC levels The main finding of the present study was that systemic nicotine not only increases extracellular DA levels in the VTA and NAc shell, but also increases the levels of ECs in both areas. To the best of our knowledge this is the first evidence of nicotine affecting extracellular EC levels in the brain. While the levels of AEA, OEA, and PEA were significantly increased (exceeding 300% of baseline) by nicotine, the levels of 2-AG in the NAc increased to 250% of baseline in the NAc shell and only showed a tendency towards an increase in the VTA. Since this is the first report presenting microdialysis data of effects of nicotine on ECs in the reward pathway, direct comparisons with existing data are not possible. However others have indirectly investigated the role of these ligands in nicotine induced processes. Blocking of the enzymatic hydrolysis of ECs by inhibiting or knocking-out fatty-acid-amide-hydrolase (FAAH) has been shown to suppress nicotine induced CPP, the development of nicotine self-administration (Scherma et al., 2008b), and suppresses the reinstatement of nicotine seeking (Forget et al., 2009). Inhibition of FAAH by URB597, blocked the effect of nicotine on the burst-firing of VTA DA neurons (Melis et al., 2008), and inhibits the stimulatory effects nicotine has on NAc shell DA levels (Luchicchi et al., 2010; Scherma et al., 2008b). In contrast, FAAH knock-out mice and URB597 administration have also showed to result in a dose-dependent increase in the rewarding effects of nicotine as measured by CPP (Merritt et al., 2008). Interestingly, increasing the dose of URB597 in that study actually diminished the increase in rewarding

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properties of nicotine. This discrepancy indicates the distinct functions of the substrates degraded by FAAH. Much of the effects elicited by URB597 have been ascribed to increased AEA levels. However, a PPAR-α antagonist has shown to block the analgesic effects of URB597, suggesting that ligands that bind to the PPAR-α receptor, like OEA and PEA, actually mediate these URB597 induced effects (Jhaveri et al., 2008). In line with this, it is shown that OEA and PEA directly inhibit nicotine reward, by suppressing the nicotine induced DAergic effects in the mesolimbic pathway (Melis et al., 2008). This finding is in line with the negative modulations of reward processes induced by URB597 administrations, which suggest that these might be OEA and/or PEA mediated. However, the nicotine induced increase in OEA and PEA levels in the VTA and NAc in the present study are contradictory to the inhibitory effects of these non-CB-1 ECs in published data (Jhaveri et al., 2008; Melis et al., 2008). In the present study nicotine had a stronger stimulatory effect on PEA and OEA when compared to AEA and 2-AG. The effect of nicotine on PEA and OEA levels shows a gradual pattern reaching high levels more than 60 minutes post-nicotine administration. The significance of this more pronounced increase of OEA and PEA when compared to the CB-1 receptor dependent ECs AEA and 2-AG, is presently unknown. When specifically looking at AEA and 2-AG, the increase in AEA was more pronounced than the effect of nicotine on 2-AG, suggesting that for nicotine reward, AEA might be a crucial agonist for the CB-1 receptor. This CB-1 receptor dependency was expected as AEA was reported to play a critical role in mediating the activity of DAergic neurons in the mesolimbic pathway after drug administration (Solinas et al., 2006). In addition, it is suggested that the by FAAH inhibition enhanced nicotine induced reward processes are AEA mediated (Serrano and Parsons, 2011). A differential effect on extracellular EC levels regarding AEA and 2-AG levels during reward has been noticed before (Caille et al., 2007; Justinova et al., 2005; Justinova et al., 2011), and showed extracellular levels of 2-AG and AEA that were differentially increased during self-administration of drugs of abuse (Caille et al., 2007). Self-administration of ethanol increased extracellular NAc shell 2-AG levels, while extracellular AEA levels were unaffected. Conversely, self-administration of heroin resulted in a strong increase in extracellular AEA levels, while 2-AG only marginally increased.

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In contrast to the systemic administration experiments direct application of nicotine into the VTA (100µM) and NAc shell (1µM) did not modify EC levels in the VTA and NAc. A possible explanation for the absence of effects of local infusion of nicotine on the ECs is to speculate that CB-1 pathways located outside the VTA-NAc reward system are involved in the CB-1 – nicotine interaction. However this conclusion is at variance with the finding that rimonabant blocked the enhanced DA release seen after local nicotine infusion (Figures 2 and 3). Effect of rimonabant on the nicotine induced extracellular EC levels The current study showed that the nicotine-induced increase of AEA and OAE levels in the VTA was CB-1 receptor dependent as rimonabant dose-dependently antagonized these effects. That the effects of OEA are modulated by the CB-1 receptor is an unexpected observation as there are no reports mentioning a direct link between this EC and the CB-1 receptor. Like other studies have reported rimonabant alone did not affect the release of ECs (Caille et al., 2007; Di et al., 2005). This was expected because ECs, unlike classical neurotransmitters, are only released upon demand and rimonabant acts as an antagonist in the concentrations used in the present study. This is the first report showing in a single experiment, a direct effect of rimonabant on both the rewardive potential of nicotine (increase in NAc shell DA) and the ECs AEA and OEA released after systemic nicotine administration. From the current results it might be concluded that AEA and OEA play a primary role in nicotine induced DA release. Whether this acts via a direct (CB-1) or indirect (via PPAR-α on other types of neurons) mechanism needs to be determined. Earlier studies in which ECs were sampled studied the effects of an antagonist not in a single experiment but combined two datasets (Alvarez-Jaimes et al., 2009; Caille et al., 2007; Giuffrida et al., 1999; Long et al., 2009; Orio et al., 2009; Villanueva et al., 2009). E.g. in these experiments it was shown that EC levels rose during ethanol self administration and that rimonabant could reduce this behavior (Alvarez-Jaimes et al., 2009; Caille et al., 2007). The technical difficulty of combining their experimental setup (behavioral experiment) with microdialysis explains the use of two separated experiments.

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It would be interesting to study the effects of local infusions of both CB-1 and PPAR-α agonists and antagonists into the VTA while sampling the NAc for EC and DA release. These experiments might result in a better understanding of the role of AEA and OEA in nicotine induced reward. To evaluate the role of the CB-1 receptor in the local nicotine infusion stimulated accumbal dopamine release, we monitored EC levels during local infusions, and monitored the effects of systemic rimonabant on these effects. Our experiments showed that the CB-1 receptor dependency of the dopaminergic effect after local nicotine infusion was probably not related to CB-1 receptors in the VTA or NAc shell since none of the monitored ECs was affected by nicotine infusions. Probably CB-1 receptors outside the VTA and NAc shell antagonize the effect of local nicotine infusions on accumbal dopamine release. We noticed an increase in PEA levels in the VTA after rimonabant administration (Figures 6 and 7). This increase was blocked during both types of nicotine infusions (in the VTA as well as in the NAc). At the present time we have no explanation for this effect. Basal values of EC Since sampling ECs is both technically and analytically challenging, there is only a limited number of studies available reporting basal EC levels. Extracellular levels of EC in the VTA are not published, as are the levels of OEA and PEA. However, there are reports of sampling AEA and 2-AG in the NAc shell (Alvarez-Jaimes et al., 2009; Caille et al., 2007; Ferrer et al., 2007; Orio et al., 2009), AEA (corrected for microdialysis recovery) was reported to be between 0.56 and 2.8nM, while 2-AG ranged from 4.4 to 10.3nM. These values are in line with the values found in the present study: the level of AEA and 2-AG in the NAc were: 0.53 (corrected for 15% AEA dialysis recovery), and 4.95 nM (corrected for 20% 2-AG dialysis recovery). These findings support our sampling method, which uses 15 times less β-cyclodextrine as dialysis-additive. However, the ratio between the level of AEA and 2-AG was 9.3, which is higher although not statistically significant, than those reported averaging 4.5 (range: 2-7.9). Remarkably, the efficiency of sampling was higher for 2-AG that for AEA, in contrast to earlier reports (Buczynski and Parsons, 2010). The difference as observed in recoveries are probably caused by the type of dialysis membrane used (polyacrylonitril vs polyethylsulfone).

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Conclusion

The present study shows that nicotine not only enhanced DA release but also stimulated the release of all ECs monitored, in both the VTA and NAc shell. The finding that systemic nicotine on extracellular levels of AEA and PEA was blocked by rimonabant suggests that both the CB-1 and PPAR-α receptors are involved in the CB1-nicotine interaction. The results of the present study might stimulate future research on the mechanism of nicotine reward and the contribution of the mesolimbic reward pathway. Studies combining microdialysis and behavioral models might provide more insight in the role that ECs play in nicotine reward and addiction.

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Chapter 7 Summary of the Thesis Jelle Kleijn

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This thesis focuses on the reward circuitry localized in the Ventral Tegmental Area (VTA) and Nucleus Accumbens (NAc). In particular the role that nicotine and CB-1 receptors play in reward processes related to drugs of abuse like nicotine and amphetamine was studied. Although it is realized that it was preclinical study, the present results might be useful to better understand reward and abuse in humans. In short First the responsiveness of dopamine (DA) release in the reward circuitry was re-evaluated, using nicotine as a stimulant (Chapter 2). Next a large series of in vitro recovery microdialysis experiments were carried in order to identify the optimal conditions for sampling endogenous cannabinoids (ECs) (Chapter 5). It is believed that these analytes act as mediators in the reward processes underlying addiction. Subsequently, we conducted a study in which both the nicotine reward model and the sampling technique were combined (Chapter 6). Two further studies during the evolvement of this thesis symbolize the versatility of the cannabinoid system in neuropharmacology. Chapter 3 describes the effect of amphetamine on DA release in four different sub-regions of the reward circuitry, focusing in particular at the role that CB-1 receptors play in these processes. In chapter 4 is it shown that the efficacy of the antidepressant citalopram to enhance serotonin release is modulated by CB-1 receptor activity. More specific After a general introduction (Chapter 1), we described in Chapter 2 the effect of nicotine in the VTA and NAc shell. The latter brain areas are considered crucial in nicotine induced reward processes. E.g. gene knock-out experiments have demonstrated the importance of an intact VTA for nicotine induced reward processes. In this chapter we studied the effect systemic nicotine administration on DA release in the NAc shell, which is regarded as being related to reward. Subsquently, local infusions of nicotine were used to determine the most sensitive brain regions (VTA or NAc shell) to nicotine. It appeared that the NAc shell responded to 1000 times lower nicotine concentrations, compared to the VTA. These results were further strengthened by electrophysiological experiments showing that only a relatively high nicotine concentration of 0.1mg/kg i.v. increased the firing of DA cells in the VTA. Apparently the NAc shell is more sensitive than the VTA for nicotine to increase the release of DA in the NAc. This indicates that not only the VTA is responsible for the nicotine induced reward but

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rather an interaction between the NAc shell and the VTA. Therefore, the NAc might be more than just a signaling center of the VTA, as it might play a crucial role in integrating the incoming DA signal into a reward stimulus. Chapter 3, describes an elaborate microdialysis study, carried out in order to identify the brain region(s) responsible for the effects of amphetamine on impulsive behavior. Impulsive behavior plays an important role in the development and reinstatement of addiction. Microdialysis probes were placed in sub-regions of the medial Prefrontal Cortex (mPFC) and the NAc, and the monoamines DA, noradrenaline (NE), and serotonin (5-HT) were monitored. Systemic amphetamine increased DA and NE levels in both the NAc (shell and core) and the mPFC (ventral and dorsal), while 5-HT levels remained at baseline. Although the CB-1 receptor antagonist/inverse agonist rimonabant alone did not affect monoamine release, it dose-dependently inhibited amphetamine-induced DA release specifically in the NAc shell. Rimonabant did not affect amphetamine-induced monoamine release in any other region. The results in this chapter might therefore provide new insights into the pharmacological mechanisms underlying CB-1 receptor involvement in amphetamine-induced behavior. Whereas it is likely that the effect of rimonabant on amphetamine-induced DA release seen in the NAc shell resulted from CB-1 receptor blockade, the region where this effect on DA release is initiated remains elusive. Therefore, future research should focus on locally inhibiting CB-1 receptor function during an amphetamine challenge. It is the author’s hypothesis that the interaction with CB-1 receptors takes place at GABA receptors in either the NAc shell itself or in the VTA. This hypothesis is based on the ability of endocannabinoids to inhibit GABAergic activity and by this action indirectly stimulate DA release. Further evaluation of the reward processes initiated by amphetamine is recommended. Future research should focus on the role of other brain areas, including the VTA, in which local infusions of cannabinoids might substantiate the postulated hypothesis. Future studies might also validate the effect of rimonabant on NAc shell DA release seen after amphetamine administration in a behavioral model, preferentially combining microdialysis and a cognitive test like the 5-choice serial reaction time test. Chapter 4 Illustrates the versatility of the CB-1 receptor. The CB-1 receptor is the most abundant G-protein coupled receptor in the brain and is localized on a variety of neurons (e.g. containing the transmitters GABA, glutamate, NE, 5-HT). Therefore, it is to be expected that stimulating or blocking the

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CB-1 receptor would influence their pharmacological responsiveness. One of such interactions is shown in this chapter. Using microdialysis in a brain region which is relevant for the clinical effects of the antidepressant citalopram, the mPFC, we studied the effect of CB-1 receptor stimulation or blockade on the effects of this selective serotonin re-uptake inhibitor (SSRI). The results showed that stimulating the CB-1 receptor inhibited the capacity of citalopram to increase 5-HT levels, while blocking the CB-1 receptor resulted in a prolongation of the effect of citalopram. Since a large percentage of depressed individuals use drugs like cannabis, while being treated with an SSRI, these results might have implication for the clinical efficacy of citalopram. Future research is needed to validate these findings in a more chronical SSRI and marijuana use model (both in animals and in humans). Moreover, the interaction between the CB-1 receptor on the effectiveness of this particular SSRI might be one of many examples in which the CB-1 receptor interferes with the pharmacology of CNS drugs. Therefore it would be relevant to study the effects of drugs other then SSRI’s. Future research might also focus on psychosis, since it is particularly this type of mental illness that is often related to marijuana use. In order to perform the studies described in Chapter 6, a sampling method for four different ECs was developed. ECs are lipophilic compounds and difficult to handle in microdialysis protocols. Chapter 5 describes the conditions that determine the dialysis recovery of the ECs. β-Cyclodextrine proved to be the most effective additive to trap the ECs in the aqueous dialysis fluid. Although β-cyclodextrine concentrations up to 30% w/v resulted in high recoveries of the ECs, they also increased the fluid recovery to levels higher than 100% which would in vivo mean drainage of extra-cellular fluid from the tissue surrounding the dialysis probe. As an acceptable compromise a relatively low concentration of 2% β-cyclodextrine was chosen. To further understand the underlying mechanism of nicotine-induced reward, four different ECs (anandamide (AEA); 2-arachidonoylglycerol (2-AG); oleoylethanolamide (OEA); palmitoylethanolamide (PEA)) were studied by microdialysis. The ECs were sampled in the NAc shell and VTA, during nicotine administration. Simultaneously the nicotine-induced increase of DA was determined. The results are described in Chapter 6, in which for the first time a sampling method is shown able to sample all four ECs. Systemic nicotine administration not only increased the level of DA in the NAc shell (as earlier described), but also increased the levels of ECs in the VTA as well

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as in the NAc. To our knowledge this is the first evidence that nicotine administration increases extracellular levels of ECs in the brain. As described earlier in Chapter 2, infusion of nicotine directly into the VTA or NAc shell increased DA levels in the NAc shell. This increase was inhibited by rimonabant, suggesting that the response of DA is CB-1 receptor mediated. However, analysis of the EC levels during co-administration of rimonabant and nicotine did not further elucidate the underlying mechanism of action as nicotine infusion did not modify the ECs in the extracellular fluid. Although both systemic and local nicotine administration resulted in a (similar) enhancement of DA release in the NAc shell, this was not the case with the ECs. Systemic nicotine increased EC levels in the VTA and NAc shell. Although local nicotine infusions did increase Accumbal DA levels the release of any EC in both the VTA and NAc shell was not. This might imply that while nicotine is able to increase EC release in the VTA and NAc, the modulating role of CB-1 receptors (shown by the inhibitory effect of rimonabant on both EC and DA release) takes place outside the VTA and NAc shell. The results described in Chapter 6 highlights some intriguing questions. First, which brain area plays the modulating role in EC driven nicotine reward? Secondly, why do the levels of ECs increase slower and last longer than the more transient increase in DA after the same stimulus? Options for future studies, that might answer these questions, include the use of local infusions of CB-1 receptor agonists and antagonists. In addition, infusion of inhibitors of individual EC degrading enzymes (such as FAAH and MAGL) into the region of interest might identify the ECs involved. This strategy could especially be worthwhile when rapid changes in EC levels that do not cope with the time resolution of the microdialysis technique are responsible for the study effects.

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Summarizing the main conclusions of this thesis: • The Nucleus Accumbens shell region might be more important in regulating the nicotine-induced reward processes than previously thought • The interaction between the amphetamine-induced increase in extracellular dopamine and CB-1 receptors is detectable in the Nucleus Accumbens shell and not in the Nucleus Accumbens core or the Prefrontal Cortex • Stimulating the CB-1 receptor inhibits the effect of citalopram on extracellular levels of serotonin in the Prefrontal Cortex • Blocking the CB-1 receptor prolongs the effect of citalopram on extracellular levels of serotonin in the Prefrontal Cortex • Adding bovine serum albumin in a concentration of 0.2% is a necessary step in preventing endogenous cannabinoids from binding to the inner wall of glassware, pipettes and tubing • Including 2% of β-cyclodextrine in the perfusion fluid of microdialysis is an efficient and save way of sampling endogenous cannabinoids • Nicotine stimulates the release of dopamine and endogenous cannabinoids in the Nucleus Accumbens shell and Ventral Tegmental Area. The dopamine response to nicotine appeared to be CB-1 receptor mediated, while the increases of endocannabinoids were not.

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Chapter 8 Nederlandse samenvatting van het proefschrift Jelle Kleijn

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Het onderzoek gepresenteerd in dit proefschrift richtte zich op het beloningscircuit in het centraal zenuwstelsel (CZS), waarvan het Ventrale Tegmentale Gebied (VTG) en de Nucleus Accumbens (NAc) deel uitmaken. Het primaire doel was om de effecten van farmaca op de cannabinoid CB-1 receptor in het beloningscircuit te onderzoeken. De meeste aandacht ging daarbij uit naar de effecten van nicotine. Hoewel het onderzoek dierexperimenteel van aard was kunnen de resultaten het inzicht in de effecten van verslavende middelen bij de mens vergroten. In het kort Als eerste is het stimulerende effect van nicotine op de afgifte van de neurotransmitter dopamine in het beloningscircuit van de rat met behulp van de microdialyse techniek bestudeerd. (Hoofdstuk 2). Vervolgens zijn, in een uitgebreid in vitro microdialyse onderzoek, de meest optimale condities voor het bemonsteren van endogene cannabinoiden (ECs) bepaald (hoofdstuk 5). Volgens een gangbare hypothese spelen deze ECs een belangrijke rol bij de beloningsprocessen die ten grondslag liggen aan verslaving. Dankzij de nieuwe bemonsteringsmethode kon in hoofdstuk 6 de rol van ECs in de nicotine-geïnduceerde beloning (DA afgifte) worden geëvalueerd. Daarnaast zijn er in dit promotieonderzoek studies uitgevoerd om meer inzicht te geven in de vele werkingsmechanismen van het endogene cannabinoid systeem. Hoofdstuk 3 richtte zich op het effect van amfetamine op de afgifte van dopamine in een viertal specifieke hersengebieden waarvan wordt aangenomen dat deze betrokken zijn bij het beloningscircuit. Hierbij is in het bijzonder gekeken naar de rol die CB-1 receptoren spelen. In hoofdstuk 4 kon worden aangetoond dat het effect van het antidepressivum citalopram (dat de afgifte van serotonine in het CZS verhoogt) wordt geremd door CB-1 receptor stimulatie en gestimuleerd door CB-1 receptor blokkade. In meer detail Na de algemene introductie (hoofdstuk 1), beschrijven we in hoofdstuk 2 het effect van systemisch en lokaal toegediend nicotine op de afgifte van dopamine in de VTG en NAc; aan deze hersengebieden wordt immers een cruciale rol toegeschreven in de door nicotine geïnduceerde beloningsprocessen. Een aanwijzing hiervoor zijn

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experimenten waarbij gericht genen zijn uitgeschakeld (“knock-out experimenten”) of receptoren worden geblokkeerd met behulp van antagonisten. Deze experimenten hadden aangetoond dat een intacte en functionerende VTG essentieel is voor de nicotine-geïnduceerde beloningsprocessen. In hoofdstuk 2 laten we zien dat nicotine de dopamine afgifte in de schil van de NAc stimuleert. De dopamine afgifte in dit deel van de NAc staat in verband met beloning. Door middel van lokale nicotine infusies, direct in de VTG en de NAc schil, werd de gevoeligheid voor nicotine in beide regio’s vergeleken. Het bleek dat de NAc schil op een veel (tot 1000 keer) lagere concentratie nicotine reageerde dan de VTG. Elektrofysiologische experimenten bevestigden deze bevinding: Pas een relatief hoge systemische dosering van nicotine (0.1mg/kg i.v.) liet een verhoging zien van de vuurfrequentie van dopamine neuronen in de VTG. Deze resultaten tonen aan dat niet alleen de VTG belangrijk is voor nicotine- geïnduceerde beloningsprocessen maar dat het eerder een samenspel is tussen de NAc en de VTG. De NAc is dus mogelijk meer dan alleen maar een projectiegebied van de VTG. De NAc zou een bepalende rol kunnen spelen in de integratie van de VTG-signalen tijdens een belonende stimulus. Hoofdstuk 3 beschrijft een uitgebreid onderzoek – uitgevoerd met behulp van microdialyse - dat gericht was op het identificeren van de hersenkern die verantwoordelijk is voor het effect van amfetamine op impulsief gedrag. Impulsief gedrag speelt een belangrijke rol bij het ontwikkelen en het onderhouden van verslaving en verslavende handelingen. Er werden microdialyse-probes geplaatst in de subregio’s van de mediale PreFrontale Cortex (mPFC) en de NAc. In deze hersengebieden werden de extracellulaire concentraties van de monoamines dopamine (DA), noradrenaline (NE), and serotonine (5-HT) bepaald. Systemisch toegediend amfetamine verhoogde de afgifte van DA en NE in de beide subregio’s van de NAc (schil en kern) en in de mPFC (ventraal en dorsaal), maar de 5-HT afgifte werd niet beïnvloed. Wanneer er kort voor het toedienen van amfetamine een CB-1 antagonist werd gegeven, werd de DA-stijging in de NAc schil dosisafhankelijk geremd: De CB-1 antagonist alleen gaf geen effect op de afgifte van DA te zien. Deze resultaten kunnen mogelijk leiden tot nieuwe inzichten in de rol van de CB-1 receptor met betrekking tot amfetamine-geïnduceerd impulsief gedrag. Het is echter niet

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uitgesloten, dat het effect van CB-1 receptorblokkade op het amfetamine-geïnduceerde effect heeft plaats gevonden buiten de NAc schil, aangezien dit hersengebied signalen van vele andere kernen kan ontvangen, waaronder de VTG. Toekomstig onderzoek zal zich moeten richten op het locaal blokkeren van de CB-1 receptor in de hersengebieden, die in direct contact staan met de NAc schil, tijdens een amfetamine toediening. Onze hypothese is, dat de interactie met de CB-1 receptoren plaatsvindt in combinatie met remming van GABAerge activiteit, in de NAc schil zelf of in de VTG. Deze hypothese is gebaseerd op de in de literatuur beschreven waarneming dat ECs de door GABA afgifte geïnduceerde effecten kunnen remmen, waardoor ze indirect DA afgifte zouden kunnen stimuleren. Hoofdstuk 4 illustreert de functionele veelzijdigheid van de CB-1 receptor. Omdat de CB-1 receptor de meest voorkomende G-eiwit gekoppelde receptor is in het CZS, en aanwezig is in een grote variëteit aan neuronen (GABA, glutamaat, NE, 5-HT, etc.) kan verwacht worden dat stimulatie van deze receptor de afgifte van al deze neurotransmitters kan moduleren en zo een grote verscheidenheid aan processen kan beïnvloeden. Een mooi voorbeeld van een dergelijke interactie wordt in hoofdstuk 4 beschreven. Met behulp van microdialyse in de mPFC (een gebied dat een belangrijke rol speelt in het klinische effect van het antidepressivum citalopram) vonden we een sturend effect van de CB-1 receptor op het effect van citalopram. Het bleek dat de 5-HT niveaus (veroorzaakt door het remmen van de heropname van deze neurotransmitter) afnamen, door het stimuleren van de CB-1 receptor met een agonist, terwijl blokkeren van de CB-1 receptor met een antagonist het effect van citalopram juist kon verlengen. Deze modulerende rol van de CB-1 receptor is mogelijk klinisch relevant aangezien relatief veel depressieve patiënten drugs zoals cannabis gebruiken, terwijl ze onder behandeling staan van een selectieve serotonine heropname remmer (SSRI) zoals citalopram. Het zou dus mogelijk zijn dat door cannabisgebruik het klinisch effect van citalopram wordt verminderd. Toekomstig onderzoek zal zich vooral moeten richten op het valideren van deze interactie in een langdurig experiment en vervolgens bij de mens. Overigens is de getoonde interactie tussen de CB-1 receptor en de SSRI waarschijnlijk slechts een van de (vele?) interacties die een geactiveerde CB-1 receptor kan

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aangaan. Het zou interessant zijn ook de interactie met andere typen antidepressiva te bestuderen, alsmede met antipsychotica, gezien de rol die cannabisgebruik speelt bij patiënten met schizofrenie. Om de rol van de verschillende endogene cannabinoiden in beloningsprocessen te kunnen bestuderen is directe bemonstering en bepaling van deze lipofiele stoffen een absolute meerwaarde. In hoofdstuk 5 worden de condities beschreven die de opbrengst van ECs tijdens bemonstering met de microdialyse methode bepalen. Door toevoegen van het hydrofiele β-cyclodextrine (een kooiverbinding die de ECs kan insluiten) aan de perfusievloeistof, werd de opbrengst (de “recovery”) van ECs sterk verhoogd. Hoe hoger de concentratie toegevoegd β-cyclodextrine, des te hoger was de opbrengst van ECs. Tegelijkertijd werd echter, door een osmotisch effect, een groter volume aan perfusievloeistof opgevangen dan er in gepompt werd, wat in vivo zou betekenen dat er drainage van de locale extracellulaire vloeistof kan optreden. Als acceptabel compromis werd een concentratie van 2% β-cyclodextrine gekozen. Om het onderliggende beloningsmechanisme van nicotine nader te bestuderen, zijn de ECs anandamide (AEA), 2-arachidonoylglycerol (2-AG), oleoylethanolamide (OEA) en palmitoylethanolamide (PEA) na systemische nicotine-toediening bemonsterd. De ECs werden zowel in de NAc schil als in de VTG bepaald, terwijl tegelijkertijd de extracellulaire DA stijging werd gemeten. Deze experimenten worden in detail besproken in Hoofdstuk 6. In dit hoofdstuk wordt voor het eerst getoond, dat met onze methode alle vier ECs met microdialyse bemonsterd kan worden. Systemisch toegediende nicotine verhoogde niet alleen de DA afgifte in de NAc schil (zoals eerder beschreven), maar ook de afgifte van de ECs, zowel in de NAc schil als in de VTG. Zover wij weten, is dit het eerste bewijs, dat nicotine in staat is de vorming van ECs in het CZS te verhogen. Zoals beschreven in hoofdstuk 2, verhoogt directe infusie van nicotine in de VTG de DA afgifte in de NAc schil. Deze afgifte werd dosis-afhankelijk geremd door de (systemisch gegeven) CB-1 receptor antagonist rimonabant, wat suggereert, dat deze afgifte CB-1 receptor gestuurd is. Echter, analyse van de extracellulaire EC niveaus na een locale infusie van nicotine plus rimonabant in de VTG gaf geen bevestiging van de mogelijke rol van de CB-1 receptor, want nicotine gaf nu geen verandering in de afgifte van ECs. Hoewel zowel

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systemische als locale toediening van nicotine eenzelfde effect gaf op de DA afgifte in de NAc schil, was dit dus niet het geval voor de ECs. Dit zou kunnen betekenen, dat hoewel systemisch nicotine de EC afgifte in de NAc schil en de VTG verhoogt, het modulerende effect van CB-1 receptoren plaatsvindt buiten de NAc schil en de VTG. De resultaten van hoofdstuk 6 roepen twee belangrijke vragen op. Ten eerste, welk hersengebied speelt de modulerende (hoofd)rol in de EC-gestuurde nicotinebeloning? En ten tweede, waarom stijgen, na een gelijke stimulus, de niveaus van de ECs langzamer en duurt deze stijging langer dan de korter durende DA vrijgiftes? Een eerste optie voor nader onderzoek dat antwoord zou kunnen geven op de gestelde vragen, is het locaal infunderen, in relevante hersengebieden, van CB-1 receptor agonisten en -antagonisten. Daarnaast zou infusie van remmers van onder meer FAAH en MAGL (enzymen die ECs kunnen afbreken) in relevante hersengebieden, mogelijk de specifieke ECs kunnen identificeren, die betrokken zijn bij nicotine-geinduceerde beloning. Deze strategie zou bijzonder informatief kunnen zijn, als de veranderingen in EC-niveaus na nicotine toediening in feite sneller zijn, dan de in hoofdstuk 6 toegepaste microdialyse techniek kan volgen.

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Samengevat zijn de belangrijkste conclusies van dit proefschrift:

• De nucleus accumbens schil zou belangrijker kunnen zijn in de regulatie van nicotine-geïnduceerde beloningsprocessen dan voorheen gedacht werd

• De interactie tussen de amfetamine geïnduceerde dopamine

vrijgifte en CB-1 receptoren vindt plaats in de schil en niet de kern van de nucleus accumbens, noch in de prefrontale cortex

• Stimulatie van CB-1 receptoren remt het effect van citalopram

op de extracellulaire serotonine concentraties in de prefrontale cortex

• Blokkeren van CB-1 receptoren verlengt het effect van

citalopram op de extracellulaire serotonine concentraties in de prefrontale cortex

• Toevoegen van bovine serum albumine in een concentratie van

0.2% is een cruciale stap in het hanteren van endogene cannabinoiden omdat het aspecifieke binding aan glas en kunststoffen voorkomt

• Het toevoegen van β-cyclodextrine aan de perfusievloeistof is

een efficiënte en veilige manier om de opbrengst (de “recovery”) van de bemonstering van endogene cannabinoiden door middel van de microdialyse techniek te verbeteren.

• Nicotine stimuleert de afgifte van dopamine en endogene

cannabinoiden in de nucleus accumbens schil en het ventrale tegmentale gebied. De dopamine reactie op nicotine blijkt gemedieerd door de locale CB-1 receptor, terwijl de stijging in endogene cannabinoiden niveaus dat niet is.

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Chapter 9 Acknowledgements / Dankwoord Jelle Kleijn

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As most of the people I would like to thank for their contribution and personal help in my thesis mainly speak Dutch I wrote this section mostly in Dutch. Al vroeg in de Master Medisch Farmaceutische Wetenschappen aan de RUG kwam ik in aanraking met de afdeling Biomonitoring en Sensoring en Brains On-line. Ik wilde daar graag een stage doen met microdialyse omdat deze techniek mijn aandacht had getrokken bij Solvay. De stage onder begeleiding van Martin en Ben beviel zo goed dat ik volmondig “Ja, ik wil” antwoordde op de vraag of ik een promotieonderzoek bij hen wilde uitvoeren. Ik heb tijdens mijn vier jaar durende onderzoek geen dag gehad dat ik niet met plezier naar het werk ging. Dit is voor mij het allerbelangrijkste en zonder de volgende mensen zou het ook lang niet zo leuk geweest zijn: Met bedanken begin ik bij het begin en daarom op de Antonius Deusinglaan 1. De belangrijkste oorzaak van de plezierige werkomgeving was de gezellige groep biotechnici, andere PhD studenten en Post-Docs. Karola, Kim, Suzan, Daphne, Annelie, Harm, Azueres en Carlos, Wahono, Si, Miranda, Donnatella en Isabella, wat heb ik van jullie genoten! Vele practical jokes waren aan de orde van de dag. Helaas werkte de helft van de opgezette grappen niet, maar de voorpret was voldoende om er weer een week tegen aan te kunnen. Zowel op de werkvloer als ernaast hebben we leuke dingen gedaan en zijn vele van jullie van collega’s opgewaardeerd naar vrienden. Ik weet zeker dat het hier niet ophoudt, al zien we elkaar minder, de volgende afspraak is al gemaakt om elkaar weer te zien. Ik heb er zin in. Carlos, we had a great time together, both starting to practice judo together with our friend and very good scientist to be: Chris. We shared laughter, frustration, and dreams on and off the lab. I’m sure you will do a good job on finishing your thesis, hang in there! Wahono, when you came, you were given a Dutch name: Piet. During your PhD period you became more and more Dutch (only the positive traits). I’m sure that with your new developed Dutch assertiveness you will be able to finish the thesis fast and efficient. Si and Miranda, I have enjoyed every bit of your company, advice and trust. I was so proud to be your “paranimf”, Miranda, thanks for offering me this position!

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Ik wil Martin, Joost en Gunnar ontzettend bedanken voor de ondersteuning die zij mij geboden hebben. Ondanks dat jullie het ontzettend druk hadden werd er altijd even tijd vrijgemaakt om mij uit te motiveren als het even in de mist liep. Martin, ik heb ontzettend veel van je geleerd. Je bent een goede wetenschapper met veel kennis, schrijftalent en geduld, waar ik als PhD student dankbaar gebruik van heb kunnen maken. Joost, je hebt me ontzettend veel geholpen met de papers en morele support wanneer ik die nodig had. Jou relativeringsvermogen heeft bij mij veel stress buiten de deur gehouden. Ik ben trots jou als een van mijn paranimfen te hebben. Gunnar, zonder jou bereidheid mij toegang te geven tot de analyse apparatuur (weliswaar in de weekenden) had ik het nooit af gekregen. Ik hoop, dat ik je vertrouwen nooit heb beschaamd, want ik realiseer me volledig dat dit een unieke situatie was. Natuurlijk was het allemaal niet zo gestroomlijnd verlopen als ik geen administratieve ondersteuning had verkregen van Margriet, Janneke, Janine, en Cathy de Haan. Tot op het laatst was ik niet op de hoogte van alle procedures en dat heeft jullie veel extra werk bezorgd. Ik heb er veel van geleerd en tracht ook bij mijn nieuwe werkgever meer energie te steken in procedurele taken. Bedankt voor jullie geduld en gezelligheid. Ik deelde de kantoorruimte met een paar collega’s van de vakgroep Farmacochemie. Hier kreeg ik te maken met Ulrike, Jeroen en Andre. Ulrike, ik heb je altijd bewonderd om je werklust en professionaliteit. Er zijn maar weinig PhD studenten die zo hard hebben gewerkt om hun proefschrift af te ronden. Je doet goed werk bij BOL, ga zo door. Jeroen, je optimisme en drive zijn erg aanstekelijk. Andre, jij hebt mij echt ondersteund, we kennen elkaar al vanaf onze eerste studie in Leeuwarden. Toen ik nog in mijn eentje was op het lab na het verhuizen van BOL heb ik erg veel aan je steun gehad. We hebben veel plezier gehad en ondanks je pogingen heb ik toch nog bijzonder weinig farmacochemische kennis opgenomen. Bedankt voor alles en ik ben blij dat je mijn paranimf en vriend bent. De eerste chromatogrammen die ik heb gegenereerd op het lab waren samen met Jan de Vries, de trouwe analist van Ben. Het speelse gemak waar jij de HPLC mee aan de praat kreeg als ik weer voor een

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storing had gezorgd, was wonderbaarlijk. Marius heeft mij ook ontzettend veel geleerd met en over de HPLC; het was leuk jullie twee verschillende leerscholen te ervaren. Als de ene oplossing niet werkte, dan deed de andere dat wel. Op een gegeven moment kreeg ik de mogelijkheid over te gaan op de LC-MS/MS om zo sneller en meer data te kunnen genereren. Dit was nooit gelukt als Corry niet het geduld en vertrouwen had, dat ze mij gegeven heeft. Wat een topper ben jij toch. De manier van lesgeven is wel zo direct, dat het af en toe erg zweten was, maar daardoor was het zeer effectief. Ik heb veel respect voor jou en hoe jij het analytisch lab hebt omgetoverd van een universitair bij elkaar geraapte inboedel tot een volledig efficiënt industrieel lab. Bedankt Corry. Ook wil ik Theo, Nynke, Robert, Wim, Daniel, Julien, Saskia, Elvira en Tietie ook bedanken voor hun gezelligheid en hulp. Ik ben Brains-Online (BOL) en haar werknemers erg dankbaar voor de middelen, kennis en infrastructuur, die mijn onderzoek mogelijk hebben gemaakt. Zonder jullie analysemethoden was het werk een stuk trager verlopen en minder interessant geweest. Voor de productie, levering en ontwikkeling van de probes die ik voor mijn onderzoek nodig had kon ik altijd bij de mensen van Brainlink terecht. Zonder Korrie, Jeanette en Sylvia was dat nooit gelukt. Kirsten wil ik toch wel even extra bedanken. We hebben een hoop plezier gehad op onze tripjes naar Neuroscience. Je bent een erg fijne collega en vriendin. Bedankt voor het meedenken, het vertrouwen dat je me gaf en de fijne samenwerking. De Neuroscience tripjes waren ook lang niet zo leuk geweest zonder de hulp van de slimme accommodatie makelaar en BOL’ler van het eerste uur Marieke. Ik heb een hoop van je geleerd met de dierregistratie en omgang met de ethische toetsingscommissie. I also want to thank Arash, although we did not work together directly I have the feeling we would work together very well. Take good care of my Dutch friends in the big US of A. Het laatste jaar zat vol met samenwerkingen die het voordeel van werken binnen TIPharma symboliseerden. Zo hebben we in de UvA

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electrophysiologie met onze glutamaatsensor proberen te combineren. Femke, Taco en Wytse bedankt voor jullie inzet en geduld. Helaas hebben we het goede resultaat van onze eerste poging niet meer kunnen reproduceren, ondanks de inspanningen van Wahono om een “royal sensor” in elkaar te zetten. Samen met het VUMC hebben we onze velden van expertise ook gedeeld. In een intensief en nachtelijk experiment is voor mij uiteindelijk duidelijk geworden dat de wetenschap in een universitaire setting niet mijn toekomst is. Toch heb ik een hoop positieve ervaringen en elementen uit deze samenwerking met Joost, Tommy en Anton gehaald. Joost, ik heb me altijd verwonderd over het niveau dat jij al vanaf het begin van je PhD traject leek te hebben. Ga zo door, jij kan heel ver komen in de wetenschap, succes! Tommy, bedankt dat je wil deelnemen in de oppositie tijdens de verdediging van dit proefschrift, een ware eer. Ook binnen de RUG hebben we nog een samenwerking opgezet. Microdialyse combineren met gedrag. Een uiterst veelbelovende en arbeidsintensieve opzet was het gevolg. Romy and Piray, I have enjoyed all our quite moments in the lab, you were both very eager to learn and taught me a lot as well. I hope that by now both of you are (almost) finished. Aansluitend wil ik natuurlijk TIPharma bedanken voor het platform dat zij bieden voor alle samenwerkingen en de flexibiliteit in richting en financiering. Natuurlijk heb ik het werk niet alleen gedaan. Ik heb een aardige groep stagiaires gehad, die voor een deel van het werk verantwoordelijk zijn. Ik wil graag Yannick, Mariam, Sibel, Iren, Kim, Iris, Jolanda, Angela, Chantal en Balaji bedanken voor hun tomeloze inzet. Het begeleiden van jullie was dankbaar werk! Bedankt voor jullie inzet en energie en ik wens jullie veel succes in jullie verdere carrière. Catriene, jou wil ik even apart bedanken voor de plezierige samenwerking die we gehad hebben. Jou manier van communiceren is erg “open” wat een laagdrempelige situatie creëert. Ik heb daar dankbaar gebruik van gemaakt in de jaarregistraties voor Biomonitoring en Sensoring en bij mijn eigen projecten. Bedankt voor de prettige samenwerking!

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Marianka, ik wil ook jou even bedanken voor de vrijheid die je me gaf in het afronden van mijn proefschrift. Ik ben ontzettend blij met de functie die ik vervul bij TEVA en het vertrouwen dat jullie vanaf het begin in mij getoond hebben. Dank je wel! Ik wil mijn paranimfen nog even apart bedanken voor de hulp rondom het proefschrift en alvast voor de steun tijdens de verdediging ervan. Met jullie aan mijn zijde komt het helemaal goed! Bedankt jongens. Ik wil de leescommissieleden graag bedanken voor de energie die zij in het doornemen en goedkeuren van dit proefschrift hebben gestoken. Prof. J.M. Koolhaas, Prof. P.G.M. Luiten en A.G.M. van der Zee, bedankt! Hans Zaagsma, ik ken niemand die zo behulpzaam en betrokken is als u. Ik ben elke keer weer verrast hoe bevlogen u bent met uw werk. Bedankt voor het corrigeren van mijn laatste hoofdstuk en samenvatting, dit waren de lastigste en laatste loodjes en natuurlijk bedankt voor het deelnemen aan de oppositie. Mijn officiële begeleiding was in de kundige handen van Thomas en Ben. Thomas, jij hebt vanaf het begin gefunctioneerd als een voorbeeld. Ooit zal ook ik een eigen bedrijf starten. Jij blijft een unieke kerel, zo jong en zo gedreven om iets op te zetten wat snelheid, flexibiliteit en hoge kwaliteit biedt met een team, dat jij altijd wist te motiveren. Ik heb veel van je geleerd. Ik wil nog altijd meer weten. Bedankt. Ben, de manier waarop jij mij begeleid hebt, heb ik als bijzonder fijn ervaren. Veel vrijheid en verantwoordelijkheid. Je bent een goede mentor, wetenschapper in de neurochemie (& archeologie), auteur van tekstboeken (& romans) en bovenal een levensgenieter. Bedankt voor jouw interesse en geduld in mijn ontwikkeling van wetenschapper en mens. Jij bent een echt voorbeeld. Ik wil mijn vrienden thuis bedanken voor het begrip dat jullie hebben gehad voor het feit dat ik minder tijd had door al die weekenden op het werk. We hebben des ondanks veel leuke tijden gehad en er gaan nog leuke tijden komen, daar ben ik zeker van. Bedankt jongens!

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Natuurlijk moet ik mijn directe familie ook bedanken voor het begrip en de steun die ik van jullie ontvangen heb. Pap en Mam, Arne, Inge, Guillaume en Mariet, ik weet nog goed hoe jullie reageerden op mijn eerste artikel, ik was zo dankbaar voor jullie reacties. Jullie moeten weten, dat ik zonder jullie steun tijdens die periode het praktische deel niet binnen de 4 jaar gehaald zou hebben. Inkie, ook bedankt voor de creatieve invulling van mijn kaft. Jouw ontwerp maakt het nog persoonlijker. Marjolijne, jij hebt waarschijnlijk het meest moeten inleveren voor mijn ambitie. Ik wilde graag promoveren maar zonder jouw steun was die investering in tijd en energie zeker niet mogelijk geweest. We gaan het qua vakanties snel goed maken. Zonder jouw steun de laatste maanden had het afronden van dit proefschrift nog lang op zich moeten laten wachten. Als laatste wil ik Nora bedanken. Hoe vermoeid of gefrustreerd ik ook thuis kwam omdat experimenten mislukten en het schrijven niet vorderde, jij wist mij direct alles te doen vergeten met een mooie glimlach. Je hebt inmiddels een zusje gekregen: Livia. Ik kan niet wachten om te zien wat jullie met de wereld gaat doen. Hij ligt aan jullie voeten! Marjolijne, Nora en Livia, bedankt.