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UNIVERSITY OF CAMERINO Department of Experimental Medicine and Public Health ________________________________________________________ The NOP receptor as a target in the treatment of alcohol abuse by Daina Economidou Supervisors: Prof.Maurizio Massi Prof. Roberto Ciccocioppo 1 List of Papers This thesis is based on the papers list below: I. Ciccocioppo R, Economidou D, Fedeli A, Massi M. The nociceptin/orphanin FQ/NOP receptor system as a target for treatment of alcohol abuse: a review of recent work in alcoholpreferring rats. Physiol Behav 79:121-128, 2003 II. Ciccocioppo R, Economidou D, Fedeli A, Angeletti S, Weiss F, Heilig, M, Massi M. Attenuation of Ethanol Self-Administration and of Conditioned Reinstatement of Alcohol-Seeking Behaviour by the Antiopioid Peptide Nociceptin/OrphaninFQ in Alcohol-Preferring Rats. Psycopharmacology 172:170-178, 2004 III. Ciccocioppo R, Economidou D, Rimondini R, Sommer W, Massi M, Heilig M. Buprenorphine reduces alcohol drinking through activation of the Nociceptin/Orphanin FQ-NOP receptor system. Biol Psychiatry Mar 11, 2006 [Epub ahead of print] IV. Economidou D, Fedeli A, Massi M, Ciccocioppo R. Effect of novel NOP receptor ligands on ethanol drinking in the alcohol-preferring msP rats. Peptides Manuscript submitted for publication V. Economidou D, Hansson A, Fedeli A, Massi M, Heilig M, Ciccocioppo R. The Central Nucleus of the Amygdala is the neuroanatomical site of action for the effects of N/oFQ on alcohol drinking. J Neurosc Manuscript submitted for publication VI. Economidou D, Ubaldi M, Lourdusamy A, Massi M, Ciccocioppo R. Activation of brain NOP receptors attenuates alcohol withdrawal symptoms in rats. Manuscript 2 CONTENTS Chapter 1 General Introduction 4 Chapter 2 The nociceptin/orphanin FQ/NOP receptor system as a target 70 for treatment of alcohol abuse: a review of recent work in alcohol-preferring rats Chapter 3 Effect of novel NOP receptor ligands on ethanol drinking in 96 the alcohol-preferring msP rats Chapter 4 Buprenorphine reduces alcohol drinking through activation of 116 the Nociceptin/Orphanin FQ-NOP receptor system. Chapter 5 Attenuation of ethanol self-administration and of conditioned 151 reinstatement of alcohol-seeking behaviour by the antiopioid peptide nociceptin/orphanin FQ in alcohol-preferring rats Chapter 6 The Central Nucleus of the Amygdala is the neuroanatomical 182 site of action for the effects of N/oFQ on alcohol drinking Chapter 7 Activation of brain NOP receptors attenuates alcohol 207 withdrawal symptoms in rats Chapter 8 Summary, conclusions and suggestions for further research Scientific Publications Acknowledgements 3 230 Chapter 1 General Introduction 4 General introduction 1. Alcoholism 1.1. Epidemiology Alcohol is the second most commonly abused psychotropic drug after caffeine in the world today (Samson & Harris, 1992), and alcoholism has emerged as a major social and health problem (Royal College of Psychiatrists, 1986). In the United States 19% of men and 8% of women have been diagnosed, at some time in their lives, with alcohol dependence as defined in the American Psychiatric Association’s (1994) Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (Grant et al., 1994). In Australia, in 1977 it was estimated that one in five hospital beds were occupied by those suffering the effects of alcohol (Commonwealth Department of Community Services and Health, 1987). In addition to causing numerous serious medical disorders (e.g., liver and heart disease), alcohol dependence is associated with costly, adverse social consequences such as disruption of families, crime, traumatic accidents, and lost productivity. As a result, the annual costs related to alcohol dependence in the United States for 1998 have been estimated at $185 billion (Harwood, 1998, 2000). Unfortunately, heavy alcohol use and alcohol dependence has been also increasing among younger people (Windle, 1990; Sobeck et al., 2000), indicating that alcohol dependence may become an ever more prominent public health problem. In fact, 31% of 12th graders in the United States reported getting drunk in the past month 5 and 6–10% of teens meet diagnostic criteria for an alcohol use disorder (Rohde et al., 1996; Clark et al., 2002) Alcoholism, is a chronically relapsing disorder characterized by compulsive alcohol seeking and use (McLellan et al., 1992; O’Brien et al., 1998). More than 80% of addicts relapse to compulsive drug use after a period of withdrawal and abstinence, during what is known as the protracted withdrawal phase. The long-lasting nature of this compulsion and the high rates of recidivism represent a challenge for effective treatment. Two major factors implicated in the resumption of drinking include subjective reactions provoked by stressful events and by environmental stimuli that have become conditioned to the pharmacological actions of alcohol (Marlatt, 1985; Ludwig et al., 1974; Rogers et al., 1979; O'Brien et al., 1992; Childress et al., 1993; Stormark et al., 1995). In addition, recent data suggest that stress and ethanol-related environmental stimuli interact to augment relapse risk as measured in an operant response-reinstatement model of relapse (Liu & Weiss, 2002a). Because of the chronically relapsing nature of alcohol addiction, relapse prevention has emerged as a central focus of treatment and medication development efforts. However, understanding the neural basis of the relapse phenomenon is still limited, and research in this area will be of critical importance toward progress with the development of effective “anti-relapse” mediations. 1.2. Genetic Vulnerability to Alcohol Abuse There is, in recent days, clear evidence that people may be genetically predisposed to alcoholism, although this does not negate the role of environmental 6 factors (Gianoulakis & de Waele, 1994; Begleiter et al., 1995; Ferguson & Goldberg, 1997). An estimated 40 to 60% of the individual variation in alcohol preference and vulnerability to alcoholism is genetic in origin, as revealed by adoption studies (Goodwin et al., 1977; Hesselbrock, 1995; Bohman et al., 1981; Cloninger et al., 1981), and by studies on large samples of cross-sectionally ascertained twin pairs (Heath et al., 1997; Kendler et al., 1995, 1997). In fact, sons of alcoholics are approximately 4 to 9 times more likely to become alcoholic that are sons of nonalcoholics (Cotton, 1979; Cloninger et al., 1981; Goodwin, 1985). Moreover, sons of alcoholics who are adopted by non-alcoholic families early in life, are still more than 3 times more likely to become alcoholic than are similarly adopted sons of nonalcoholics (Bohman, 1978; Bohman et al., 1981; Cloninger et al., 1981; McGue, 1997). In addition, twin studies reported a 50200% greater concordance rates for alcoholism among identical, compared to non identical twins (McGue, 1997) It is nowadays well consolidated the important role of the mesocorticolimbic dopamine (DA) system in the brain reward pathway (Imperato & Di Chiara, 1986; Wise & Rompre, 1989; Weiss et al., 1993; Doyon et al., 2003), and the DA neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc) are considered to play a key role in mediating the rewarding actions of various drugs of abuse, including alcohol (Koob & Bloom, 1988). The dopamine D2 receptor (DRD2) gene has been proposed to be a primary candidate in drug addiction, because disturbances in dopamine availability and its receptors have been implicated in neural reward mechanisms (Koob & Swerdlow, 1988). In a study conducted by Konishi et al., (2004a) a significant difference of the genotype frequency for the DRD2-141C insertion/deletion (Ins/Del) allele was found between alcoholic and control Mexican-Americans (Konishi et al., 2004a). In accordance to these data, Ishiguro et al., (1998) also reported that the frequency of the 7 DRD2-141C Ins allele was significantly higher in alcoholics (88%) than in control subjects (77%) in a Japanese population (Ishiguro et al., 1998). In addition, Blum et al., (1990) reported a higher prevalence of the TaqI A1 allele of the DRD2 gene in alcoholics compared with control subjects (Blum et al., 1990). However, despite numerous subsequent case-control studies, the role of the A1 allele as a candidate marker in predisposing alcoholism/drug dependence has remained controversial. Some investigators have found significant associations (Comings et al., 1991; Parsian et al., 1991; Amadeo et al., 1993; Arinami et al., 1993), whereas others have not replicated the finding (Gelernter et al., 1991; Goldman et al., 1992; Turner et al., 1992). However, when the results of 15 published studies, with 1015 alcoholics and 898 control subjects, were combined and compared, the TaqI A1 allele was more prevalent in the alcoholic group than in the control group (Noble, 1998). The neurotransmitter serotonin (5-HT) is known to play an important role in many physiologic and behavioral functions, such as motor activity, food intake, sleep, and reproductive activity, as well as in mood, cognition, and emotion (LeMarquand et al., 1994; Owens & Nemeroff, 1994). The serotonin transporter (5-HTT) affects serotonergic neurotransmission by reuptake of synaptic serotonin, resulting in termination of serotonergic neurotransmission. Rates of serotonin reuptake vary among individuals, and such variability is partly under genetic control (Meltzer & Arora, 1988). The 5-HTT–linked polymorphic region (5-HTTLPR) has a 44 base pair (bp) insertion/deletion (Ins/Del) functional polymorphism, resulting in short (S) or long (L) alleles. Positive associations were found between the short variant and severely affected alcoholics (Sander et al., 1997), alcohol dependence (Hammoumi et al., 1999; Lichtermann et al., 2000; Thompson et al., 2000), and type II alcoholism (Hallikainen et al., 1999). In addition, Konishi et al., (2004b) demonstrated that the frequency of the 5-HTTLPR (S) allele was significantly higher in alcoholic Mexican-Americans (61.5%) 8 than in nonalcoholic control subjects (52.8%) (Konishi et al., 2004b). In contrast, however, there are some reports showing that the S allele was significantly less frequent in severe alcoholics (Ishiguro et al., 1999) and that the L allele was associated with type II alcoholism (Parsian & Cloninger, 2001). Twitchell et al., (2001) reported that more children with the LL genotype than with the SS/SL genotype had a history of alcohol consumption (Twitchell et al., 2001). However, results of some studies have indicated no relation between the 5-HTTLPR polymorphism and alcohol dependence (Matsushita et al., 2001; Kranzler et al., 2002). The gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the brain. It interacts at three classes of receptor sites, designated GABAA, GABAB, and GABAC. Thus, the subunit composition of the GABAA receptor is known to importantly determinate the response to different drugs (Levitan et al., 1988; Ymer et al., 1990). The 3 gene (GABR 3) encodes a major subunit of the GABAA receptor and is clustered with other 5 and 3 subunit genes on human chromosome 15 (Wagstaff et al., 1991). Noble, (1998) reported that variants (G1, 181 bp) of GABR3 contributed to the risk for alcoholism, and, especially when they were combined with DRD2 TaqI A allele, the risk for alcoholism became more robust in Caucasian subjects of European (non-Hispanic) descent (Noble, 1998). Glutamate, is the major excitatory neurotransmitter in the brain and is known to also play an important role in the pathogenesis of alcohol dependence. The ionotropic glutamatergic N-methyl-D-aspartate receptors (NMDAR) have been implicated as primary target sites for acute and chronic effects of ethanol and, the NR1asplice variant of NMDAR1 subunit and the NMDAR2B subunit were reported the most sensitive to ethanol (Lovinger, 2000). In a study by Wernicke et al., (2003) an association between the A allele of the NMDAR1 gene and alcoholism was showed, whereas, Sander et al., 9 (2000) demonstrated that genetic variation of the astroglial glutamate transporter (EAAT2) gene confers vulnerability to risk-taking behavior in alcoholics (Sander et al., 2000; Wernicke et al., 2003). Genetically determined differences in opioid activity have also been found to confer vulnerability for alcoholism. In fact, in a series of studies Gianoulakis et al. showed that individuals with a family history of alcoholism (high risk) have lower basal plasma -endorphin levels, but greater release of -endorphin after exposure to a 0.5g/kg dose of ethanol than do individuals without this history (low risk) (Gianoulakis et al., 1989; Gianoulakis, 1996, 2001). Further support for the hypothesis that -endorphin response to ethanol may represent a biomarker for increased genetic risk for alcoholism was provided by findings of a recent study of twins confirming that this response has significant heritability (Froehlich et al., 2000). Into the causes of alcoholism attention has also drawn to the potentially important role of polymorphisms of two major enzymes of alcohol metabolism, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). In fact, in alcoholics functional polymorphisms have been observed at genes encoding these enzyme proteins, inducing a variation in the rate of elimination of ethanol and the rate of formation and elimination of acetaldehyde. For example, among the Atayal the group with alcohol use disorders (alcohol dependence and alcohol abuse) had a significantly lower frequency of the ADH2*2 allele (0.82) than those without alcohol use disorders (0.91) (Chen et al., 1997). In addition, in Asian populations the ADH2*2 allele was found at higher frequencies in controls than in alcoholics (Thomasson et al., 1991; Chen et al., 1997; Shen et al., 1997; Tanaka et al., 1997). Also the ADH3*1 allele is generally found at higher frequencies in controls than in alcoholics (Chen et al., 1997). 10 Taken together, above data suggest that the predisposition to alcohol misuse and dependence varies greatly between individuals and may be influenced by a number of genes (Radel & Goldman, 2001). In fact, as described above, candidate genes associated with alcoholism risk have been identified. 1.3. Neurobiology of Alcohol Abuse The reinforcing and rewarding properties of ethanol and ethanol-seeking behaviour, are known to be mediated by several neurochemical systems and in a widespread neuroanatomical sites in the brain (for review see, Koob et al., 1998). In this section, the mechanisms underlying ethanol’s reinforcing properties in the brain and alcohol seeking behaviour will be discussed. Ethanol Reinforcement The DAergic mesolimbic system has been proposed to be one of the main neural networks through which the rewarding and reinforcing effects of drugs of abuse, and of alcohols’, are mediated (Self & Nestler, 1995; Wise, 1996, 1998; Koob & Le Moal, 1997; Robbins & Everitt, 1999). The mesolimbic dopaminergic system is comprised of a group of neuronal cell bodies that originates in the VTA in the midbrain. These cells possess axonal processes that terminate in several forebrain regions including the NAcc, prefrontal cortex, amygdala, olfactory tubercle, hippocampus (Fallon & Moore, 1978a, 1978b; Fallon et al., 1978; Swanson, 1982). However, the dopaminergic projection from the VTA to the NAcc consisting mainly by the A10 group of DA 11 neurons (Deutch et al., 1993; Herz, 1997), and the increase of extracellular DA concentrations in the NAcc, are known to be the key function for mediating alcoholinduced reinforcement (Koob, 1992). The first reports that systemic ethanol administration stimulates DA release in the NAcc, appeared in the literature almost 2 decades ago (Gessa et al., 1985; Imperato & Di Chiara, 1986). Successive studies confirmed and extended this data. In rats, an increased firing of DAergic neurons in the VTA, has been reported both in vivo and in vitro after acute and subchronic exposure to ethanol (Gessa et al., 1985; Brodie et al., 1990; Diana et al., 1992, 1993b). Moreover, ethanol given systemically (Imperato & Di Chiara, 1986; Di Chiara & Imperato, 1988a; Yoshimoto et al., 1991, 1992; Diana et al., 1993b; Heidbreder & De Witte, 1993) or microinjected directly into the VTA (Yoshimoto et al., 1991) induced a dose-dependent release of DA in the NAcc. Microinjections of ethanol into the NAcc (Wozniak et al., 1991; Yim et al., 1998) have also been shown to induce a dose-dependent release of DA in this brain area; however, the high concentrations required suggest that the systemic effect of ethanol on DA release in the NAcc is probably mediated via the VTA (Samson et al., 1997; Yim et al., 1998). In addition, ethanol self-administration both in the nonselected Wistars and ethanol-preferring (P) rats induced a significant increase in DA levels in the NAcc (Weiss et al., 1992, 1993). Conversely, withdrawal from ethanol has been found to induce a significant decrease in DA release in the NAcc (Rossetti et al., 1992; Diana et al., 1993a). However, ethanol-induced increased DA release has been seen in some other brain areas as well, suggesting an important role of these in the reinforcing effects of ethanol. For instance, in a study by Yoshimoto et al., (2000) intraperitoneal injection of 2 g/kg of ethanol significantly increased by 270% over baseline the extracellular levels of DA in the central nucleus of the amygdala (CeA) (Yoshimoto et al., 2000). 12 Moreover, systemic injection of ethanol also increased DA release in the ventral pallidum (VP) of Wistar rats (Melendez et al., 2003). The importance of the mesolimbic DAergic transmission in the brain in inducing alcohol reinforcement, is also supported by pharmacological studies. Systemic treatment with bromocriptine, a D1 and D2 receptor agonist, and GBR 12909, a dopamine reuptake inhibitor, decreased ethanol intake in the P rats (McBride et al., 1990). In addition, Silvestre et al., (1996) reported that the D1-selective agonist, SKF 38393, decreased the intake of a sweetened ethanol solution in a 23-hr, 2-bottle-choice procedure using food-deprived rats, whereas, the selective D2/D3 agonist, 7-OH-DPAT increased the consumption. In contrast, the D2-selective antagonist, raclopride, did not affect sweetened ethanol consumption in these studies (Silvestre et al., 1996). On the other hand, Samson and Hodge (1993), reported that local microinjections of the D2 receptor antagonist, raclopride, to the NAcc dose-dependently decreased ethanol self administration, which was most likely due to post-synaptic blockade (Samson & Hodge, 1993). However, the D2/D3 receptor agonist quinpirole, when locally injected into the VTA, also dose-dependently decreased ethanol self-administration, probably due to inhibition of the dopaminergic neurons in this area (Samson & Hodge, 1993). In addition, El-Ghundi et al., (1998) demonstrated the importance of the D1 receptor in alcohol reinforcement, as D1 receptor deficient (D1-/-; ‘knockout’) mice showed markedly decreased ethanol consumption and preference respect to wild-type (D1+/+) controls (El-Ghundi et al., 1998). Treatment with the D2 receptor antagonist decreased residual drinking in the knockout mice and caused a small decrease in ethanol consumption in the wild type mice, thus, the authors suggested that the D1 receptor was more important in mediating “alcohol-seeking behaviour” than the D2 receptor (ElGhundi et al., 1998). Lastly, treatment with the selective D3 receptor antagonist, SB277011-A, induced a significant attenuation in ethanol preference, intake and lick 13 responses in the P rats suggesting an important role of this receptor in alcohol reinforcement (Thanos et al., 2005). In recent days, is also well consolidated the importance of the brain opioidergic system in controlling and mediating the reinforcing and rewarding properties of ethanol, both in humans and in rodents (Reid et al., 1991; Gianoulakis, 1996; Herz, 1997; Oswald & Wand, 2004). It has been proposed, in fact, that alcohol may stimulate the release of endogenous opioid peptides in the brain, which in turn could interact with areas associated with reward and positive reinforcement (Jamensky & Gianoulakis, 1997; Koob et al., 1998). Moreover, it has been suggested that increased activity of brain enkephalin or -endorphin opioid peptide systems, in response to ethanol exposure may be important for initiating and maintaining high alcohol consumption (Gianoulakis et al., 1989; Rasmussen et al., 1998). Likewise, increased density of or (or both) opioid receptors in brain regions involved in the reinforcing effects of drugs of abuse, may also be important in initiating and maintaining high alcohol consumption (DeWaele & Gianoulakis, 1997; Marinelli et al., 2000). Conversely, increased dynorphin activity or increased binding site density may inhibit high ethanol consumption (McLearn & Rodgers, 1959; Herz, 1997). The administration of nonspecific opioid receptor antagonists, such as naltrexone, as well as of and selective opioid receptor antagonists has been shown to decrease alcohol consumption in a dose-dependent manner in a number of animal species and in a number of experimental paradigms (Altshuler et al., 1980; Myers et al., 1986; Froehlich et al., 1990, 1991; Weiss et al., 1990; Kornet et al., 1991; Hubbell et al., 1993; Hyytia, 1993; Krishnan-Sarin et al., 1995a, 1995b; Honkanen et al., 1996; Davidson & Amit, 1996; Franck et al., 1998; Heyser et al., 1999). Different studies also demonstrated that -opioid receptor agonists may also attenuate alcohol consumption 14 (DiChiara & Imperato, 1988b; Sandi et al., 1988; Spanagel et al., 1990, 1992). Naltrexone has been reported to be an effective treatment for individuals with alcohol dependence (Volpicelli et al., 1992; O’Malley, 1996). Combined with behavioural therapy, naltrexone, has been shown to reduce craving and alcohol intake in alcoholic patients (O’Malley, 1996). Important neurochemical targets for the acute effects of alcohol are also the facilitation of inhibitory GABAergic (widely accepted to underlie the acute sedative effects of alcohol), and inhibition of excitatory glutamatergic neurotransmission. Longterm adaptive changes in these two neurotransmitter systems to the sedative effects of alcohol are thought to underlie the development of alcohol dependence. In response to chronic exposure to alcohol, there is a compensatory up-regulation of the glutamatergic system and a down-regulation of the GABAergic system resulting in an increased tolerance for alcohol (Grobin et al., 1998). However, when alcohol is abruptly withdrawn, a state of hyper-excitability emerges, perceived by the subject as a disagreeable state of arousal, anxiety and sleeplessness. This is the core of the negative affective state from which the alcoholic patient will drink to relieve. These plastic changes in the brain, brought about by changes in protein synthesis, are only slowly reversible. This may explain the persistence of negative craving during alcohol withdrawal, and why stable abstinence after acute detoxification is so difficult to achieve. Pharmacological studies also support the important role of the GABAergic and glutamatergic neurotransmission in the reinforcing properties of alcohol. Treatment with GABAA antagonists, for instance, decreased operant alcohol self-administration. Using an operant model of ethanol self-administration, pretreatment with RO 15-4513, a benzodiazepine inverse agonist, at low doses selectively decreased responding for ethanol but not for water (Samson et al., 1987). Isopropylbicyclophosphate, a 15 picrotoxinin site ligand, selectively decreased responding for ethanol at very low doses in alcohol-preferring, alcohol nonpreferring, and Wistar rats (Rassnick et al., 1993). Above data, suggest that acute blockade of GABAA receptor function can block the motivation for responding for ethanol, supporting the hypothesis that activation of GABA is an important component in the acute reinforcing effects of ethanol. On the other hand, treatment with a selective GABAB agonist, decreased ethanol selfadministration in nondependent animals (Janak & Gill, 2003) and the alcohol deprivation effect in alcohol preferring rats (Colombo et al., 2003a, 2003b). Several clinical studies also have shown a potential efficacy of baclofen in reducing alcohol craving and ethanol withdrawal (Addolorato et al., 2002a, 2002b). These studies and evidence that GABAB receptor agonists may modulate mesolimbic dopamine neurons have provided a rationale for the hypothesis that activation of GABAB receptors may decrease the reinforcing actions of ethanol (Cousins et al., 2002). In addition, a very potent GABAA antagonist, SR 95531, when microinjected into the basal forebrain, significantly decreased ethanol consumption (Hyytia & Koob, 1995). SR 95531, was injected bilaterally into the NAacc, bed nucleus of the stria terminalis (BNST) and CeA in rats trained to self-administer alcohol in a limited access procedure. The most sensitive site of the effect of this drug was, however, the CeA (Hyytia & Koob, 1995). The GABAergic neurotransmission into the CeA has been reported to be an important target in alcohols’ reinforcing properties also by other researchers (Koob, 2003, 2004; Koob & Le Moal, 2001). Acute intraperitoneal injection of ethanol increased c-fos immunoreactivity in the CeA and over 70% of these cells were GABAergic neurons (Morales et al., 1998). In addition, electrophysiological studies showed an increased GABA release in the CeA after ethanol superfusion, an effect blocked by treatment with a GABAA receptor antagonist (Roberto et al., 2003). Moreover, in a study conducted by June et al., (2003), it was reported that microinjections of a GABA A receptor antagonist into the VP produced marked reductions in alcohol-reinforced 16 behaviors in two selectively bred rodent models of chronic alcohol drinking, the high alcohol drinking (HAD) and alcohol-preferring (P) rats (June et al., 2003). The glutamatergic system is also a target for the actions of ethanol. N-methyl-Daspartate (NMDA) receptor antagonists decreased ethanol intake in rats selectively bred for high ethanol preference (McMillen et al., 2004). Moreover, neramexane and acamprosate, an uncompetitive and a functional, respectively, NMDA receptor antagonists suppressed the rewarding effects of ethanol as measured in conditioned place preference (Kotlinska et al., 2004; McGeehan & Olive 2003). In addition, the mGluR5 antagonist MPEP decreased operant ethanol self-administration in the P and Wistar rats (Backstrom et al., 2004; Schroeder et al., 2005) and in the alcoholpreferring inbred C57BL6/J mice (Hodge et al., 2006). Cue-Induced Reinstatement of Alcohol Seeking Behavior Research utilizing reinstatement models of relapse points predominantly to a role for dopamine and opioid systems in the motivating effects of ethanol-associated environmental stimuli. However, there is also growing evidence for a role of glutamatergic (Glu) neurotransmission as well. In rats, re-exposure to environments associated with ethanol availability increases extracellular DA levels in the NAcc (Katner et al., 1996; Gonzales & Weiss, 1998; Bespalov et al., 1999; Weiss & Porrino, 2002), and ethanol-related visual cues activate the ventral striatum in abstinent alcoholics (Braus et al., 2001), suggesting that mesolimbic DA transmission may have an important function in the incentivemotivational effects of ethanol and, by extension, ethanol craving and relapse. Indeed, appetitively motivated behavior preceding delivery of an ethanol solution is more 17 sensitive to reversal by DA antagonists than behavior maintained by ethanol itself (Czachowski et al., 2001b). A role of DA in relapse associated with exposure to alcohol cues has been confirmed more directly by pharmacological findings, where selective blockade of either D1 or D2 receptors dose-dependently increased latency to initiate responding and reduced the number of responses at a previously active ethanol-paired lever (Ludwig et al., 1974; Liu & Weiss, 2002b). A role of opioid systems in relapse has also been implicated. In fact, clinical findings suggest that the nonselective opiate antagonist, naltrexone, attenuates craving associated with exposure to ethanol cues (Gerrits et al., 1999; Monti et al., 1999; Rohsenow et al., 2000) and reduces relapse rates in abstinent alcoholics (Krystal et al., 2001; O'Brien et al., 1996). Experimental support for this hypothesis comes from preclinical findings that naltrexone as well as - and delta-selective opiate antagonists reverse conditioned reinstatement of ethanol-seeking by ethanol-associated contextual stimuli (Katner et al., 1999; Ciccocioppo et al., 2002). With respect to glutamate transmission, evidence is accumulating for the role of this excitatory amino acid in processing of drug-associated stimuli. In fact, presentation of alcohol-associated stimuli has been found to increase accumbal glutamate levels in laboratory animals (Hotsenpiller et al., 2001). Treatment with acamprosate, a functional NMDA receptor antagonist reported to act through a partial interplay with the mGlu5 receptors (Harris et al., 2002), dose-dependently reduced reinstatement of ethanolseeking behaviour (Bachteler et al., 2005), whereas, in the same study, pretreatment with neramexane, a noncompetitive NMDA receptor antagonist, did not significantly modify cue-induced alcohol-seeking, suggesting an involvement of the mGlu5 receptor in this behaviour (Bachteler et al., 2005). In respect to this data, pretreatment with MPEP, a noncompetitive mGlu5 receptor antagonist significantly attenuated in a doserelated manner ethanol-seeking behavior (Backstrom et al., 2004). In another study, 18 treatment with the mGlu2/3 receptor agonist LY379268 and the mGlu8 receptor agonist (S)-3,4-DCPG, also blocked alcohol reinstatement (Backstrom & Hyytia, 2005). Moreover, in a more recent study, a significant reduction of cue-induced alcoholseeking behaviour was also seen with the AMPA receptor antagonist GYKI 52466 (Sanchis-Segura et al., 2006). Stress-Induced Reinstatement of Alcohol Seeking Behavior Another major factor implicated in the resumption of compulsive drinking includes the subjective reactions provoked by stressful events (Brown et al., 1995; Sinha et al., 1999, 2000; Weiss et al., 2001; Breese et al., 2005). The significance of stress as a factor for relapse risk is also well documented in the animal literature where footshock stress and intracranial administration of CRF produces strong reinstatement of ethanol-seeking behavior (Le et al., 1999, 2000, 2002; Martin-Fardon et al., 2000; Liu & Weiss, 2002a; Funk et al., 2003), while administration of CRF antagonists prevents drug-seeking behavior induced by these manipulations (Funk et al., 2003; Le et al., 2000, 2002). Data from the literature, proposes two CRF-rich brain regions that are part of the non-neuroendocrine brain stress system, to be mainly implicated in alcohol-seeking responses induced by footshock stress; the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). The preponderance of existing evidence suggests that the BNST which receives CRF-containing neurons from the CeA (Sakanaka et al., 1986; Gray, 1993), and, like the CeA, contains a dense network of CRF immunoreactive cells, appears to be the most sensitive site for the response-reinstating effects of CRF and footshock. Direct administration of CRF into the BNST, but not into the CeA, elicits cocaine-seeking behavior (Erb et al., 2001). Consistently, local injection of the CRF receptor antagonist D-Phe-CRF(12-41) into the 19 BNST but not the CeA antagonized foot-shock-induced reinstatement of cocaineseeking behaviour (Erb & Stewart, 1999; Erb et al., 2001). Thus, activation of CRF receptors in the BNST may be a critical substrate for stress-related unconditioned responses in general, including stress-induced alcohol-seeking behavior. It is, however, important to consider that the BNST receives a CRF-containing projection from the CeA (Gray, 1990; Sakanaka et al., 1986) such that stress-induced activation of the CeA may, via this pathway, be responsible for the effects of stress on drug-seeking behavior. Therefore, both the CeA and BNST may participate in the regulation of stress-induced drug-seeking and other unconditioned responses, although the BNST may perhaps represent a critical output pathway for these effects. Alcohol Withdrawal Up to 71% of individuals presenting for alcohol detoxification, manifest significant symptoms of alcohol withdrawal (Myrick & Anton, 1998). Alcohol withdrawal, is a clinical syndrome that affects alcoholic patients who either decrease their alcohol consumption or stop drinking completely. The affective withdrawal symptoms, make alcohol abstinence difficult and increase the risk of relapse in recovering alcoholics (Anton, 1999; Spanagel, 2003). Alcohol withdrawal syndrome is mediated by a variety of mechanisms. The brain is known to maintain neurochemical balance through inhibitory and excitatory neurotransmitters. The main inhibitory neurotransmitter is GABA which acts through the GABAA receptor, while, one of the major excitatory neurotransmitters is glutamate, which acts through the NMDA receptor. Alcohol enhances the effect of GABA on GABAA receptors, resulting in decreased overall brain excitability. Chronic exposure to alcohol results in a compensatory decrease of GABAA receptor response to GABA, 20 evidenced by increasing tolerance of the effects of alcohol. Alcohol inhibits NMDA receptors, and chronic alcohol exposure results in up-regulation of these receptors. Abrupt cessation of alcohol exposure results in brain hyperexcitability, because receptors previously inhibited by alcohol are no longer inhibited. Brain hyperexcitability manifests clinically as anxiety, irritability, agitation, and tremors. Severe manifestations include alcohol withdrawal seizures and delirium tremens. Benzodiazepines are the treatment of choice for alcohol withdrawal syndrome in humans (Mayo-Smith, 1997) and they are able to prevent most of the clinical manifestations of this condition. NMDA receptor antagonists have also been reported to markedly reduce ethanol withdrawal signs in rodents (Morriset et al., 1990; Liljequist, 1991; Thomas et al., 1997). 2. N/OFQ-NOP receptor brain system N/OFQ, is a 17-aminiacid peptide that shows high structural homology with opioid peptides, in particular with dynorphin A (Meunier et al., 1995; Reinscheid et al., 1995, 1998). However, N/oFQ lacks the N-terminal tyrosine residue necessary for affinity at classic opioid receptors (Meunier et al., 1995; Reinscheid et al., 1995, 1998). Thus, N/OFQ selectively binds at nanomolar concentrations to the NOP receptor (previously referred to as ORL1 or OP4 receptor), but not to MOP, KOP, or DOP opioid receptors, nor do opioid peptides activate the NOP receptor (Reinscheid et al., 1996). Activation of membrane NOP receptors by N/OFQ results in the same sequence of intracellular events induced by opioid receptors, namely negative coupling with adenylyl cyclase, activation of inwardly rectifying K+ channels, and inhibition of Ca2+ 21 current in a pertussis toxin-sensitive manner (Reinscheid et al., 1995; Meunier et al., 1995; Meunier, 1997; Henderson & McKnight, 1997; Darland et al., 1998). However, these cellular responses to N/OFQ are insensitive to naloxone (Henderson & McKnight, 1997; Darland et al., 1998), confirming that the pharmacological actions of this peptide are not mediated by the classic opioid receptors. Neuroanatomical and immunohistochemical studies have shown that N/OFQ and the NOP receptor are widely distributed in brain areas involved in reward processes (Fig. 1). In fact, a high density of the NOP receptor has been found in the amygdala, particularly in its central nucleus (CeA), whereas a dense distribution of these receptors has also been reported in the frontal cortex, VTA, VP, BNST, and lateral hypothalamus. A light density of the NOP receptors has been reported also in the NAcc, olfactory tubercle and hipocampus. The same brain regions also reveal moderate to high levels of mRNA for NOP receptors. The distribution of mRNA for the N/OFQ precursor differs from that of the NOP receptors, but again moderate to high density of mRNA for the N/OFQ precursor has been reported in the frontal cortex, BNST, and CeA. On the other hand, the VTA and VP exhibit only a light density, while in the NAcc low levels of mRNA for the N/OFQ precursor have been found (Anton et al., 1996; Florin et al., 1997; Sim & Childers, 1997; Darland et al., 1998; Ikeda et al., 1998; Neal et al., 1999). 22 Figure 1. Distribution of N/OFQ and NOP receptor in the brain (taken from, Darland et al., 1998) Electrophysiological data, reports that N/OFQ prevents glutamate-mediated postsynaptic neuronal activation in the hippocampus and amygdala (Tallent et al., 2001; Marti et al., 2002), and inhibits the firing of -endorphin cells in the hypothalamic arcuate nucleus (Wagner et al., 1998). These arcuate neurons project among other brain regions to VTA and NAcc, where they interact with mesolimbic DA transmission and influence motivated behaviours (DiChiara & North, 1992; Johnson & North, 1992; Devine et al., 1993a, 1993b; Herz, 1997). Moreover, it has been shown that 91% of tyrosine hydroxylase-positive cells in the VTA co-express NOP receptors, and that N/OFQ can directly and indirectly (via GABA interneurons) modulate neural activity of VTA DA neurons (Maidment et al., 2002; Norton et al., 2002; Zheng et al., 2002). 23 2.1. N/OFQ and neurotransmitter release in the brain reward system 2.1.1. Dopamine Murphy et al., (1996) have reported that intracerebroventricular (ICV) injections of N/OFQ reduce DA levels in the NAcc of anesthetized rats (Murphy et al., 1996). The same authors observed that administration of N/OFQ in the VTA of anesthetized rats evokes a pronounced reduction of extracellular DA levels in the NAcc, suggesting that the VTA may be the site of action for this effect (Murphy et al., 1996; Murphy & Maidment, 1999). Administration into the VTA of the GABAA receptor antagonist bicuculline prevented the effect of N/OFQ on NAcc DA levels, suggesting that GABAergic interneurons located in the VTA may mediate the effect of N/OFQ (Murphy & Maidment, 1999). However, the results obtained in microdialysis studies carried out in freely moving rats are somehow different from those obtained in anesthetized rats. In freely moving rats, it has been reported that ICV injections of N/OFQ do not modify basal DA levels, but completely abolish morphine-induced DA release in the NAcc (Di Giannuario et al., 1999). Altogether these observations support the idea that N/OFQ may be involved in the control of DA release in the NAcc. It is likely that the different results reported above for the effect of N/OFQ on basal DA levels are related to the use of anesthetized rats (halotane) in the first study and of conscious rats in the latter. 2.1.2. Opioids Electrophysiological studies have demonstrated that N/OFQ inhibits the activity of -endorphinergic neurons in the hypothalamic arcuate nucleus (ARC), by activating 24 a subset of inwardly rectifying K+ channels (Wagner et al., 1998). These neurons project to many cerebral areas, including the VTA and the NAcc, where they can influence reward processes and interact with the mesolimbic DAergic system (Herz, 1997). N/OFQ, has been found to reverse several of the actions of opiate drugs, which has given rise to the hypothesis that N/OFQ may act as a functional "anti-opioid" agent. Specifically, N/OFQ blocks the analgesic effects of morphine (Samson & Harris, 1992; Mogil et al., 1996) prevents the development of morphine-induced conditioned place preference (Ciccocioppo et al., 1999a; Murphy et al., 1999) and at the neurochemical level, inhibits morphine-induced DA release in the NAcc (Di Giannuario et al., 1999). 2.1.3. GABA N/OFQ, has been reported to increase GABA overflow in the VTA (Murphy & Maidment, 1999), and this effect is apparently responsible for the effect of the peptide on NAcc DA levels (see Section 2.1.1.). However, N/OFQ was reported to decrease, by a presynaptic mechanism, GABA release in different brain areas, such as the lateral and central nucleus of the amygdala (Meis & Pape, 1998, 2001). 2.1.4. Glutamate N/OFQ, has been shown to evoke glutamate release in the VTA (Murphy & Maidment, 1999). This effect may contribute to the modulatory effect of N/OFQ on DA neurotransmission in the NAcc. Moreover, N/OFQ has been reported to facilitate glutamate release through NOP receptors in the substantia nigra as well (Marti et al., 25 2002). On the other hand, N/OFQ inhibits K+-evoked glutamate release in cerebrocortical slices in a naloxone insensitive manner (Nicol et al., 2002). 2.2 Alcohol-abuse related behaviours: Putative significance of the N/OFQ-NOP receptor system Alcohol Reinforcement As mentioned above (section 1.4.), it is in recent days proposed that the rewarding and reinforcing effects of ethanol may be mediated by stimulation of the mesocorticolimbic DAergic system (Gessa et al., 1985; Imperato & Di Chiara, 1986; Weiss et al., 1993). The effects of ethanol on the mesolimbic DAergic system may, in turn, be dependent upon the effects of this drug on the endogenous opioid system (Johnson & North, 1992; Di Chiara, 1995; Herz, 1997). In fact, activation of endorphinergic neurons of the ARC may represent an important mechanism by which ethanol stimulates reward processes (Herz, 1997). These -endorphin neurons project, in part, to the NAcc where they may stimulate DAergic neurotransmission by activating -opioid receptors located on DAergic terminals. Other -endorphin neurons project from the ARC to the VTA, thus stimulating -opioid receptors located on GABA interneurons of the VTA, which inhibit the DAergic neurons (Johnson & North, 1992). The electrophysiological study of Wagner et al., (1998) demonstrated that N/OFQ inhibits the activity of -endorphinergic neurons of the ARC, and this effect may contribute to reduce the rewarding properties of ethanol (Wagner et al., 1998). Alternatively, it is possible that N/OFQ may inhibit DAergic neurotransmission in the 26 NAcc, by inhibiting the DAergic neurons in the VTA (Murphy & Maidment, 1999). The VTA expresses relatively high levels of intermediate-size N/OFQ neurons, and shows high density of NOP receptors (Ikeda et al., 1998). Therefore, it is possible that, in this nucleus, N/OFQ modulates the activity of DAergic neurons. Cue-Induced Reinstatement of Alcohol Seeking Behavior As discussed above (see Section 1.4.), several lines of evidence suggest a prominent role of opioid neurotransmission in the control of ethanol-seeking behaviour induced by environmental stimuli. In fact, treatment with the non-selective opioidergic antagonist naltrexone as well as with - and -selective opiate antagonists blocked cueinduced ethanol relapse (Ciccocioppo et al., 2002; Katner et al., 1999). Therefore, considering the functional anti-opioid actions of N/oFQ in conjunction with the growing evidence on “anti-craving” and “anti-reinstatement” actions of opiate antagonists, we’d like to speculate that the NOP receptor represents a promising target for treatments aimed at relapse prevention. In addition, N/oFQ has been also shown to modulate brain glutamatergic neurotransmission, by inhibiting, via a presynaptic mechanism, glutamate transmission in several brain regions including the hippocampus and amygdala (Meis & Pape, 2001; Tallent et al., 2001; Marti et al., 2002). As discussed above, however (see Section 1.4.), the glutamatergic neurotransmission has an important role in drug-related learning and behaviour and, therefore, in cue-induced relapse. Subsequently, the reduction of glutamatergic activity by N/OFQ may be another mechanism by which activation of the NOP receptor may reduce reactivity to alcohol cues. 27 Stress-Induced Reinstatement of Alcohol Seeking Behavior N/OFQ, is known to exert pronounced anxiolytic actions and to reduce responsiveness to stress in rodents (Griebel et al., 1999; Jenck et al., 2000a, 2000b; Ciccocioppo et al., 2001). In rats, ICV injection of the peptide increases the time spent in the open arms of the elevated plus maze and, in mice, reverses the suppressant effect of urocortin, a CRF analogue, on spontaneous exploration of an unfamiliar environment while increasing the time spent in the light compartment of a light-dark box (Jenck et al., 1997). In addition, mice deficient for the N/OFQ gene consistently show impaired adaptation to repeated stress, and both basal and post-stress plasma corticosterone levels are elevated in these animals compared to wild type controls (Koster et al., 1999). Recent data, strongly suggests that the anxiolytic actions of the peptide is related to a functional antagonist action on the brain CRF system. Although devoid of affinity for CRF receptors (Imaki et al., 1995; Jenck et al., 1997), N/OFQ completely blocked the anorexic effect of restraint and footshock stress as well as of intracranial CRF administration (Ciccocioppo et al., 2002, 2003). Furthermore, microinjection studies have revealed that the BNST is a particularly sensitive site for the “anti-CRF” effect of N/oFQ on reversal of stress-induced inhibition of food intake (Ciccocioppo et al., 2003). Consistent with the apparent functional CRF antagonist action of N/OFQ, central administration of this peptide was found to antagonize the effects of footshock stress on reinstatement of ethanol-seeking (Martin-Fardon et al., 2000). In spite of the recognition that stress is a major factor in the resumption of drinking in detoxified alcoholics no effective treatment medicaments are currently available for ameliorating relapse risk associated with stress. In fact, preclinical data from animal studies reported that naltrexone (in clinic treatment for alcohol abuse) fails to prevent footshock-induced reinstatement (Le et al., 1999; Liu & Weiss, 2002a) suggesting, that opiate antagonists will be of little benefit in this regard. Stress-induced 28 reinstatement of ethanol-seeking is, however, effectively blocked by selective CRF receptor antagonists, such as D-Phe-CRF, or CP-154,526 (Liu & Weiss, 2002a). Considering, the putative dual action of N/OFQ to act as a functional CRF antagonist, in addition to its functional opiate antagonist action, we speculate that the N/OFQ-NOP receptor system may be a highly promising target for “anti-relapse” medications. Alcohol Withdrawal N/OFQ, is known to possess (like benzodiazepines) anxiolytic properties and to reduce responsiveness to stress in rodents (Griebel et al., 1999; Jenck et al., 2000a, 2000b; Ciccocioppo et al., 2001). In addition, N/OFQ is known to act in the brain as a presynaptic neuron inhibitor controlling, among other, the glutamatergic neurotransmission in different brain areas (Schlicker & Morari, 2000). Therefore, taking into consideration above properties of N/OFQ we predict an effect of this peptide on alcohol withdrawal signs induced by chronic ethanol drinking. 3. Behavioural models for the investigation of alcohol abuse 3.1. Animal models In regard to the important contribution of genetic factors in the predisposition to alcohol abuse and alcoholism (see Section 1.2.), selective breeding for high and low alcohol-drinking preference has produced animal lines useful for studying the nature of excessive alcohol-drinking and -seeking behaviour (Nevo & Hamon, 1995). Until 29 recent days, different sets of alcohol-preferring and non-preferring lines of rats have been developed through selective breeding programs. The ALKO alcohol/nonalcohol (AA/ANA) lines (Eriksson, 1968), the Sardinian alcohol-preferring/nonpreferring (sP/sNP) lines (Colombo, 1997), the alcohol-preferring/nonpreferring (P/NP) lines (Lumeng et al., 1977), and the high/low alcohol-drinking (HAD/LAD) lines (Li et al., 1993). Compared with the alcohol-nonpreferring line, the preferring animals have been found to exhibit differences in various neurotransmitter neuromodulator systems involved in reward- and alcohol-related behaviors. Dopamine: When compared with the alcohol-nonpreferring counterparts, the alcohol-preferring rats have been found to exhibit lower DA levels in the NAcc as well as in other forebrain areas (Murphy et al., 1987). In particular, the HAD rats have 1030% lower levels of DA in the NAcc compared with the LAD rats (Gongwer et al., 1989). Zhou et al., (1995) found a decreased number of neuronal projections from the VTA to the NAcc in the P rats relative to the NP rats, indicating these animals have fewer dopamine fibres in the accumbens shell (Zhou et al., 1995). In accordance to this data, McBride et al., (1995) demonstrated a 25% lower content of DA in the NAcc in the P compared to the NP offsprings (McBride et al., 1995). However, when neuronal activity of the VTA DA neurons was compared in P and Wistar rats, VTA DA neurons were found to have more frequent burst firing in the P rats (Morzorati, 1998), suggesting that this increased activity in the alcohol-preferring P rats could be a compensatory mechanism for the reduced DA neurons in this animal line. Quantitative autoradiography revealed a 20-25% lower binding of tritiated sulpiride in the caudate putamen, the medial and lateral nucleus accumbens, and the VTA of P rats, compared with NP rats (McBride et al., 1993a). Similar findings where revealed in the sP and sNP rats (Stefanini et al., 1992; Casu et al., 2002). 30 Oral self-administration of ethanol was found to increase DA overflow above baseline values to a greater extent in the NAcc of the P rats respect the unselected Wistars (Weiss et al., 1993), suggesting that the VTA DA system of the P line of rats is more sensitive to the reinforcing actions of ethanol. Moreover, anticipation for ethanol access increased extracellular NAcc DA levels in the P but not Wistar rats (Katner et al., 1996). In another study, Katner and Weiss, (2001) compared rats from the HAD, LAD, AA, ANA, and Wistar lines for the release of DA from the NAcc, and found that rats predisposed to high ethanol intake have a greater DA release in this brain area in response to ethanol (Katner and Weiss, 2001). Autoradiografic studies, in diverse limbic regions, showed a higher density of the D1 receptor sites in the sNP compared to the sP rats (DeMontis et al., 1993), but no difference was seen in the P and HAD respected to the NP and LAD lines, respectively (McBride et al., 1997a, 1997b). For the D2 receptor, a higher density was shown in the sNP and NP respect their counterparts (Stefanini et al., 1992; McBride et al., 1993a), but not in the HAD/LAD and AA/ANA lines (Syvalahti et al., 1994; McBride et al., 1997a). No differences in the density of the D3 receptor were found in the alcoholpreferring compared to the non-preferring rat lines (McBride et al., 1997a, 1997b). Serotonin (5-HT): An association between high ethanol preference and lower 5HT contents in several CNS areas was found. Compared with NP rats, the P rats have 12-26% lower levels of 5-HT and its primary metabolite 5-HIAA in the cerebral cortex, frontal cortex, whole corpus striatum, anterior striatum, NAcc, hippocampus, thalamus, and hypothalamus (Murphy et al., 1982, 1987). HAD rats also have lower 5-HT and/or 5-HIAA levels in the cerebral cortex, striatum, NAcc, septal nuclei, hippocampus, and hypothalamus compared with LAD rats (Gongwer et al., 1989). The above mentioned 31 lower contents of brain 5-HT observed, seem to be due to a reduced 5-HT innervation. In fact, Zhou et al., (1991, 1994a) reported that immunoreactive 5-HT fibres were lower in several CNS regions of the P rat line compared to the NP line, e.g., frontal cortex, NAcc, hippocampus. This, may be a result of fewer 5-HT neurons in the medial and dorsal raphe of the P rats (Zhou et al., 1991, 1994b). However, lower serotonin levels have not been seen in all rats bred for high ethanol preference. In fact, higher levels of 5-HT were found in certain brain regions in the AA compared with ANA rats (Ahtee & Eriksson, 1972, 1973; Korpi et al., 1988). Autoradiografic studies in diverse limbic regions, showed an increased density of the 5-HT1A receptor in the P compared to the NP rats (McBride et al., 1994, 1997b), but no differences were seen in the HAD and ANA lines compared to the LAD and ANA rats, respectively (Kopri et al., 1992; McBride et al., 1997a). In the contrary, for the 5HT1B receptor a lower density was found in the P compared to the NP rats (McBride et al., 1997b). In regard to the 5-HT2 receptor, a lower density was found in the P compared to the NP rats (McBride et al., 1993b), a result also found in the sNP compared the sP line (Ciccocioppo et al., 1999b). No differences in the expression of this receptor (5-HT2) were seen in the HAD and ANA lines compared to their counterparts (Korpi et al., 1992; McBride et al., 1997a). In addition, no differences in the 5-HT3 receptor levels were found in the AA compared to the ANA rat line (Ciccocioppo et al., 1998), whereas, data regarding the 5-HT3 receptor levels is controversial. In fact, McBride et al., (1997b) reported no differences in this receptor in the P compared to the NP rats, whereas, Ciccocioppo et al., (1998) reported a decreased density of the 5-HT3 receptor in the amygdala of the P rats compared to the NP animals (McBride et al., 1997b; Ciccocioppo et al., 1998). 32 GABA: An increase in GABAergic terminal density was found in the NAcc of the P rats, when compared with the NP rats. Similarly, there were more GABAergic terminals in the NAcc of HAD rats than of the LAD rats (Hwang et al., 1990). Thielen et al., (1997) by employing quantitative autoradiography demonstrated a significantly greater GABA-enhanced flunitrazepam binding in P than in NP rats in several cortical areas, whereas lower binding was found in entorhinal cortex, the mediodorsal thalamus, and the posterior hippocampus (Thielen et al., 1997). A result also shown, by Wong et al., (1996) in the AA compared to the ANA rats (Wong et al., 1996). Opioids: Comparisons of ethanol-naive P and NP rats revealed no line differences in preproenkephalin mRNA contents in the Nacc, striatum, amygdala, or hypothalamus. However, Li et al., (1998) showed that intragastric infusion of 2.5 g/kg ethanol increased the mRNA content in the NAcc of P rats, but not in NP rats (Li et al., 1998), suggesting that the accumbens enkephalinergic system of the P line is more sensitive to the effects of ethanol. Moreover, a higher hypothalamic proopiomelanocortin (POMC) mRNA expression has been seen in the AA respect the ANA rats (Gianoulakis et al., 1992; Marinelli et al., 2000). Using quantitative autoradiographic methods, a higher density of -opioid receptors has been seen in various limbic areas of the P relative to NP rats, including the NAcc shell and core (McBride et al., 1998). These differences are in general agreement with differences observed in the NAcc and prefrontal cortex between the AA and ANA lines (DeWaele et al., 1995; Marinelli et al., 2000). A lower density of opioid receptors in the ventromedial hypothalamus has been found in the AA respect the ANA rats (Marinelli et al., 2000). Finally, in relation to the -opioid receptors data are controversial. In the AA rats, a higher density compared to the ANA rats was found 33 (DeWaele et al., 1995; Soini et al., 1998), whereas in the P rats, Strother et al., (2001) reported a lower density respect the NP line (Strother et al., 2001). 3.2. Behavioural models Voluntary alcohol drinking The most widely used method of assessing alcohol drinking is to measure consumption under the two bottle choice paradigm. Under this model animals are offered, in their home cages, free choice between alcohol and water in graduate tubes from which the intakes are measured by reading the volume consumed from the graduated burettes. When scheduled ethanol availability is limited each day, animals drink at a higher rate and achieve higher blood ethanol concentrations than when it is available continuously, even though higher doses are consumed under unlimited access conditions. Operant self-administration Self-administration procedures have been used to model patterns of human drug abuse. Operant self-administration requires that animals emit a specified behavior, typically a bar press, to obtain brief access to an ethanol solution (Fig. 2). Operant studies assess the motivation to procure alcohol, and reveal medication effects on within session ingestion patterns. Solutions with similar reinforcing effects to alcohol can be identified to ascertain whether medication effects are specific to alcohol reinforcement. 34 Figure 2. Picture of self-administration chamber The vast majority of the selfadministration experiments that have been carried out to study alcohol intake and reinforcement with rats have employed fixed ratio (FR) schedules of reinforcement and progressive ratio (PR). Fixed ratio schedule: Under a FR schedule of reinforcement animals must press a lever an invariable number of times (FR1, 1 time; FR2, 2 times; etc) to receive one dose of the reward. Progressive ratio schedule: Under the PR schedule of reinforcement the animal must progressively increase the number of lever pressings to receive a single dose of the reward. The self-administration session is considered finished when the animal fails to complete the ratio within the time-period the session lasts (e.g., 30 min). Break point is the last ratio successfully completed by the animal and is a measure of the reinforcing value of the drug. Cue-induced drug seeking behaviour: Under this experimental paradigm, animals learn to associate and discriminate between cues or stimuli predictive of alcohol vs water availability. Then, if after a period of extinction (no cue presentation) and in the 35 absence of the reinforcer (e.g., alcohol) the drug-paired cues are re-presented to the animal, a seeking behaviour (a significant increase of the number of lever pressings respect extinction levels) is observed. This behaviour is not observed after re-exposure to the water-paired cues. Stress-induced drug seeking behaviour: Here, the animal after an extinction period (self-administration in the absence of the reinforcer) and before the extension of the levers, receives into the self-administration chamber footshock stress for 15 min. Then, the levers come out into the chamber and the animals’ behaviour is measured (always in the absence of the reinforcer). Alcohol withdrawal: Animals are made alcohol dependent after a period (5-6 days) of alcohol intoxication, during which they receive 12-13 g/kg of 25% w/v of ethanol per day at cycles of 12-h. 8-24 hours after the last ethanol intoxication withdrawal symptoms are measured in the animals. 36 3. Summary, Research Objectives, and Significance Alcohol abuse and persistent vulnerability to relapse following withdrawal and abstinence represents a great challenge for the treatment of alcohol addiction. The main objective of the studies present in this thesis, was to investigate and provide information on the potential pharmacotherapeutic use of the NOP receptor as a promising target for the treatment of alcohol abuse. The first study (Chapter 2) summarises previous work concerning the possible role of the N/OFQ-NOP receptor system in the control of alcohol abuse. In the following study (Chapter 3), it was examined whether the ability of N/OFQ to reduce home-cage (two bottle choice) ethanol consumption in the alcohol-preferring msP rats, also extends to treatment with different newly synthesized peptidergic NOP receptor agonists. Buprenorphine, is an analgesic and is a maintenance treatment of opioid dependence. Recently, it was found that buprenorphine is a full agonist at the NOP receptor (Bloms-Funke et al., 2000; Lutfy et al., 2003; Wnendt et al., 1999). In Chapter 4, we evaluated whether buprenorphine would reduce voluntary ethanol consumption in the msP rats via activation of NOP receptors. In a successive study (Chapter 5), the ability of N/OFQ to reduce alcohol reinforcement under operant conditions of self-administration was investigated. In this study, we also tested whether administration of this peptide affects cue-induced reinstatement of alcohol-seeking behaviour in the msP rats. Overall investigations were performed in the alcohol-preferring msP rats. In another study, we aimed at evaluating the ability of N/OFQ to reduce ethanol selfadministration also in the nonselected Wistar rats. In this study, the site of action of the effects of N/OFQ on operant alcohol intake was also investigated (Chapter 6). 37 As mentioned above, the abrupt cessation of alcohol intake causes to the patients withdrawal symptoms that are often associated with relapse in recovering alcoholics. 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Alcohol 12:403-12. 69 Chapter 2 The nociceptin/orphanin FQ/NOP receptor system as a target for treatment of alcohol abuse: a review of recent work in alcohol-preferring rats 70 The nociceptin/orphanin FQ/NOP receptor system as a target for treatment of alcohol abuse: a review of recent work in alcohol-preferring rats Abstract The intracerebroventricular administration of the 17 aminoacid peptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor (previously referred to as ORL-1 or OP4 receptor), reduces voluntary 10% ethanol intake in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats. Studies aimed at the pharmacological characterization of the receptor, which mediates the effect, have shown that the C-terminal 13 aminoacid sequence is crucial for activity and that the selective NOP receptor antagonist [Nphe1]N/OFQ(1–13)NH2 blocks the effect of N/OFQ on ethanol drinking. In place conditioning studies, N/OFQ abolishes the conditioned place preference (CPP) induced by ethanol in msP rats, or by morphine in nonselected Wistar rats; these findings suggest that N/OFQ is able to abolish the rewarding properties of ethanol and morphine. Moreover, N/OFQ inhibits reinstatement of alcohol-seeking behavior induced to electric footshock stress, as well as reinstatement of alcohol-seeking behavior induced by ethanol-paired cues. Together, these findings suggest that N/OFQ and its receptor may represent an interesting target for pharmacological treatment of alcohol abuse. Keywords: Nociceptin/orphanin FQ; NOP receptor; Alcohol intake; Reward; Alcohol-seeking behavior; Relapse 71 1. Introduction Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor, previously referred to as opioid receptor-like1 (ORL1) receptor (Meunier et al., 1995; Reinscheid et al., 1995; Cox et al., 2000), is a 17 aminoacid neuropeptide structurally related to the opioid peptide dynorphin A (Nothacker et al., 1996; Reinscheid et al., 1998). Despite its structural homology with opioid peptides, N/OFQ does not bind to MOP, DOP or KOP opioid receptors (previously referred to as , or receptors), nor do opioid peptides activate the NOP receptor (Reinscheid et al., 1996). Nevertheless, activation of membrane NOP receptors by N/OFQ results in the same sequence of intracellular events induced by classic opioid receptors, namely, negative coupling with adenylyl cyclase, activation of inwardly rectifying K+ channels and inhibition of Ca2+ current in a pertussis toxin sensitive manner (Reinscheid et al., 1995; Henderson & McKnight, 1997; Meunier, 1997; Darland et al., 1998; Hawes et al., 2000). These cellular responses to N/OFQ are insensitive to naloxone (Henderson & McKnight, 1997; Darland et al., 1998), providing evidence that the pharmacological actions of this peptide are not mediated by the classic opioid receptors. Brain mapping studies show that the neuroanatomical distribution of N/OFQ and its receptor differs from that of other opioid peptides and opioid receptors (Anton et al., 1996; Florin et al., 1997; Sim & Childers, 1997; Darland et al., 1998; Ikeda et al., 1998; Neal et al., 1999). Interestingly, these studies have shown from moderate to high density of N/OFQ and its receptor in brain areas such as the amygdala (AMY), the medial prefrontal cortex (mPFC), the ventral tegmental area (VTA), the lateral hypothalamus (LH), the bed nucleus of the stria terminalis (BNST) and the nucleus accumbens (NAc), that represent important neuroanatomical elements of the brain reward circuits and are involved in the regulation of motivational processes. 72 From a functional point of view, N/OFQ possesses antiopioid properties (Mogil et al., 1996a, 1996b; Mogil & Pasternak, 2001) and, acting as a presynaptic neuron inhibitor, it is able to control dopaminergic, noradrenergic and glutamatergic neurotransmission in different brain sites (Schlicker & Morari, 2000). Altogether, these findings point at the N/OFQ–NOP receptor as a system potentially involved in the regulation of reward and drug abuse processes. Indeed, several studies demonstrate that activation of this system results in reduction of the rewarding properties of ethanol, morphine and cocaine (Ciccocioppo et al., 1999b, 2000a 2000b; Murphy et al., 1999). Lastly, recent data suggest that central administration of N/OFQ reduces reinstatement of alcohol-seeking behavior elicited by stress and by environmental conditioning factors (Martin-Fardon et al., 2001; Ciccocioppo et al., 2002b). This article is aimed at summarizing previous work, and presenting some new data of our group, concerning the possible role of the N/OFQ–NOP receptor system in the control of alcohol abuse. 2. N/OFQ and voluntary alcohol intake It is well known that the mesocorticolimbic dopaminergic system is involved in mediating the rewarding and reinforcing effects of ethanol (Gessa et al., 1985; Imperato & Di Chiara, 1986; Weiss et al., 1993), and that this effect depends, at least in part, on the interaction with the endogenous opioid system (Johnson & North, 1992; Di Chiara, 1995; Herz, 1997). Pretreatment with opioid receptor antagonists, like naloxone or naltrexone (Grahame et al., 2000; Volpicelli et al., 1992), or with selective dopaminergic antagonists (Panoca et al., 1995), significantly reduces ethanol intake. 73 N/OFQ, even though it does not bind to the classic opioid receptors, has been proposed to represent a brain ‘‘anti-opioid’’ peptide for its property to reverse morphine-induced analgesia (Mogil et al., 1996a, 1996b; Mogil & Pasternak, 2001). Microdialysis data have shown that central administration of N/OFQ can modulate extracellular dopamine levels in anesthetized rats, as well as in freely moving animals (Eriksen & Gotestam, 1984; Di Giannuario et al., 1999). These evidences prompted us to begin studies aimed at evaluating whether N/OFQ might reduce ethanol consumption in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats (Gessa et al., 1991; Ciccocioppo et al., 2000a), an animal model of high ethanol intake. In these studies, ethanol availability was restricted to 30 min/day after N/OFQ administration, while water and food were available ad libitum over the 24 h. N/OFQ was given into the left lateral cerebroventricle (icv) just prior to the presentation of 10% (v/v) ethanol. In these studies, subchronic (7 days) intracerebroventricular administration of 0.5–1.0 g/rat of N/OFQ significantly reduced ethanol intake in msP rats (Ciccocioppo et al., 1999b). While chronic experiments have consistently shown reduction of ethanol intake in response to N/OFQ, variable findings have been obtained following acute administration of the peptide. In some experiments, the acute intracerebroventricular injection of N/OFQ, 0.5 g/rat, resulted in a modest but significant increase in ethanol intake (Ciccocioppo et al., 1999b, 2000a); it was proposed that the increased intake in response to the first administration of N/OFQ might be due to an attempt of the animal to overcome the inhibitory effect of N/OFQ on the rewarding properties of ethanol. Interestingly, the subchronic administration of N/OFQ reduced ethanol intake, but did not modify the concomitant intake of water and food (Ciccocioppo et al., 1999b). Several studies have shown that N/OFQ stimulates feeding in freely feeding rats (Pomonis et al., 1996; Leventhal et al., 1997, 1998; Stratford et al., 1997; Polidori et al., 2000a, 2000b), however, the hyperphagic effect is observed at doses higher than those 74 which reduce ethanol intake. In freely feeding msP rats, a statistically significant hyperphagic effect is observed in response to intracerebroventricular doses of 3–4 g/rat of N/OFQ, while the effect on ethanol intake is observed at 0.5 or 1 g/rat. The intracerebroventricular injection of 0.5–1.0 g/rat of N/OFQ did not modify blood alcohol levels ( Ciccocioppo et al., 2000a), determined by gas chromatography following intragastric administration by gavage of 0.7 g/kg of 10% ethanol. These findings indicate that the effect on ethanol intake is not the consequence of unselective interference with the rats’ ingestive behavior or of interference with the pharmacokinetics of ethanol. Further experiments were aimed at the pharmacological characterization of the NOP receptor involved in this effect of N/OFQ on ethanol intake (Ciccocioppo et al., 2002d). For this purpose, the effect on ethanol drinking of intracerebroventricular injections of N/OFQ(1–17)NH2, N/OFQ(1–13)NH2 or N/OFQ(1–12)NH2 was studied. The effect on ethanol intake of the heptadecapeptide amide, N/OFQ(1–17)NH2, was similar in intensity to that of N/OFQ; however, N/OFQ(1–17)NH2 was slightly less potent than the endogenous ligand, since it significantly reduced ethanol intake at 1.0, but not 0.5 g/rat. The tridecapeptide, N/OFQ(1–13)NH2, induced a statistically significant, but less pronounced attenuation of ethanol intake only at 2.0 g/rat, whereas the dodecapeptide N/OFQ(1–12)NH2 was ineffective at intracerebroventricular doses up to 4.0 g/rat. Thus, the tridecapeptide is apparently crucial for the effect of N/OFQ on ethanol consumption. This finding is similar to those reported by our group for the pharmacological characterization of the NOP receptor controlling food intake (Polidori et al., 2000a, 2000b), as well as to those obtained in a variety of other in vitro and in vivo tests (Calò et al., 2000a, 2002). Lastly, to confirm that the effect of N/OFQ is mediated by NOP receptors, the ability of the selective NOP receptor antagonist [Nphe1]N/OFQ(1–13)NH2 (Calò et al., 75 2000b), and of the nonselective opioid receptor antagonist naloxone to prevent N/OFQinduced reduction of ethanol intake was investigated. As shown in Fig. 1, an 8-day treatment with intracerebroventricular N/OFQ, 0.5 g/rat, significantly reduced ethanol intake in comparison to controls [F(1,14) = 7.64, P < .01]; but following pretreatment with [Nphe1]N/OFQ(1–13)NH2, the same dose of N/OFQ did not significantly modify ethanol intake in comparison to controls [F(1,14) = 1.46, P>.05]. On the other hand, pretreatment with subcutaneous naloxone, 1 mg/kg, did not abolish the reduction of ethanol intake induced by N/OFQ, 0.5 g/kg, in comparison to controls [F(1,14) = 5.9, P < .01]. Indeed, rats receiving N/OFQ following naloxone pretreatment showed a trend towards an increased reduction of ethanol intake, in comparison to that evoked by N/OFQ following vehicle pretreatment. Fig. 1. Upper panel: 30 min ethanol intake in msP rats following intracerebroventricular injection of isotonic saline (V) or N/OFQ, 0.5 g/rat preceded by intracerebroventricular injection of vehicle or [Nphe1]N/OFQ(1–13)NH2 (NPHE), 66 g/rat. Values represent the mean ± S.E.M. of six to seven subjects. Lower panel: 30 min ethanol intake in msP rats following intracerebroventricular injection of isotonic saline (V) or N/OFQ, 0.5 g/rat preceded by subcutaneous injection of vehicle or naloxone (NAL), 1 mg/kg. Values represent the mean ± S.E.M. of six to seven subjects. Difference from controls: * P < .05; * * P < .01; where not indicated, difference from controls was not statistically significant. 76 These findings together provide further evidence of the involvement of NOP receptors in mediating N/OFQ-induced reduction of ethanol intake. Interestingly, the work of other authors have shown that basal levels of N/OFQ are markedly different in mouse lines that exhibit different alcohol preference, and are markedly influenced by repeated ethanol administrations (Lindholm et al., 2002; Ploj et al., 2000). 3. N/OFQ and ethanol- or morphine-induced conditioned place preference (CPP) The place conditioning paradigm is widely used to assess the rewarding properties of drugs, and to study the biological mechanisms subserving them. Drugs abused by humans, such as cocaine, amphetamines, alcohol and morphine can evoke CPP in the place conditioning paradigm (Ciccocioppo et al., 1999a; Swerdlow et al., 1989). Several studies have investigated the effect of N/OFQ on ethanol- and morphineinduced CPP. The results of these studies have shown that intracerebroventricular injections of N/OFQ (0.5 or 1.0 g/rat), just before the intragastric administration of 0.7 g/kg of ethanol or the subcutaneous injection of 3.0 mg/kg of morphine, prevented the development of CPP by these two drugs of abuse (Ciccocioppo et al., 1999b, 2000a, 2000b). Conversely, intracerebroventricular injection of N/OFQ (0.25–1.0 g/rat) induced neither preference nor aversion. These findings suggest that the peptide is devoid of intrinsic motivational properties per se (Devine et al., 1996; Ciccocioppo et al., 1999b). The effects of N/OFQ on the acquisition of morphine-induced CPP was also reported by another laboratory (Murphy et al., 1999), whereas several research groups reported that intracerebroventricular administration of N/OFQ at doses as high as 200 g/rat does not influence place conditioning per se (Devine et al., 1996; Murphy et al., 1999). 77 These data support the idea that N/OFQ may act as a functional antiopioid peptide, also in regard to the motivational properties of opioids. More recently, Kotlinska et al. (2002) have reported that N/OFQ inhibits also the expression of cocaine-induced CPP, however, the effect on the rewarding properties of cocaine was observed at doses of N/OFQ (10-20 g/rat) higher that those effective on ethanol- and morphine-induced CPP (Kotlinska et al., 2002). In keeping with the results of our group, Kuzmin et al. (2003) have reported data showing that N/OFQ inhibits the acquisition, the expression and the reinstatement of ethanol-induced CPP in NMRI mice (Kuzmin et al., 2003). The mechanisms by which N/OFQ prevents ethanol- and morphine-induced CPP have not been fully elucidated yet. It is well known that drugs that induce CPP share the common characteristic of increasing extracellular dopamine concentration in the NAc. Accordingly, blockade of dopamine D1 receptors inhibits CPP induced by drugs of abuse (Di Chiara, 1995). Indeed, microdialysis experiments have shown that intracerebroventricular administration of N/OFQ inhibits morphine-induced dopamine release in the NAc (Di Giannuario et al., 1999), and suggest that this finding may account for the effect on place conditioning. Murphy and Maidment (1999) have reported that administration of N/OFQ in the VTA of anesthetized rats evokes a pronounced reduction of extracellular dopamine levels in the NAc, suggesting that the VTA may be the site of action for this effect. Moreover, they showed that administration of N/OFQ in the VTA results in increased extracellular levels of GABA in this area. Administration into the VTA of the GABAA receptor antagonist bicuculline prevented the effect of N/OFQ on NAc dopamine levels, suggesting that GABAergic interneurons located in the VTA may mediate the effect of N/OFQ (Murphy & Maidment, 1999). Rewarding stimuli, including morphine, are known to increase c-fos expression in the shell of the NAC, through the activation of dopaminergic neurons (Bontempi & 78 Sharp, 1997). In a recent experiment conducted in our laboratory, it has been shown that intracerebroventricular N/OFQ, 1.0 g/rat, completely blocked morphine-induced c-fos expression in the shell of the NAc (Fig. 2). This finding may represent a biochemical correlate to the effects of the peptide on morphine-induced CPP. Fig. 2. Fos-immunoreactive nuclei in rats treated with morphine (M), 3 mg/kg, and/or N/OFQ, 1.0 g/rat, or their vehicles (V). Values are ± S.E.M. of 6 rats/group. Difference from controls as in Fig. 1. 4. N/OFQ effect on stress-induced reinstatement of alcohol-seeking behavior It is well known that in humans stress represents a major contributing factor to psychiatric disorders, such as anxiety, anorexia, depression and also drug dependence (Brown et al., 1995; Shaham et al., 2000; Weiss et al., 2001). Studies in transgenic N/OFQ knockout mice showed that these animals exhibit increased stress-related anxiety and stress-induced alteration of nociceptive threshold (Koster et al., 1999). Moreover, mice lacking N/OFQ show elevated glucocorticoid levels, indicating a chronic activation of the hypothalamic–pituitary–adrenal axis, which might contribute to the observed phenotypic changes. In addition, N/OFQ may be important for adaptation to stress, because N/OFQ-deficient mice failed to habituate to repeated 79 exposure to stressful stimuli (Koster et al., 1999). Consistent with data obtained in these knockout mice, evidence has been provided that intracerebroventricular administration of N/OFQ results in marked anxiolytic and anti-stress action. For example, central administration of this peptide or peripheral injection of the heterocyclic analogue Ro 64-6198 increases the time spent in the open arms of an elevated plus maze in rats, while it inhibits the effect of urocortin on spontaneous exploration in unfamiliar environment and increases the time spent in the light compartment of a light–dark box in mice (Jenck et al., 1999, 2000). The major mediator of stress in mammals is the corticotropin-releasing factor (CRF) and studies conducted in our laboratory have shown that activation of NOP receptors by N/OFQ or Ro 64-6198 results in marked inhibition of CRF-induced anorexia in rats, which suggests a potent functional antagonism of CRF by N/OFQ (Ciccocioppo et al., 2001, 2002a). Clinical studies indicate that stress increases alcohol consumption and facilitates relapse in detoxified former alcoholics (Brown et al., 1995). The significance of stress as a factor for risk of relapse is also well documented in preclinical studies, where electric footshock stress has consistently been shown to elicit reinstatement of ethanolseeking behavior in drug-free animals (Le et al., 1998, 1999, 2000, 2002; Liu & Weiss, 2002a; Martin-Fardon et al., 2001). This effect of stress is apparently under the control of the brain CRF system, because reinstatement of ethanol-seeking behavior elicited by electric footshock stress is blocked by central administration of selective CRF receptor antagonists (Le et al., 2000). Based on these data and considering the functional anti-CRF activity of N/OFQ, several experiments were conducted to investigate the ability of this peptide to prevent stress-induced reinstatement of ethanol-seeking behavior in rats. The results obtained show that exposure to intermittent unpredictable electric footshock elicits reinstatement of ethanol-seeking behavior. The intracerebroventricular administration of 0.1–2.0 80 g/rat of N/OFQ significantly reduced stress-induced reinstatement of ethanol responding (Martin-Fardon et al., 2001). Lever pressing at the inactive lever was never modified by stress or N/OFQ treatment, suggesting that the effect of the peptide is not a consequence of unspecific changes in the animal’s behavior. Recent experiments have replicated these findings and have shown that the effect evoked by 4 g/rat is not significantly different from that evoked by 2 g/rat (Fig. 3). Fig. 3. Effect of N/OFQ on stress-induced reinstatement of extinguished ethanol responding in rats trained to self-administer 10% ethanol. Data represent the mean (±S.E.M.) number of responses at the previously active lever after 15 min of intermittent footshock. Rats (n = 11) were injected intracerebroventricularly with 2.0 or 4.0 g/rat of N/OFQ or its vehicle (0), 5 min before exposure to footshock. Difference from controls as in Fig. 1. 81 5. N/OFQ and cues-induced reinstatement of alcohol-seeking behavior The conditioning of drugs of abuse with discrete environmental stimuli has been proposed as another major factor in the abuse potential of these drugs (O’Brien et al., 1992, 1998). Consistent with this view, conditioned stimuli predictive of ethanol availability can evoke intense drug desire that may lead to resumption of its abuse in abstinent detoxified alcoholics (Eriksen & Gotestam, 1984; Kaplan et al., 1985; Laberg, 1986; Cooney et al., 1987, 1997; Monti et al., 1987, 1993). Studies in animals have recently confirmed the important role played by environmental conditioning factors in resuming extinguished ethanol responding in rats trained to self-administer alcohol (Katner et al., 1999; Ciccocioppo et al., 2002c; Liu & Weiss, 2002a, 2002b). Clinical and preclinical data obtained with naltrexone suggest a critical role of the opioid neurotransmission, possibly through interaction with dopamine neurons, in the control of ethanol-seeking and relapse. Opioid mechanisms may be involved in the regulation of both the direct reinforcing effects of ethanol, and the appetitive effect of stimuli predictive of its availability. This is suggested by studies showing that naltrexone reduces not only ethanol consumption, but also the urge to drink, elicited by presentation of alcohol cues in human alcoholics (Monti et al., 1999; Rohsenow et al., 2000), and decreases the efficacy of alcohol cues to reinstate extinguished responding at a previously drug-paired lever in rats (Katner et al., 1999). One mechanism of action, by which opiate receptor antagonists reduce the primary reinforcing effects of ethanol in rats self-administering alcohol, is by interfering with ethanol-dependent dopaminergic activation (Benjamin et al., 1993; Gonzales & Weiss, 1998). Therefore, inhibition of the motivating effects of ethanol-related stimuli by opiate receptor antagonists may involve an opioid–dopamine link, as well. This hypothesis is supported by two pieces of evidence. First, exposure to drug-associated contextual stimuli can increase dopamine release in the NAc (Weiss et al., 1993, 2000; Katner et al., 1999), 82 and treatment with a selective dopamine D1 receptor antagonist reduces ethanolseeking behavior elicited by an alcohol-predictive discriminative stimulus (Liu & Weiss, 2002b). Secondly, it is known that DOP and MOP1 opioid receptors have a tonic stimulatory role on mesolimbic dopamine neurons projecting from the VTA to the NAc (Devine et al., 1993a, 1993b; Di Chiara & North, 1992; Johnson & North, 1992). Therefore, blockade of opioid receptors may result in inhibition of meso-accumbal dopamine neurotransmission that may reduce the ability of cues to reinstate ethanolseeking behavior. As mentioned earlier, N/OFQ possesses functional antiopioid activity (Mogil & Pasternak, 2001). This property may be accounted for by its ability to inhibit the firing of -endorphinergic neurons in the hypothalamic arcuate nucleus (Wagner et al., 1998). These arcuate neurons project to the VTA and the NAc, where they interact with mesolimbic dopamine neurons involved in motivated behaviours (Di Chiara & North, 1992; Devine et al., 1993a, 1993b; Herz, 1997). Moreover, it has been shown that 91% of tyrosine hydroxylase-positive cells in the VTA coexpress NOP receptors, and that N/OFQ can directly and indirectly (via GABA interneurons) modulate neural activity of VTA dopaminergic neurons (Murphy & Maidment, 1999; Maidment et al., 2002; Norton et al., 2002). Based on these evidences, we hypothesized that N/OFQ, like naltrexone or dopamine receptor antagonists, may be able to reduce reinstatement of ethanol-seeking behavior by environmental conditioning factors. In keeping with this hypothesis, a recent study of our group showed that cues predictive of ethanol availability trigger reinstatement of extinguished ethanol responding in genetically selected msP rats, and this effect was markedly reduced by intracerebroventricular administration of 4.0 g/rat of N/OFQ given just prior to the reinstatement session (Fig. 4). 83 Fig. 4. Self-administration: Responses in the presence of distinct olfactory discriminative stimuli predictive of the availability of 10% (v/v) ethanol (S +) or water (S-) during 20 days of self-administration training. Extinction: Extinction responses during the 20 days of this phase. Reinstatement: Responses in rats exposed to the S+ and S- conditions (in the absence of ethanol or water) and treated intracerebroventricularly with 4.0 g/rat of N/OFQ or its vehicle (0). Responding in the S + test differed from S- responses (P < .01). Treatment with N/OFQ significantly reduced responses under S+ but not under S- conditions. Values represent the mean (±S.E.M.) of 8 subjects/group. Difference from extinction as in Fig. 1. 6. Conclusions Studies conducted in our laboratory have shown that N/OFQ controls a variety of ethanol-related behaviors in rats. Specifically, this peptide reduces ethanol consumption and reinstatement of ethanol-seeking behavior induced by environmental conditioning factors and by stress. These effects could be accounted for by a modulatory (inhibitory) role of NOP receptor on the regulation of brain dopamine and opioid function, on the one hand, and of brain CRF activity, on the other hand. At present, only a few drugs have been approved for treatment of alcohol abuse. Among these drugs, the opioid receptor antagonist naltrexone seems to possess the most promising profile of action. However, clinical data indicate that naltrexone may 84 have adverse side effects and can give compliance problems. Consistently, in rats, naltrexone administration results in development of conditioned place and taste aversion, and of stress-like reactions. N/OFQ is devoid of motivational effects per se and stimulation of NOP receptor by this peptide does not evoke aversion (Ciccocioppo et al., 1999b; Devine et al., 1996). In addition, naltrexone does not block stress-induced reinstatement of ethanol-seeking behavior in rats (Le et al., 1999), whereas N/OFQ does (Martin-Fardon et al., 2001). Recent data suggest that, in situations in which both stress- and alcohol-paired cues are present, neither naltrexone nor the CRF receptor antagonist, D-Phe-CRF, is sufficient to prevent reinstatement when they are administered alone (Liu & Weiss, 2002a). Because most situations encountered by alcoholics are likely to be both stressful and to have cues strongly associated with drinking, NOP receptor agonists might be expected to have greater therapeutic efficacy than either naltrexone or CRF antagonists alone. Altogether these considerations suggest that agents targeting the NOP receptor may have pharmacotherapeutic potential for treatment of alcoholism and may offer some advantages over other classes of drugs (i.e. opioid or CRF receptor antagonists). 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Ann NY Acad Sci 937:1-26. 95 Chapter 3 Effect of novel NOP receptor ligands on ethanol drinking in the alcoholpreferring msP rats 96 Effect of novel NOP receptor ligands on ethanol drinking in the alcohol-preferring msP rats Abstract Activation of the NOP receptor by the endogenous ligand nociceptin/orphanin FQ (N/OFQ), reduces alcohol consumption in the alcohol preferring msP rats. The present study, evaluated the effect of three newly synthesized peptidergic and of one brainpenetrating heterocyclic NOP receptor agonists on alcohol drinking under the two bottle choice paradigm of voluntary intake. MsP rats were intracerebroventricularly (ICV) injected with the NOP receptor agonists OS-462, UFP-102 or UFP-112, or received by intraperitoneal (IP) injection Ro 64-6198, and tested for 10% w/v alcohol consumption. Results, showed a decreased alcohol consumption after treatment with all three peptidergic NOP receptor agonists (OS-462, UFP-102 and UFP-112) while Ro 64-6198 was ineffective. The present findings, in a first place confirmed that activation of brain NOP receptors results in a reduction of alcohol drinking in the msP rats, and we demonstrated that OS-462, UFP-102 and UFP-112 act as potent NOP receptor agonists. At present it is unclear why Ro 64-6198 fails to exert the same effect. However, binding and electrophysiological studies have shown that this compound possesses a pharmacological profile that is quite different from that of N/OFQ. Overall, the results indicate that OS-462, UFP-102 and UFP-112 may represent valuable pharmacological tools to investigate the role of the brain N/OFQ system. Keywords: Nociceptin/orphanin FQ; NOP receptor; Alcohol intake 97 INTRODUCTION Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the NOP opioid receptor, previously referred to as ORL-1 or OP4 receptor (Meunier et al., 1995; Reinscheid et al., 1995), is known to be structurally related to the opioid peptide dynorphin A (Meunier et al., 1995, 2000; Reinscheid et al., 1995, 1998). However, N/OFQ does not bind to MOP, DOP and KOP opioid receptors nor do opioid peptides activate the NOP receptor (Reinscheid et al., 1995, 1998). On the contrary, N/OFQ activates with high selectivity the NOP receptor (Reinscheid et al., 1995) eliciting, intracellular responses with the same intracellular mechanisms as classic opioid receptors (Reinscheid et al., 1995). N/OFQ, has been found to block opioid-induced supraspinal analgesia and it has been proposed to act in the brain as a functional antiopioid peptide (Mogil et al., 1996a, 1996b; Morgan et al., 1997). Moreover, brain mapping studies showed a neuroanatomical distribution of N/OFQ and the NOP receptor different from that of other opioid peptides and receptors (Anton et al., 1996, Darland et al., 1998, Florin et al., 1997, Ikeda et al., 1998, Neal et al., 1999, Sim & Childers, 1997, Mollereau & Mouledous, 2000). A wide distribution of this peptidergic system has been found in various corticomesolimbic structures, including the amygdala, the bed nucleus of the stria terminalis, the nucleus accumbens (NAcc) and various fronto-cortical areas. Interestingly, such brain areas are known to be involved in the regulation of the motivational properties of drugs of abuse (Koob et al., 1998; Wise, 1998; Everitt & Wolf, 2002) and, an important involvement of the N/OFQ–NOP receptor system in the control of reward and drug abuse processes has been now well established (Ciccocioppo et al., 2000a). In fact, pre-treatment with N/OFQ blocked ethanol-, morphine- and cocaine-induced conditioned place preference (Ciccocioppo et al., 1999, 2000b; Murphy et al., 1999; Kotlinska et al., 2002), whereas, microdialysis studies 98 demonstrated that central administration of N/OFQ significantly attenuates the increase of extracellular dopamine (DA) induced in the Nacc after administration of cocaine or morphine (Di Giannuario et al., 1999; Luffy et al., 2001). Moreover, previous studies, conducted in our laboratory, have shown that chronic intracerebroventricular (ICV) injections of the peptide significantly reduced ethanol intake in the genetically selected Marchigian Sardinian alcohol-preferring (msP) rats both in the two bottle choice (free choice ethanol vs water) and oral self-administration paradigms (Ciccocioppo et al., 1999, 2004), whereas, pre-treatment with a selective NOP receptor agonist blocked this effect (Ciccocioppo et al., 1999). In relation to the above mentioned well documented role of the N/OFQ-NOP receptor system, in controlling the rewarding and reinforcing processes induced by abused drugs, and even more, due to the particularity of this opioid peptide to exert its effects without activating the classic opioid receptors (MOP, DOP and KOP), great interest is at present devoted to the development of novel NOP receptor agonists with interesting pharmacodynamic and pharmacokinetic properties, particularly with the property of influencing brain NOP receptors following peripheral administration. Recently, several petidergic NOP receptor agonists, as well as, one non-peptidergic brain-penetrating NOP receptor agonist, Ro 64-6198, have been synthesized (Wichmann et al., 2000). The present study, was sought to evaluate the effect of three newly synthesized peptidergic NOP receptor agonists (OS-462, UFP-102 and UFP-112) and of Ro 646198, a non-peptidergic brain-penetrating agent, in relation to the ability of these compounds to reduce home-cage voluntary ethanol drinking in the msP rats. 99 2. Methods 2.1. Animals Male genetically selected alcohol-preferring rats were employed. They were bred in the Department of Pharmacological Sciences and Experimental Medicine of the University of Camerino (Marche, Italy) for 50 generations from Sardinian alcoholpreferring (sP) rats of the 13th generation, provided by the Department of Neurosciences of the University of Cagliari (Fadda et al., 1990; Gessa et al., 1991). These animals are referred to as Marchigian sP (msP) rats. At the time of the experiments their body weight ranged between 400 and 450 g. They were kept in a room with a reverse 12:12 h light/dark cycle (lights off at 8:00 a.m.), temperature of 2022oC and humidity of 45-55%. Rats were offered free access to tap water and food pellets (4RF18, Mucedola, Settimo Milanese, Italy). All procedures were conducted in adherence with the European Community Council Directive for Care and Use of Laboratory Animals. 2.2. Surgical Procedures For intracranial surgery, rats were anaesthetized by intramuscular injection of 100-150 l/rat of a solution containing tiletamine cloridrate (58.17 mg/ml) and zolazepam chloridrate (57.5 mg/ml). A guide cannula for injections into the lateral cerebroventricle was stereotaxically implanted and cemented to the skull. The following coordinates, taken from the atlas of Paxinos and Watson (1986), were used: antero-posterior = -1.0 mm behind the bregma, lateral = 1.8 mm from the sagittal suture, ventral = 2 mm from the surface of the skull. 100 Peptides were injected through a stainless-steel injector protruding 2.5 mm beyond the cannula tip. Experiments began one week after surgery. Before the beginning of the experiments, animals received ICV injections of saline to habituate them to the drug administration procedure. Cannula placement was verified before the experiment by ICV injection of 100 ng/rat of angiotensin II; only animals that showed a clear dipsogenic response (at least 6 ml of water in 5 min) were used for further testing. 2.3. Drugs The NOP receptor agonist OS-462 (PM=823), corresponds to the Example 25 of the European Patent (Ishiyama et al., 2003; Oyama & Sakano, 2003) and was provided by Nippon Shinyaku, Co Ltd, Kyoto, Japan. The NOP receptor agonists UFP-102 (PM=2725) and UFP-112 (PM=2738) were a generously gift of Dr. Remo Guerrini of the Department of Pharmaceutical Sciences, University of Ferrara, Italy, while Ro 646198 was provided by Hoffmann-La Roche (Basel, Switzerland). Ro 64-6198 was dissolved in a solution containing 10% DMSO, 10% Tween 80, 80% distilled water and was given by IP injection (1 ml/kg). All the other NOP receptor agonists, were dissolved in sterile isotonic saline and were injected ICV in a volume of 1 l/rat. 2.4. Experimental procedure At the age of 3 months msP rats were selected for their preference for 10% ethanol solution (w/v), offering them free choice between water and 10% ethanol 24-h a day for 15 days. Water and 10% ethanol were offered in graduated drinking tubes 101 equipped with metallic drinking spouts and their consumption was measured by reading the volume consumed from the graduated burettes. Food intake was measured by weighing the food containers and taking into account spillage. Ethanol, water and food intakes are expressed as g/kg to reduce the influence of differences in body weight. The rats employed in the following experiments had a 24-h ethanol intake of 6-7 g/kg with a percent ethanol preference [ml of ethanol solution/ml of total fluids (water + 10% ethanol) ingested in 24 h x 100] higher than 90. Animals had free access to 10% ethanol, water and food for 24-h a day until acquisition of stable baseline of alcohol intake. Subsequently, ethanol availability was restricted to 30 min/day, at the beginning of the dark phase (9:30 a.m.) of the reverse light/dark cycle. All animals received 3-4 IP or ICV injections before initiation of the experiments (pre-treatment period) to familiarize them with the experimental procedure. All the experiments were carried out according to a between subject design, in which each group of rats received a single dose of a single compound. 2.4.1. Effect of subchronic ICV treatment with OS-462 on voluntary ethanol intake in msP rats A group of msP rats (n=22) was divided into 3 groups (n=6-8) with similar baseline ethanol intake during the pre-treatment period (3 days). Animals were then ICV treated, for 6 consecutive days, 10 min before access to ethanol with OS-462 at the doses of 0.5 and 1.0 g/rat, or its vehicle (controls). Ethanol, water and food intake were measured for 30 min after access to ethanol. Intakes were measured for other 4 102 days after the end of the treatment where all animals were ICV injected with saline (post-treatment period). 2.4.2. Effect of subchronic ICV treatment with UFP-102 on voluntary ethanol intake in the msP rats MsP rats (n=23) were divided into 3 groups (n=7-8) with similar baseline ethanol intake during the pre-treatment period (3 days). At this point and for 6 consecutive days animals were ICV treated, 10 min before access to ethanol, with UFP-102 (0.25 or 1.0 g/rat) or its vehicle (controls), respectively. Ethanol, water and food intake was measured for 30 min after access to ethanol. Intakes were measured for other 4 days after the end of the treatment where all animals were ICV injected with saline. 2.4.3. Effect of subchronic ICV treatment with UFP-112 on voluntary ethanol intake in the msP rats A group of msP rats (n=21) were divided into 3 groups (n=7) with similar baseline levels of ethanol consumption during the pre-treatment period (3 days). At this point and for 6 consecutive days animals were treated, 30 min before access to ethanol, with UFP-112 (0.01 or 0.05 g/rat, ICV) or its vehicle (controls), respectively. Ethanol, water and food intake was measured for 30 min after access to ethanol. Intakes were measured for other 4 days after the end of the treatment where all animals were ICV injected with saline. 103 2.4.4. Effect of subchronic IP treatment with Ro 64-6198 on voluntary ethanol intake in the msP rats To evaluate the effect of Ro 64-6198 on voluntary ethanol intake, 27 msP rats were divided into 3 groups (n=8-10) with similar baseline ethanol consumption during the pre-treatment period. At this point, for 7 consecutive days, the first group of animals received IP isotonic saline (controls), while the other two groups received 0.3 and 1.0 mg/kg of Ro 64-6198, respectively. Ethanol was given 30 min after injections, and ethanol, water and food intake was recorder for the subsequent 60 min. At completion of drug testing ethanol, water and food intake were monitored for another 4 days (posttreatment), during which all animals received only IP injections of saline. Statistical analysis Statistical analysis of data was performed by means of two-way analysis of variance (ANOVA) with between-groups comparisons for drug treatment and withingroups comparisons for day of treatment. Post-hoc comparisons were carried out by Newman-Keuls test. Statistical significance was set at *p < 0.05 and **p < 0.01. 104 3. Results 3.1. Effect of subchronic ICV treatment with OS-462 on voluntary ethanol intake in the msP rats The overall ANOVA revealed a significant treatment effect [F(2,19) =7.28; P < 0.01]. As shown in Fig. 1A, post-hoc comparisons demonstrated a significant difference between controls and animals treated with both doses (0.5 and 1.0 g/rat; ICV) of OS462 (P < 0.01). Neither food nor water intake were modified by OS-462 treatment (data not shown). 3.2. Effect of subchronic ICV treatment with UFP-102 on voluntary ethanol intake in the msP rats The overall ANOVA revealed a significant treatment effect [F(2,20) = 11.45; P < 0.01]. As shown in Fig. 1B, post-hoc comparisons demonstrated a significant difference between controls and animals treated with both doses (0.25 and 1.0 g/rat; ICV) of UFP-102 (P < 0.01). Neither food nor water intake were modified by UFP-102 treatment (data not shown). 3.3. Effect of subchronic ICV treatment with UFP-112 on voluntary ethanol intake in the msP rats The overall ANOVA revealed a significant treatment effect [F(2,18) = 4.23; P < 0.05]. As shown in Fig. 1C, post-hoc comparisons demonstrated a significant difference 105 between controls and animals treated with both doses (0.01 and 0.05 g/rat; ICV) of UFP-102 (P < 0.05). Neither food nor water intake were modified by UFP-102 treatment (data not shown). Fig. 1 Voluntary alcohol intake (10% w/v) following subchronic (6 days) ICV treatment with: (A) OS-462 (0.0, 0.5 and 1.0 g/rat), (B) UFP-102 (0.0, 0.25 and 1.0 g/rat) and, (C) UFP-112 (0.0, 0.01 and 0.05 g/rat) in msP rats. Data are means ± S.E.M. *P < 0.05, **P < 0.01 vs. controls. 106 3.4. Effect of subchronic IP treatment with Ro 64-6198 on voluntary ethanol intake in the msP rats The analysis of variance did not reveal an overall effect of treatment [F(2,24) = 2.57; p > 0.05]. Post-hoc comparisons showed a significant increase of ethanol intake following administration of 1.0 mg/kg of Ro 64-6198 (p<0.01), whereas, injection of 0.3 mg/kg of the drug did not modify alcohol consumption. In particular, as shown in Fig. 2, administration of 1.0 mg/kg of Ro 64-6198 significantly increased ethanol intake on days 3, 7 and 8 of drug treatment. Moreover, treatment with Ro 64-6198 elicited a significant increase of food intake [F(2,24) = 9.822; p < 0.05]. Post-hoc comparisons showed a significant increase of food intake (p<0.01) only at the highest dose (1.0 mg/kg, IP) tested (data not shown). In addition the overall ANOVA demonstrated a significant treatment effect on water intake [F(2,24) = 7.033; p < 0.05]. Post-hoc tests revealed a significant decrease of water intake (p<0.01) following administration of both 0.3 and 1.0 mg/kg of Ro 646198 (data not shown). Ethanol Intake (30 min) (g/kg) 2.5 Pre-Treat. Treatment Post-Treat. * Veh Ro 64-6198 0.3 Ro 64-6198 1.0 mg/kg * 2.0 1.5 Fig. 2 Voluntary alcohol intake (10% w/v) following subchronic (9 days) treatment with Ro 64-6198 (0.0, 0.03 and 1.0 mg/kg, IP) in msP rats. Data are means ± S.E.M. *P < 0.05 vs. controls. 1.0 0.5 0.0 107 Discussion The results of the present study showed ICV administration of OS-462 (0.5 or 1.0 g/rat), UFP-112 (0.01 or 0.05 g/rat) and UFP-102 (0.25 or 1.0 g/rat) induced a pronounced and statistically highly significant decrease in voluntary ethanol intake in the msP rats, after subchronic (6 days) treatment. Interestingly, this effect was similar in intensity to the one described before with N/OFQ (Ciccocioppo et al., 1999). Moreover, discontinuation of treatment with all above mentioned NOP receptor agonists resulted in resumption of ethanol intake that returned at pre-treatment levels; an effect also seen with N/OFQ (Ciccocioppo et al., 1999). On the other hand, subchronic IP treatment with Ro 64-6198, not only failed to decrease ethanol consumption but when administrated at the highest dose of 1.0 mg/kg, induced a significant increase in ethanol drinking in msP rats. Furthermore, treatment with 1.0 mg/kg of Ro 64-6198 also elicited a significant increase of food intake, whereas, both doses (0.3 and 1.0 mg/kg) of the compound significantly decreased water intake in the msP rats. There is large evidence that N/OFQ acts in the brain as a functional anti-opioid agent. Foe example, N/OFQ has been found to block the analgesic effects of morphine (Mogil et al., 1996a, 1996b; King et al., 1998; Mogil & Pasternak, 2001), to prevent the development of morphine-induced conditioned place preference (Murphy et al., 1999; Ciccocioppo et al., 2000b) and to inhibit morphine-induced DA release in the NAcc (Di Giannuario et al., 1999). In addition, electrophysiological data demonstrated that the N/OFQ system inhibits the firing of -endorphin cells in the hypothalamic arcuate nucleus (Wagner et al., 1998). These arcuate neurons project, among other brain regions to the ventral tegmental area (VTA) and the NAcc, where they interact with mesolimbic DA transmission and influence motivated behaviour (Di Chiara & North, 1992; Johnson & North, 1992; Devine et al., 1993a, 1993b; Herz, 1997). Moreover, it 108 has been shown that 91% of tyrosine hydroxylase-positive cells in the VTA co-express NOP receptors, and that N/OFQ can directly and indirectly (via GABA interneurons) modulate (inhibit) neural activity of VTA DA neurons (Maidment et al., 2002; Norton et al., 2002; Zheng et al., 2002). Based on these data showing the modulatory role that N/OFQ has on corticomesolimbic DA and opioid activity and taking into consideration the important role these systems in the regulation of ethanol-related behaviours, it could hypothesised that N/OFQ-NOP receptor system may reduce the motivational value of alcohol by modulating the brain DAergic and opioidergic functions. One of the main challenges in the research field concerning the N/OFQ-NOP receptor system, is to generate brain-penetrating molecules able to selectively bind the brain NOP receptors after peripheral administration. Therefore, in the present study the ability of Ro 64-6198, a non-peptidergic brain-penetrating NOP receptor agonist (Wichmann et al., 2000), to reduce ethanol intake after peripheral injection, was evaluated. Unfortunately, results showed that Ro 64-6198 does not decrease voluntary ethanol consumption in the msP rats, but, on the contrary, when injected at the highest dose of 1.0 mg/kg this compound induced a significant increase on ethanol drinking. At present, it is unclear why Ro 64-6198 fails to exert the same effects on ethanol intake as the endogenous N/OFQ and the three NOP receptor agonists tested in this study. Interestingly, binding and electrophysiological studies have shown that Ro 646198 possesses a pharmacological profile that is quite different from that of N/OFQ. For instance, at high doses, it exhibits moderate affinity also for the MOP opioid receptor and compared to N/OFQ it controls the electrophysiological activity of a specific neuronal subpopulation only (Jenck et al., 2000, Rizzi et al., 2001). The unspecificity of Ro 64-6198, and in particular its residual agonistic activity at classical opioid receptor may also explain why this compound also increased food consumption. 109 In fact, it is known that stimulation of central opioid activity, and in particular activation of the -receptor subtype potently stimulates feeding in rodents. In conclusion, the results of the present study clearly demonstrate that OS-462, UFP-102 and UFP-112 act as potent and selective NOP receptor agonists, to reduce ethanol intake. On the contrary, this effect does not seem to be shared by Ro 64-6198. Data, suggest that OS-462, UFP-102 and UFP-112 may represent valuable pharmacological tools to further evaluate the role of the N/OFQ-NOP receptor system Conversely, Ro 64-6198 seems to posses a pharmacological profile of action that does not seems to be superimposable to that of the endogenous ligand as well as of the other agonist tested here. Overall, the present findings suggest that activation of NOP receptors may represent a promising approach for pharmacological treatment of alcohol abuse, however Ro 64-6198 does not appear to a useful tool to evaluate treatment efficacy following peripheral drug administration. Acknowledgements The authors thank Marino Cucculelli for technical assistance and animal care. 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Br J Pharmacol 136:1065-71. 115 Chapter 4 Buprenorphine reduces alcohol drinking through activation of the Nociceptin/Orphanin FQ-NOP receptor system 116 Buprenorphine Reduces Alcohol Drinking Through Activation of the Nociceptin/Orphanin FQ-NOP Receptor System Abstract Background: Activation of the NOP receptor by its endogenous ligand nociceptin/orphanin FQ reduces ethanol intake in genetically selected alcohol preferring Marchigian Sardinian alcohol preferring (msP) rats. Here we evaluated whether buprenorphine, a partial agonist at-opioid and NOP receptors, would reduce ethanol consumption in msP rats via activation of NOP receptors. Methods: Marchigian Sardinian alcohol preferring rats trained to drink 10% alcohol 2 hours/day were injected with buprenorphine (0.03, 0.3, 3.0, or 6.0 mg/kg intraperitoneally [IP]) 90 min before access to ethanol. Results: Similar to prototypical -agonists, the two lowest doses of buprenorphine significantly increased ethanol consumption (p < 0.01); in contrast, the two highest doses reduced it (p < 0.05). Pretreatment with naltrexone (0.25 mg/kg, IP) prevented the increase of ethanol intake induced by 0.03 mg/kg of buprenorphine (p < 0.001) but did not affect the inhibition of ethanol drinking induced by 3.0 mg/kg of buprenorphine. Conversely, pretreatment with the selective NOP receptor antagonist UFP-101 (10.0 or 20.0 g/rat) abolished the suppression of ethanol drinking by 3.0 mg/kg of buprenorphine. Conclusions: Buprenorphine has dualistic effects on ethanol drinking; low doses increase alcohol intake via stimulation of classic opioid receptors, whereas higher doses reduce it via activation of NOP receptors. We suggest that NOP agonistic properties of buprenorphine might be useful in the treatment of alcoholism. Key Words: Buprenorphine; nociceptin/orphanin FQ; alcohol abuse; addiction 117 INTRODUCTION Buprenorphine has long been in clinical use for treatment of moderate-to-severe pain (Hayes et al., 1979; Murphy & MacEvilly, 1984; Gundersen et al., 1986; Vanacker et al., 1986; Finco et al., 1995; Picard et al., 1997; Maunuksela et al., 1998). More recently, evidence has accumulated in support of its efficacy for maintenance treatment of heroin dependence (Mello et al., 1993; Ling et al., 1996, 1998; Litten & Allen, 1999; Johnson & McCagh, 2000; Kakko et al., 2003), and the drug has been approved for this indication in numerous countries, including the United States, Australia, Sweden, and France. Observational data from France, where it has been in widespread use, suggest an advantageous safety profile of buprenorphine compared with methadone (Auriacombe et al., 2001), whereas its efficacy seems to be comparable when used in an optimal manner (Mattick et al., 2003). An attractive safety profile of buprenorphine, including reduced risk for overdose death due to respiratory suppression and lower street value leading to diminished risk for diversion, is predicted by its complex preclinical pharmacology. Thus buprenorphine has long been known to be a partial agonist at -opioid receptors (Cowan et al., 1977; Martin et al., 1976; Magnan et al., 1982; Sadee et al., 1982; Rosenbaum et al., 1985; Lattanzi et al., 2001) but has also antagonistic or agonistic properties at - and -opioid receptors (Cowan et al., 1977; Tyers, 1980; Sadee et al., 1982; Leader, 1987; Rovati et al., 1987; Pick et al., 1997; Negus et al., 2002). In an unexpected development, it has recently been realized that buprenorphine is also agonist/partial agonist at the NOP nociceptin/orphanin FQ (N/OFQ) receptors (Wnendt et al., 1999; Bloms-Funke et al., 2000; Lutfy et al., 2003; Huang et al., 2001). As a result of the aforementioned agonist/antagonist opioidergic properties, and principally owing to the partial stimulation of the -opioid receptor, buprenorphine induces most of the known opioid effects like pain relief, feelings of wellbeing and 118 pleasure, respiratory depression, and so forth, but with less intensity than heroin, morphine, methadone, and other opiates that fully stimulate the receptor (Johnson & Strain, 1999). Consequently, unlike other opiates, buprenorphine produces modest physical dependence (Fudala et al., 1990; Kosten et al., 1990; San et al., 1992) or respiratory depression (Walsh et al., 1994), has a lower addiction potential (Kawamoto et al., 1999), and gives only mild withdrawal symptoms even after prolonged treatment and abrupt withdrawal (Jasinski et al., 1978; Fudala et al., 1990). The lack of pronounced withdrawal symptoms is also likely related to the very slow kinetics of the drug. In addition to a long half-life, buprenorphine penetrates rapidly into the brain and binds to -receptors but dissociates from these only at a slow rate (Lewis, 1985). The role of opioids in modulating reinforcing properties of ethanol has been well documented. First of all, an altered opioidergic system has been described in animals genetically selected for high ethanol preference (Weiss et al., 1990; Gianoulakis et al., 1992; De Waele et al., 1995; Jamensky & Gianoulakis, 1997; Fadda et al., 1999; Marinelli et al, 2000). In addition, low doses of opioid agonists (e.g., morphine, methadone) increase ethanol consumption (Hubbell et al., 1986, 1993; Zhang & Kelley, 2002), whereas higher doses decrease it (Sinclair et al., 1973; Sinclair, 1974; Vacca et al., 2002). This latter effect is not specific, and it is associated with sedation, hypomotility, and simultaneous suppression of food intake (Vacca et al., 2002). Nonselective opioid antagonists such as naloxone and naltrexone, instead, dose dependently decrease alcohol intake (Samson & Doyle, 1985; Weiss et al., 1990; Volpicelli et al., 1992, 1995). Most importantly, despite some negative results (Krystal et al., 2001), a meta-analysis of available studies unequivocally supports an efficacy of naltrexone for treatment of alcohol dependence (Bouza et al., 2004). An important drawback in the use of full opioid agonists such as methadone in the treatment of opioid dependence is the increase in ethanol intake often reported during maintenance treatment programs with these compounds (Bickel et al., 1987; 119 Ottomanelli, 1999; Backmund et al., 2003). This effect is attributed to the agonistic activity of these drugs at the -opioid receptors. Buprenorphine, in contrast, only partially activates the -opioid receptor. Furthermore, during our studies of buprenorphine for heroin dependence (Kakko et al., 2003), we repeatedly encountered patient reports that motivation to consume ethanol was reduced, as were consumption frequencies and quantities. A review of the preclinical literature did not provide a clear basis for evaluating these observations. Buprenorphine has been found to reduce intravenous and oral ethanol self-administration in rats under some conditions, but reported effects have been complex and not easy to interpret (Martin et al., 1983; June et al., 1998). Recent data might shed new light on the complex actions of buprenorphine in relation to alcohol intake. It has been recently found that, in addition to its activity at classical opiate receptors, buprenorphine also acts as agonist/partial agonist at N/OFQ NOP receptors (Wnendt et al., 1999; Bloms-Funke et al., 2000; Huang et al., 2001; Lutfy et al., 2003;). Interestingly, activation of the NOP receptor system results in a marked functional anti-opioid action (Mogil et al., 1996; Ciccocioppo et al., 2000b; Mogil & Pasternak, 2001) and, as shown in previous studies by our group, administration of N/OFQ reduces ethanol consumption in the genetically selected Marchigian Sardinian alcohol-preferring (msP) rats (Ciccocioppo et al., 1999). This effect is mimicked by other NOP receptor agonists and is abolished by pretreatment with the selective NOP antagonists (Ciccocioppo et al., 2002). Taken together, these data led us to hypothesize that NOP agonism might provide a component of buprenorphine’s actions on ethanol motivational properties that confers suppression of alcohol drinking and thus counteracts classical opioid actions of this drug, normally expected to increase drinking. Furthermore, because we had observed clinically that suppression of alcohol drinking by buprenorphine was most pronounced at high doses of the drug (16–32 mg daily), we postulated that this component might be 120 preferentially expressed at the higher end of the dose–response range. Here, we therefore investigated the effect of buprenorphine on voluntary 10% w/v ethanol intake in genetically selected alcohol-preferring msP rats across a wide range of doses. We then used the nonselective opioid receptor antagonist naltrexone and the selective NOP receptor agonist UFP-101 (Calò et al., 2005) to pharmacologically dissect the complex actions of buprenorphine and reveal the respective postulated component of its actions. Finally, to evaluate whether the effect of buprenorphine at the higher doses used in our experiments is selective to ethanol consumption and not due to unspecific actions (i.e., suppression of locomotor activity), the effect of this drug alone or in combination with UFP-101 was analyzed in the open-field test. Methods and Materials Animals Male, genetically selected, alcohol-preferring rats were used. They were bred at the Department of Pharmacological Sciences and Experimental Medicine of the University of Camerino (Marche, Italy) for 53 generations from Sardinian alcohol-preferring rats of the 13th generation, provided by the Department of Neurosciences of the University of Cagliari, Italy (Agabio et al., 1996; Lobina et al., 1997). These animals are referred to as Marchigian Sardinian alcohol-preferring (msP) rats. At the time of the experiments their body weight ranged between 350 and 400 g. They were housed in a room on a reverse 12-hour light/dark cycle (lights off at 9:00 AM), temperature of 20°–22°C, and humidity of 45%–55%. Rats were offered free access to tap water and food pellets (4RF18, Mucedola, Settimo Milanese, Italy). Experiments took place at 9:30 AM, at the 121 beginning of the dark phase of the light/dark cycle. Separate groups of animals were used in each experiment. All the procedures were conducted in adherence with the European Community Council Directive for Care and Use of Laboratory Animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Intracranial Surgery For intracranial surgery, msP rats were anesthetized by intramuscular injection of 100–150 L/rat of a solution containing tiletamine hydrocloridrate (58.17 mg/mL) and zolazepam cloridrate (57.5 mg/mL). A guide cannula for intracerebroventricular (ICV) injections into the lateral cerebroventricle was stereotaxically implanted and cemented to the skull. The following coordinates, taken from the atlas of Paxinos and Watson (1986), were used: AP = 1 mm behind the bregma, L = 1.8 mm from the sagittal suture, and V = 2 mm from the surface of the skull. Drug Injections Buprenorphine and naltrexone were purchased from Tocris (Ellisville, Missouri), and UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ NH[2]) was a generous gift of Dr. G. Calò, Department of Pharmaceutical Sciences of the University of Ferrara, Italy. Naltrexone and UFP-101 were dissolved in sterile isotonic saline. Naltrexone was given by intraperitoneal (IP) injection, whereas UFP-101 was injected ICV in a volume of 1 l/rat by means of a stainless-steel injector 2.5 mm longer than the guide cannula, so that its tip protruded into the ventricle. Buprenorphine was diluted with distilled water and given by IP injection. 122 Histology At completion of the experiments, to evaluate the correct cannula placement, immediately before the rat was killed, 1 l of black India ink was injected ICV and ink diffusion into the ventricles was evaluated. Experimental Procedures Ethanol Intake. At the age of 3 months msP rats were selected for their preference for 10% ethanol solution (w/v), offering them free choice between water and 10% ethanol 24 hours/day for 10 days. Water and 10% ethanol were offered in graduated drinking tubes equipped with metallic drinking spouts. The rats used in the following experiments had a 24-hour ethanol intake of 6-7 g/kg with a percent of ethanol preference [mL of ethanol solution/mL of total fluids (water + 10% ethanol) ingested in 24 X 100] higher than 90. Starting on day 11, while maintained with food and water available during the entire day, rats received 10% ethanol for only 2 hours/day, at the beginning of the dark phase (9:30 AM). Before experiments, rats were acclimated to the limited 2-hour ethanol access for 7 days. All the experiments were carried out according to a within-subject design, in which each animal received, in a counterbalanced order, all doses and compound tested in that specific experiment (see following experiment descriptions). Before the experiments rats received at least three mock IP and/or ICV injections to familiarize them with the injection procedure. Water and ethanol intakes were measured by reading the volume consumed from the graduated burettes and were always recorded 30, 60, 90, and 120 min after ethanol 123 was offered to the animals. Food intake was measured by weighing the food containers and taking into account spillage and was measured only at 30, 60, and 120 min. Ethanol, water, and food intakes are expressed as g/kg to reduce the influence of differences in body weight. Open-Field Behavior. The open-field (66 cm X 66 cm X 20 cm) arena was used to analyze the locomotor effects of buprenorphine (3.0 mg/kg) and UFP-101 (20.0 g/rat given twice) or their combination. Rats were gently placed in the center of the open-field apparatus and left to explore the arena for 30 min for two consecutive days (habituation trials). Immediately after arena exploration, the animals were taken back to their home cage. On day 3 animals received the respective treatments (see also Experiment 7) and were subjected again to an open-field session (test trial). The numbers of beam brakes at the center and at the periphery of the arena were automatically recorded for a total testing time of 30 min. The floor of the open field was completely cleaned and dried after each trial. Experiment 1: Effect of Acute IP Injections of Buprenorphine on Voluntary Alcohol Intake To evaluate the effect of buprenorphine on voluntary 10% ethanol intake, msP rats (n = 10) were injected IP with different doses of buprenorphine (0.03, 0.3, 3.0, and 6.0 mg/kg) or its vehicle (control subjects) at intervals of 3–4 days, 90 min before access to alcohol. Baseline ethanol drinking was re-established between different dose-treatments. 124 Experiment 2: Effect of Acute IP Injections of Naltrexone on Voluntary Alcohol Intake To evaluate the effect of naltrexone on voluntary 10% ethanol intake, according to a within-subject design, msP rats (n = 10) received naltrexone (0.25, 1.0, and 2.5 mg/kg, IP) or vehicle (control subjects) at intervals of 3–4 days, 95 min before access to ethanol. Baseline ethanol drinking was re-established between different dose-treatments. Experiment 3: Effect of Acute ICV Injections of UFP-101 on Voluntary Alcohol Intake To evaluate the effect of the selective N/OFQ receptor antagonist UFP-101 on voluntary 10% ethanol intake, msP rats (n = 7) were injected ICV with 5.0, 10.0, and 20.0 g/rat or its vehicle, given twice at 95 and 15 min before access to ethanol (10% w/v). In a Latin square design rats received all drug doses or its vehicle. An interval of 3–4 days was imposed between drug treatments, and baseline ethanol drinking was reestablished during these periods. Experiment 4: Effect of IP Injections of Naltrexone on Buprenorphine-Induced Increase of Ethanol Intake To evaluate the effect of naltrexone on buprenorphineinduced increased ethanol intake, a group of msP rats (n = 10) was treated IP with naltrexone (0.25 mg/kg) or its vehicle. Five minutes later, animals received an IP injection of 0.03 mg/kg of buprenorphine or its vehicle. Drug doses were chosen on the basis of the results obtained 125 in Experiment 1 and Experiment 2. Specifically, the dose of buprenorphine was chosen that selectively increased ethanol intake, and the dose of naltrexone was chosen that was ineffective per se. Ethanol was given to the animals 90 min after buprenorphine injection, and alcohol, water, and food intake were measured for 2 hours. Tests were carried out at intervals of 3–4 days, and in a Latin square design rats received all drug treatments. Baseline ethanol drinking was re-established between different dose-treatments. Experiment 5: Effect of IP Injections of Naltrexone on Buprenorphine-Induced Decrease of Ethanol Intake To evaluate the effect of naltrexone on the reduction of ethanol drinking induced by high doses of buprenorphine, msP rats (n = 8) were injected IP with 0.25 mg/kg of naltrexone or its vehicle. After 5 min, rats received an IP injection of 3.0 mg/kg of buprenorphine or its vehicle. Drug doses were chosen on the basis of the results obtained in Experiment 1 and Experiment 2. Specifically, the dose of buprenorphine that selectively decreased ethanol intake and the dose of naltrexone that was ineffective per se were chosen. Rats were offered access to 10% ethanol 90 min after buprenorphine injection. Tests were carried out at intervals of 3–4 days, and in a Latin square design rats received all drug treatments. Baseline ethanol drinking was re-established between different dosetreatments. 126 Experiment 6: Effect of ICV Injections of UFP-101 on Buprenorphine-Induced Decrease of Ethanol Intake To evaluate the effect of UFP-101 on the reduction of ethanol drinking induced by high doses of buprenorphine, according to a within-subject design, a group of msP rats (n = 10) was injected ICV with UFP-101 or its vehicle at the doses of 10.0 and 20.0 g/rat, 95 and 15 min before access to 10% ethanol. Buprenorphine (3.0 mg/kg) or its vehicle was given 90 min before access to ethanol. Two injections of UFP-101, a peptidergic NOP receptor antagonist, were given in an attempt to better antagonize the effects of buprenorphine, a non-peptidic, long lasting opioidergic agent. Drug doses were chosen on the basis of the results obtained in Experiment 1 and Experiment 3. Specifically, the dose of buprenorphine that selectively decreased ethanol intake and the doses of UFP-101 that were ineffective per se were chosen. Tests were carried out at intervals of 3–4 days, and in a Latin square design rats received all drug treatments. Baseline ethanol drinking was re-established between different dose-treatments. Experiment 7: Effect of Buprenorphine and UFP-101 on Open-Field Behavior In this experiment, we evaluated the locomotor effects of 3.0 mg/kg of buprenorphine (IP), the selective NOP receptor antagonist UFP-101 (20.0 g/rat, ICV), or their combination. According to a between-subject design, four groups (n = 6/group) of msP rats were injected ICV with UFP-101 (20.0 g/rat or its vehicle) 95 and 15 min before exposure to the open field. Buprenorphine (3.0 mg/kg) or its vehicle was given 90 min before exposure to the open field. Drug doses were chosen on the basis of the results obtained in Experiment 6. 127 Statistical Analysis Statistical analysis of data for ethanol, food, and water intake was performed by means of two-way analysis of variance (ANOVA) with repeated measures, one factor for treatment and one factor for time. Statistical analysis for the open-field experiment was performed by means of two-way ANOVA with between-subject comparisons for drug treatment and within-subject comparisons for time (habituation vs. test trial). The distance travelled and the time spent resting were analyzed separately. Post-hoc comparisons were carried out by Newman-Keuls Test. Statistical significance was set at p < 0.05. Results Experiment 1: Effect of Acute IP Injections of Buprenorphine on Voluntary Alcohol Intake The overall ANOVA revealed a highly significant treatment effect [F(4,9) = 22.31, p < 0.001]. Post-hoc analysis showed a significant dualistic effect with an increase of ethanol consumption after administration of 0.03 and 0.3 mg/kg of buprenorphine (p < 0.01) and a decrease of drinking after treatment with 3.0 and 6.0 mg/kg of the drug (p < 0.05). As shown in Figure 1, at the two lowest doses, buprenorphine significantly increased ethanol drinking throughout the 2-hour observation, whereas injection of 6.0 mg/kg of buprenorphine resulted in a significant decrease of drinking at all time points recorded. Similarly, administration of 3.0 mg/kg of the drug induced a significant inhibition of drinking at 30, 60, and 120 min. Difference from control subjects was barely above statistical significance at 60 min. Buprenorphine treatment elicited a 128 significant decrease of food intake [F(4,9) = 5.15, p < 0.001]. Post-hoc test revealed a significant decrease only at the highest dose (6.0 mg/kg) tested (Table 1). Water intake was not modified by drug treatment (Table 1). Figure 1. Effect of intraperitoneal injection of buprenorphine (Bup) (0.0, 0.03, 0.3, 3.0, and 6.0 mg/kg) on ethanol intake in Marchigian Sardinian alcohol-preferring rats. The drug was given 90 min before ethanol access, and alcohol consumption was monitored at 30, 60, 90, and 120 min. Values represent the mean (±SEM) of 10 subjects. Difference from control subjects: *p < 0.05; **p < 0.01. Veh, vehicle. Experiment 2: Effect of Acute IP Injections of Naltrexone on Voluntary Alcohol Intake The overall ANOVA demonstrated a significant effect of naltrexone [F(3,9) = 4.257, p < 0.05]. As shown in Figure 2, post-hoc comparisons revealed a significant inhibition of ethanol intake after administration of 1.0 or 2.5 mg/kg of naltrexone, whereas injection of 0.25 mg/kg of the drug did not significantly modify alcohol consumption. Neither food intake nor water intake were modified by naltrexone (Table 1). 129 Experiment 3: Effect of Acute ICV Injections of UFP-101 on Voluntary Alcohol Intake The overall ANOVA showed that treatment with UFP-101 (5.0, 10.0, or 20.0 g/rat) given twice at 95 and 15 min before access to ethanol (Figure 3) did not modify ethanol drinking in msP rats [F(3,6) = 2.264, p = ns]. Neither food intake nor water intake were modified by UFP-101 treatment (Table 1). This is in line with previous studies in which other selective NOP antagonists were used (Ciccocioppo et al 2002). Experiment 4: Effect of IP Injections of Naltrexone on Buprenorphine-Induced Increased Ethanol Intake The overall ANOVA revealed a highly significant treatment effect [F(3,9) = 9.50, p < 0.01]. Confirming the results of Experiment 1, the dose of 0.03 mg/kg buprenorphine significantly increased ethanol drinking (p < 0.01). This effect was abolished by pretreatment with 0.25 mg/kg of naltrexone. Consistent with the data obtained in Experiment 2, administration of 0.25 mg/kg of naltrexone alone did not significantly affect ethanol drinking (Figure 4). Water and food intake were not influenced by drug treatments (Table 1). Experiment 5: Effect of IP Injections of Naltrexone on Buprenorphine-Induced Decreased Ethanol Intake The overall ANOVA showed a significant effect of treatment [F(3,7) = 5.90, p < 0.01]. As shown in Figure 5 and consistent with the result of Experiment 1, 130 administration of 3.0 mg/kg of buprenorphine significantly reduced ethanol drinking (p < 0.05). Pretreatment with naltrexone (0.25 mg/kg, IP) did not block high-dose buprenorphine-induced decrease of ethanol intake, which remained significantly lower compared with control subjects (p < 0.01). Administration of naltrexone alone reduced ethanol drinking slightly but not significantly. Water and food intake were not modified by drug treatments (Table 1). Figure 2. Effect of intraperitoneal injection of naltrexone (Ntx) (0.0, 0.25, 1.0, and 2.5 mg/kg) on ethanol intake in Marchigian Sardinian alcoholpreferring rats. Drug injections were given 95 min before ethanol access, and alcohol consumption was monitored at 30, 60, 90, and 120 min. Values represent the mean (±SEM) of 10 subjects. 131 Table 1. Effects of Buprenorphine, Naltrexone, UFP-101, or their combinations on food and water intake in marchigian sardinian alcohol-preferring rats Treatment 30 Food Intake Time (min) 60 120 30 Water Intake Time (min) 60 90 120 Buprenorphine (mg/kg, IP) Veh Bup 0.03 Bup 0.3 Bup 3.0 Bup 6.0 2.90 ± 0.56 2.51 ± 0.65 1.36 ± 0.56 1.00 ± 0.34 0.50 ± 0.27a 4.05 ± 0.62 2.97 ± 0.66 3.83 ± 1.13 3.01 ± 0.64 1.11 ± 0.41a 5.91 ± 0.91 8.69 ± 0.92 6.67 ± 1.79 7.51 ± 1.14 2.18 ± 0.76a 0.07 ± 0.04 0.00 ± 0.00 0.01 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.07 ± 0.04 0.00 ± 0.00 0.02 ± 0.01 0.00 ± 0.00 0.03 ± 0.02 0.08 ± 0.41 0.02 ± 0.01 0.02 ± 0.01 0.05 ± 0.03 0.06 ± 0.05 0.08 ± 0.41 0.03 ± 0.02 0.08 ± 0.06 0.05 ± 0.03 0.10 ± 0.05 1.08 ± 0.50 1.46 ± 0.83 0.72 ± 0.51 1.50 ± 1.06 1.60 ± 0.56 1.93 ± 0.87 0.81 ± 0.52 3.19 ± 1.21 4.39 ± 1.16 3.12 ± 0.91 3.36 ± 0.93 4.53 ± 1.27 0.46 ± 0.37 0.06 ± 0.06 0.46 ± 0.46 0.25 ± 0.21 0.50 ± 0.37 0.06 ± 0.06 0.50 ± 0.46 0.46 ± 0.42 0.56 ± 0.38 0.75 ± 0.39 0.50 ± 0.46 0.71 ± 0.62 0.86 ± 0.36 1.39 ± 0.46 0.69 ± 0.45 0.84 ± 0.75 2.70 ± 0.94 5.05 ± 1.20 2.23 ± 0.83 4.30 ± 1.21 3.81 ± 1.04 5.59 ± 0.74 3.01 ± 0.71 5.07 ± 0.98 8.20 ± 0.96 7.34 ± 1.15 9.63 ± 1.51 10.88 ± 50 0.94 ± 0.94 2.32 ± 1.50 1.19 ± 0.84 0.00 ± 0.00 0.94 ± 0.94 2.39 ± 1.49 1.33 ± 0.89 0.48 ± 0.48 1.45 ± 1.45 2.40 ± 1.49 2.48 ± 1.72 0.48 ± 0.48 1.45 ± 1.45 3.21 ± 1.98 2.48 ± 1.72 1.58 ± 1.58 1.71 ± 0.83 2.76 ± 1.03 2.80 ± 1.14 2.00 ± 0.75 0.00 ± 0.00 0.00 ± 0.00 0.04 ± 0.02 0.06 ± 0.06 0.07 ± 0.01 0.05 ± 0.03 0.05 ± 0.03 0.06 ± 0.06 0.07 ± 0.01 0.05 ± 0.03 0.05 ± 0.03 0.16 ± 0.08 0.09 ± 0.03 0.07 ± 0.20 0.10 ± 0.09 0.39 ± 0.16 4.75 ± 1.15 1.90 ± 1.11 2.84 ± 1.73 4.57 ± 1.40 0.24 ± 0.24 0.11 ± 0.07 0.20 ± 0.14 0.00 ± 0.00 0.45 ± 0.45 0.29 ± 0.18 0.31 ± 0.16 0.04 ± 0.04 0.61 ± 0.45 0.45 ± 0.29 0.74 ± 0.26 0.12 ± 0.06 0.65 ± 0.44 0.58 ± 0.38 0.82 ± 0.27 0.12 ± 0.06 5.50 ± 1.35 2.86 ± 1.51 7.34 ± 1.60 6.84 ± 1.67 0.09 ± 0.03 0.22 ± 0.09 0.87 ± 0.73 0.10 ± 0.05 0.14 ± 0.09 0.35 ± 0.35 0.98 ± 0.89 0.28 ± 0.28 0.14 ± 0.09 0.35 ± 0.35 1.05 ± 0.92 0.28 ± 0.28 0.18 ± 0.18 0.42 ± 0.40 1.13 ± 1.03 0.30 ± 0.29 Naltrexone (mg/kg, IP) Veh Nltx 0.25 Nltx 1.0 Nltx 2.5 UFP-101 (g/rat, ICV) Veh UFP-101 5.0 UFP-101 10.0 UFP-101 20.0 Naltrexone (mg/kg, IP) + Buprenorphine 0.03 (mg/kg, IP) Veh + Veh Veh + Bup 0.03 Nltx 0.25 + Bup 0.03 Nltx 0.25 + Veh 1.48 ± 0.83 2.45 ± 1.04 2.77 ± 1.15 2.13 ± 1.72 2.83 ± 1.33 5.01 ± 1.17 4.81 ± 1.39 2.32 ± 0.70 Naltrexone (mg/kg, IP) + Buprenorphine 3.0 (mg/kg, IP) Veh + Veh Veh + Bup 3.0 Nltx 0.25 + Bup 3.0 Nltx 0.25 + Veh 3.52 ± 1.36 0.29 ± 0.29 0.79 ± 0.55 2.49 ± 1.15 3.83 ± 1.31 0.75 ± 0.62 1.24 ± 0.91 2.65 ± 1.13 UFP-101(g/rat, ICV) + Buprenorphine 3.0 (mg/kg, IP) Veh + Veh Veh + Bup 3.0 UFP 10.0 + Bup 3.0 UFP 20.0 + Bup 3.0 2.35 ± 0.95 0.89 ± 0.48 3.43 ± 0.78 2.63 ± 0.56 3.37 ± 0.82 1.57 ± 0.93 5.34 ± 1.20 4.20 ± 1.18 IP, intraperitoneal; Veh, vehicle; Bup, buprenorphine; Nltx, naltrexone; UFP, UFP-101; ICV, intracerebroventricular. Treatments were conducted as described above for the respective experiments. Each value represents the mean (± SEM) of intake corrected for body weight (g/kg). ap < 0.01, difference from vehicle. 132 Experiment 6: Effect of ICV Injections of UFP-101 on Buprenorphine-Induced Decreased Ethanol Intake The overall ANOVA showed a significant effect of treatment [F(3,9) = 9.16, p < 0.001]. As in previous experiments, administration of buprenorphine at the dose of 3.0 mg/kg significantly reduced alcohol intake (p < 0.05). Pretreatment with UFP-101 at the dose of 10.0 g/rat (given twice) completely blocked this effect of buprenorphine. Administration of 20.0 g/rat of the N/OFQ antagonist (given twice) not only blocked buprenorphine- induced reduction of ethanol drinking but resulted in a significant increase of drinking, compared with control subjects (p < 0.05). As shown in Figure 6, significant differences from control subjects were observed throughout the observation period. Water and food intake were never modified by drug treatments (Table 1). Figure 3. Effect of UFP-101 (0.0, 5.0, 10.0, and 20.0 g/rat) on ethanol intake in Marchigian Sardinian alcoholpreferring rats. The drug was given intracerebroventricular twice at 95 and 15 min before ethanol access, and alcohol consumption was monitored at 30, 60, 90, and 120 min. Values represent the mean (± SEM) of seven subjects. Difference from control subjects was not statistically significant. Veh, vehicle. 133 Figure 4. Effect of intraperitoneal (IP) injection of naltrexone (Ntx), at a dose ineffective per se (0.25 mg/kg), on buprenorphine-induced increased ethanol intake (0.03 mg/kg) in Marchigian Sardinian alcohol-preferring rats. Buprenorphine (Bup) was injected IP 5 min after Ntx administration, and ethanol was given to the animals 90 min after Bup. Alcohol consumption was monitored at 30, 60, 90, and 120 min. Values represent the mean (±SEM) of 10 subjects. Difference from control subjects: *p < 0.05. Veh, vehicle. Experiment 7: Effect of Buprenorphine and UFP-101 on Open-Field Behavior The general locomotor activity of the animals was not influenced by drug treatments. The overall ANOVA showed nonsignificant differences on time spent resting [F(3,20) = 0.11, p = ns] and distance traveled [F(3,20) = 0.14, p = ns] (Table 2). Comparisons between pretreatment and treatment provided further within-subject evidence of the absence of locomotor effects due to drug injections. Table 2. Locomotor activity in naïve marchigian sardinian alcohol-preferring rats treated with buprenorphine, UFP101, or their combination Treatment Veh + Veh UFP + Veh Veh + Bup UFP + Bup Time Resting (min) Pre-Treat. Treatment 23.6 ± 1.4 22.9 ± 1.2 23.6 ± 1.0 24.6 ± 1.5 23.0 ± 1.4 23.7 ± 1.3 24.2 ± 0.7 21.9 ± 0.2 Distance Traveled (cm) Pre-Treat. Treatment 3717.6 ± 1232.5 3618.6 ± 615.5 3575.0 ± 709.1 3415.0 ± 1008.3 3859.6 ± 972.0 3050.3 ± 715.0 2997.6 ± 393.2 3961.0 ± 706.3 UFP-101 (20.0 g/rat, intracerebroventricular) was injected twice, 95 and 15 min before placing the animal into the open-field chambers. Buprenorphine (3.0 mg/kg, intraperitoneal) was given 90 min before placing the animal into the open-field chambers. Each value represents the mean (± SEM) of six subjects. Veh, vehicle; UFP, UFP-101; Bup, Buprenorphine. 134 Figure 5. Effect of intraperitoneal (IP) injection of naltrexone (Ntx), at a dose ineffective per se (0.25 mg/kg), on buprenorphine-induced decreased ethanol intake (3.0 mg/kg) in Marchigian Sardinian alcohol-preferring rats. Five minutes after Ntx administration animals received an IP injection of buprenorphine (Bup), and ethanol was given to the animals 90 min after Bup. Alcohol consumption was monitored at 30, 60, 90, and 120 min. Values represent the mean (± SEM) of eight subjects. Difference from control subjects: *p < 0.05. Veh, vehicle. Figure 6. Effect of intracerebroventricular (ICV) injection of UFP-101, at doses ineffective per se (10.0, 20.0 g/rat), on buprenorphine-induced decreased ethanol intake (3.0 mg/kg) in Marchigian Sardinian alcohol-preferring rats. The UFP-101 was ICV injected 95 and 15 min before access to ethanol. Buprenorphine (Bup) was given 90 min before access to ethanol. Alcohol consumption was monitored at 30, 60, 90, and 120 min. Values represent the mean (± SEM) of 10 subjects. Difference from control subjects: *p < 0.05. Veh, vehicle. 135 DISCUSSION We report a dualistic action of buprenorphine on ethanol consumption. At low doses (0.03 and 0.3 mg/kg), this drug increased ethanol consumption, whereas at the dose of 3.0 mg/kg, it markedly and selectively decreased it. In fact, food and water consumption as well as motor behavior as assessed in the open-field were not influenced in these animals after treatment with 3.0 mg/kg of buprenorphine. If the drug is given at higher doses (6.0 mg/kg), it further reduces ethanol consumption but food intake is also decreased concomitantly, indicating that non-specific inhibition of ingestive behavior might occur at this highest dose. The increase of ethanol drinking observed after administration of low doses of buprenorphine can be explained on the basis of the ability of this drug to activate the opioid receptor subtype. In fact, previously published studies have demonstrated that treatment with low doses of morphine or the selective agonist DAMGO increases ethanol consumption in rats (Hubbell et al., 1986, 1993, Zhang & Kelley, 2002). Consistent with this notion, in the present study we have shown that pre-treatment with naltrexone, at a dose that does not modify ethanol drinking per se, completely abolishes the increase of alcohol consumption evoked by low doses of buprenorphine. Surprisingly, however, naltrexone was unable to block the reduction of ethanol consumption induced by higher doses of buprenorphine. This demonstrates that the inhibition of ethanol drinking observed after administration of higher doses of buprenorphine is not mediated by classical opioidergic mechanisms. Searching for a mechanism that might mediate the high-dose suppressive effects of buprenorphine on alcohol intake, we tested the hypothesis that these could be mediated by its ability to activate the NOP receptors (Wnendt et al., 1999; Bloms-Funke et al., 2000). Therefore, we tested the effect of the highly selective NOP receptor antagonist UFP-101 on high-dose buprenorphine-induced reduction of ethanol drinking. The results 136 of this experiment seem to confirm our hypothesis. Central administration of the NOP antagonist at doses that did not influence ethanol drinking per se fully blocked the inhibitory effect of buprenorphine. In addition, at the highest dose of UFP-101, the NOP antagonist inverted the high-dose action of buprenorphine (i.e., whereas animals receiving 3.0 mg buprenorphine alone drank less than vehicle-treated control subjects, animals receiving the same dose of buprenorphine after UFP-101 pretreatment paradoxically drank more than control subjects receiving neither of the drugs). This seemingly paradoxical effect likely reflects that a complete blockade by UFP-101 of NOP receptors unmasks opposite buprenorphine effects mediated through activation of -opioid receptors. Under these circumstances, the activation of -opioid receptors and the subsequent increase of ethanol consumption are not longer counterbalanced by the concomitant stimulation of the NOP receptors. In a number of other studies, biphasic or even triphasic dose-related effects have been described for buprenorphine (Rance et al., 1979; Tyers, 1980; Dum & Herz, 1981; Pick et al., 1997). In a detailed investigation by (Huang et al., 2001), it was shown that buprenorphine binds at nanomolar concentration to -, -, and -receptors and at micromolar concentration to the NOP receptors. At functional level it acts as a partial agonist at -, -, or NOP receptors and as an antagonist at -receptors. In contrast, its major metabolite, norbuprenorphine, is a full agonist at - and NOP receptors, and partial agonist at - and -receptors. This complex pharmacology might account for the fact that at low doses (1.0 mg/kg, IP) buprenorphine is an effective analgesic but at higher doses its antinociceptive effects are diminished (Dum & Herz, 1981; Lizasoain et al., 1991; Lutfy et al., 2003). In general, researchers attributed both the safety and the biphasic actions of buprenorphine to its partial agonist activity at -opioid receptors and to its agonistic/antagonistic properties at -opioid receptors (Kamei et al., 1995; Lattanzi et al., 2001). 137 Our present data, however, prompt a reanalysis of buprenorphine’s pharmacological profile and an examination of whether activation of NOP receptors plays a major role in shaping it (Wnendt et al., 1999; Lutfy et al., 2003). That this might be the case is suggested by the fact that co-administration of UFP-101 (see present results) and J-113397 (Lutfy et al., 2003), two selective NOP receptor antagonists (Kawamoto et al., 1999; McDonald et al., 2003), completely eliminates the biphasic effect of buprenorphine on ethanol intake (present data) and on analgesia (Lutfy et al., 2003), respectively. It is known that activation of brain NOP receptors by the endogenous ligand N/OFQ results in an anti-opioid action (Ciccocioppo et al., 2000a, 2000b). For example, N/OFQ injected intracranially blocks the analgesic effects of morphine (Mogil et al., 1996; King et al., 1998; Mogil & Pasternak, 2001), prevents the development of morphine-induced conditioned place preference (Murphy et al., 1999; Ciccocioppo et al., 2000b), and inhibits morphine-induced dopamine release in the nucleus accumbens (Di Giannuario & Pieretti, 2000). In addition, it has been demonstrated that activation of NOP receptors by N/OFQ results in a marked inhibition of ethanol self-administration and ethanol-seeking in rodents (Ciccocioppo et al., 1999, 2004). On the basis of these data it is conceivable to hypothesize (see also Wnendt et al., 1999) that the low abuse liability, the relatively safe profile of buprenorphine, and its efficacy in reducing alcohol drinking might be due to the activation of NOP receptors induced by this drug. The results of the present study suggest a potential new application of buprenorphine in pharmacological treatment of alcohol dependence. We propose that a combination of buprenorphine with naltrexone could be particularly beneficial in this regard. First, on the basis of the documented efficacy of naltrexone for this indication (Bouza et al 2004), simultaneous blockade of the -opioid and activation of the NOP receptors should result in a synergistic inhibition of alcohol drinking. Second, coadministration of naltrexone and buprenorphine would eliminate concerns of giving an 138 opioid agonist to subjects without opioid dependence. In particular, co-administration of buprenorphine with a novel depot naltrexone preparation (Kranzler et al., 2004) seems attractive in this regard, because it would ensure compliance with naltrexone treatment before administration of buprenorphine. A second potential application is prompted by the observation that concomitant use of different drugs of abuse is on the rise and increases the likelihood of overdose and suicide (Ruttenber & Luke, 1984; Roy et al., 1990; Risser & Schneider, 1994) and participation in HIV risk behaviors (Petry, 1999) and reduces the treatment outcomes (Schuckit, 1985; Rounsaville et al., 1987). Alcohol is the drug most frequently coabused with illicit substances (Hesselbrock et al., 1985; Helzer & Pryzbeck, 1988). In the United States, approximately 50% of heroin addicts applying to methadone programs are also regular users of alcohol (Ball & Ross, 1991). Alcohol consumption further increases under methadone-maintenance therapy, and this represents a serious limitation in the long term clinical use of this compound (Hunt et al., 1986; Stastny & Potter, 1991; Backmund et al., 2003). The agonistic activity of methadone at -opioid receptors could be at the origin of this effect. In this respect, buprenorphine might offer important advantages over methadone, because owing to its ability to simultaneously activate the NOP receptors, it should reduce rather than increase alcohol consumption. Clinical studies are urgently needed to evaluate the efficacy of buprenorphine to control ethanol abuse in alcoholic patients, possibly in association with naltrexone, and to systematically evaluate its efficacy in the treatment of concomitant opiate and alcohol dependence. Finally, the present observations raise an intriguing possibility in relation to the therapeutic efficacy of buprenorphine in heroin dependence. As reviewed by Mattick et al., (2004), clinical studies of buprenorphine that have used high doses of buprenorphine have consistently shown superior outcomes to those where low doses have been used. Clinical experience indicates further improvements beyond the doses studied systematically. Yet a recent study with positron emission tomography and carfentanyl 139 displacement indicates that there is virtually no increase in -opioid occupancy by buprenorphine between the maximal clinically used dose, 32 mg daily, and one-half of that dose. This prompts the question of whether some of the additional efficacy of high buprenorphine doses might also be mediated through an activation of NOP receptors. If this proves to be the case, it would demonstrate a novel treatment principle for this disorder that lacks addictive properties. Acknowledgments This study was supported by the European Union’s Fifth Framework Program, grant QLRT-2001-01048 (to MH and RC); National Institute on Alcohol Abuse and Alcoholism, grant AA01435 (to FW subcontract to RC); and by a Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) grant 2004 to (MM). We wish to thank Mr. Marino Cucculelli for his skilful technical assistance. 140 References Agabio R, Cortis G, Fadda F, Gessa GL, Lobina C, Reali R, Colombo G (1996) Circadian drinking pattern of Sardinian alcohol-preferring rats. Alcohol Alcohol 31:385-8. Auriacombe M, Franques P, Tignol J (2001) Deaths attributable to methadone vs buprenorphine in France. JAMA 285:45. Backmund M, Schutz CG, Meyer K, Eichenlaub D, Soyka M (2003) Alcohol consumption in heroin users, methadone-substituted and codeine-substituted patients--frequency and correlates of use. Eur Addict Res 9:45-50. Ball J, Ross A (1991) The Effectiveness of Methadone Maintenance Treatment. 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Chapter 5 Attenuation of ethanol self-administration and of conditioned reinstatement of alcoholseeking behaviour by the antiopioid peptide nociceptin/orphanin FQ in alcohol-preferring rats 151 Attenuation of ethanol self-administration and of conditioned reinstatement of alcohol-seeking behaviour by the antiopioid peptide nociceptin/orphanin FQ in alcoholpreferring rats Abstract Rationale: Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid-like orphan receptor NOP, was shown to reduce home-cage ethanol consumption, ethanol-induced conditioned place preference and stress-induced reinstatement of alcohol-seeking behaviour. Objectives: The present study, using genetically selected Marchigian Sardinian alcohol-preferring (msP) rats, was designed to evaluate the effect of this opioid peptide on 10% ethanol and 10% sucrose selfadministration, under a fixed-ratio 1 (FR 1) or a progressive-ratio (PR) schedule of reinforcement. Furthermore, using an experimental model of relapse in which rats were trained to lever press for ethanol in the presence of the discriminative stimulus of an orange odour (S+) and a 1-s cue light (CS+) or for water in the presence of anise odour (S-) and 1-s white noise (CS-), the effect of N/oFQ on cue-induced reinstatement of extinguished ethanol responding was investigated. Results: Sub-chronic (6 days) intracerebroventricular (i.c.v.) injection of 0.5 g or 1.0 g N/OFQ per rat significantly reduced alcohol self-administration under both the FR 1 and PR schedules of reinforcement. Conversely, i.c.v. administration of 0.5, 1.0 or 4.0 g of the peptide per rat did not affect sucrose self-administration. In addition, i.c.v. N/OFQ (1.0–2.0 g per rat) significantly inhibited the reinstatement of extinguished ethanol responding under 152 an S+/CS+ condition, whereas lever pressing under S-/CS- was not altered. Conclusions: The present study demonstrates that the reinforcing effects of ethanol are markedly blunted by activation of the opioidergic N/OFQ receptor system. Moreover, the data provide evidence of the efficacy of N/OFQ to prevent reinstatement of ethanol seeking behaviour elicited by environmental conditioned stimuli. Key Words: Nociceptin/orphanin FQ, self-administration, alcohol abuse, addiction INTRODUCTION Nociceptin (N/OFQ), also referred to as orphanin FQ, is a 17-aminoacid peptide that shows structural homology with opioid peptides, particularly with dynorphin A, but is lacking the N-terminal tyrosine necessary for activation of traditional opioid receptors (Meunier et al., 1995; Reinscheid et al., 1995, 1998). The N/OFQ peptide binds with high affinity the opioid receptor-like 1 (ORL1) receptor, recently included in the opioid receptor family and renamed NOP, whereas it does not activate the classical opioid receptors (, , and ). At the intracellular level, however, the activation of membrane NOP receptors exerts actions similar to those induced by activation of the other opioid receptors, namely, inhibition of cAMP production, closure of voltagesensitive Ca++ channels and enhancement of an outward K+ conductance (Meunier et al., 1995; Reinscheid et al., 1995, 1998). Nevertheless, naloxone, a non-selective opioid antagonist, does not block N/OFQ intracellular events (Henderson & McKnight, 1997; Darland et al., 1998), confirming that the pharmacological actions of this peptide are not mediated by the classic opioid receptors. Neuroanatomical and immunohistochemical studies (Darland et al., 1998; Mollereau & Mouledous, 2000) have shown a wide distribution of N/OFQ and its 153 receptor in various corticomesolimbic structures, including the amygdala, the bed nucleus of the stria terminalis, the nucleus accumbens (Nacc) and various frontocortical areas involved in the regulation of the motivational effect of drugs of abuse (Koob et al., 1998; Wise, 1998; Everitt & Wolf, 2002). In recent studies using genetically selected Marchigian Sardinian alcoholpreferring rats (msP), it has been demonstrated that chronic intracerebroventricular (i.c.v.) N/OFQ injections significantly reduced home-cage ethanol intake in the two bottle choice and ethanol-induced conditioned place-preference paradigms (Ciccocioppo et al., 1999, 2002b). In addition, N/OFQ has been shown to inhibit stressinduced reinstatement of alcohol-seeking behaviour in rats trained to self-administer ethanol (Martin-Fardon et al., 2000). Lastly, several lines of evidence suggest that although it does not bind to the opioid receptors, N/OFQ is able to function as an “antiopioid” peptide that inhibits the rewarding properties of morphine. For instance, it has been shown that pretreatment with N/OFQ inhibits morphine-induced conditioned place preference (Murphy et al., 1999; Ciccocioppo et al., 2000), whereas microdialysis studies demonstrated that central administration of this peptide can block morphineinduced dopamine (DA) release in the NAcc of freely moving rats (Di Giannuario et al., 1999). To further investigate the involvement of the N/OFQ in the control of alcohol abuse, in the present work, the effect of the peptide on ethanol consumption under operant conditions was studied. For this purpose, using msP rats, the peptide was tested on ethanol-self-administration under both fixed ratio 1 (FR 1) and progressive ratio (PR) contingences. Moreover, using an animal model of relapse, the ability of N/OFQ to prevent reinstatement of ethanol-seeking behaviour elicited by environmental conditioning factors was investigated. Lastly, in order to evaluate the selectivity of the effects of the peptide, its ability to affect 10% sucrose self administration under FR 1 and PR schedules of reinforcement was studied. 154 MATERIALS AND METHODS Animals Male genetically selected Marchigian Sardinian alcohol-preferring rats were employed. They were bred in the Department of Pharmacological Sciences and Experimental Medicine of the University of Camerino (Marche, Italy) for 38 generations from Sardinian alcohol-preferring rats (sP) of the 13th generation, provided by the Department of Neurosciences of the University of Cagliari (Fadda et al., 1990; Gessa et al., 1991). These animals are referred to as Marchigian sP (msP) rats. At the beginning of the experiments, their body weight ranged between 200 g and 250 g. They were kept in a room with a reverse 12-h/12-h light/dark cycle (lights off at 0930 hours), temperature of 20–22°C and humidity of 45–55%. All animals were handled once daily for 5 min for 1 week before the beginning of the experiments. All procedures were conducted in adherence with the European Community Council Directive for Care and Use of Laboratory Animals. During the experiments, rats were offered free access to tap water and food pellets (4RF18, Mucedola, Settimo Milanese, Italy) except during the first 3 days of training to establish operant responding (see below). Intracranial surgery For intracranial surgery, each msP rat was anaesthetized by i.m. injection of 100– 150 l of a solution containing tiletamine cloridrate (58.17 mg/ml) and zolazepam cloridrate (57.5 mg/ml). A guide cannula for i.c.v. injections aimed at the left lateral cerebroventricle was stereotaxically implanted and cemented to the skull. The following co-ordinates, taken from the atlas of Paxinos and Watson (1986), were used: antero-posterior = 0.8 mm behind the bregma, lateral = 1.8 mm from the sagittal suture, ventral = 2 mm from the surface of the skull. 155 Self-administration apparatus The self-administration stations consisted of operant conditioning chambers (Med Associate, Inc) enclosed in sound-attenuating, ventilated environmental cubicles. Each chamber was equipped with a drinking reservoir (volume capacity: 0.2 ml) positioned 4 cm above the grid floor in the centre of the front panel of the chamber, and two retractable levers located 3 cm (one to the right and the other to the left) of the drinking receptacle. An infusion pump was activated by responses on the right, or active, lever, while responses on the left, or inactive, lever were recorded but did not result in activation of the pump. Activation of the pump resulted in a delivery of 0.1 ml fluid (either ethanol, sucrose or saccharin). During the infusion of ethanol or sucrose (10% w/v), a house light located on the front panel was turned on for 1.0 s (which corresponded to the duration of the syringe-pump activation). Lever presses during this period were counted but did not lead to further infusions. An IBM-compatible computer controlled the delivery of fluids, presentation of visual stimuli and recording of the behavioural data. Drug injections Nociceptin (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala- Asp-Glu) was a generous gift of Dr. R. Guerrini, Department of Pharmaceutical Sciences of the University of Ferrara, Italy. It was dissolved in sterile isotonic saline and injected i.c.v. in a volume of 1 l per rat by means of a stainless-steel injector 2.5 mm longer than the guide cannula, so that its tip protruded into the ventricle. To verify the cannula placement, immediately before the rat was sacrificed, 1 ml of black India ink was injected i.c.v., and ink diffusion into the ventricles was evaluated using a histological method. 156 Alcohol self-administration training procedures Animals were trained to self-administer 10% (w/v) ethanol in 30-min daily sessions under a FR 1 schedule of reinforcement where each response resulted in delivery of 0.1 ml fluid, as previously described (Weiss et al., 1993). During the first 3 days of training, the rats were placed under a restriction schedule limiting water availability to 2 h per day in order to facilitate acquisition of operant responding maintained by a liquid reinforcer. During this time, responses at the lever were reinforced by delivery of a 0.2% (w/v) saccharin solution into the drinking receptacle on a FR 1 schedule, throughout daily 30-min sessions. During all subsequent training and testing, water was freely available in the home cages. After successful acquisition of saccharin-reinforced responding, rats were trained to self-administer ethanol using a modification of the “sucrose-fading procedure” (Samson, 1986), which employed saccharin instead of sucrose (Weiss et al., 1993). During the first 2 days of training, responses at the lever were reinforced by a 0.2% saccharin solution containing 5.0% (w/v) ethanol. Beginning on day 3, the concentration of ethanol was gradually increased from 5.0% to 8.0% and finally 10% (w/v), while the concentration of saccharin was correspondingly decreased to 0%. From the first day, rats began to press for 10% ethanol, the house light located on the front panel was turned on for 1.0 s. Sucrose self-administration training procedures For sucrose self-administration, the training procedures were identical to that described for alcohol self-administration, except that lever pressing during the first 3 days of water deprivation was reinforced by water delivery and, after successful acquisition of operant responding, animals received immediately 10% (w/v) of sucrose. From the first day, rats begun to press for 10% sucrose, the house light located on the front panel was turned on for 1.0 s. 157 Ethanol PR In this experimental paradigm, the breaking point (BP) of ethanol was evaluated under a PR schedule of reinforcement. For this purpose, animals were first trained to self-administer 10% alcohol under a FR 1 schedule of reinforcement. Following acquisition of a stable baseline of responding with 10% ethanol, animals were tested under the PR condition where the response requirement (i.e., the number of lever responses or “ratio” required to receive one dose of 0.1 ml of 10% ethanol) was increased in the following manner: for each of the first 5 ethanol deliveries, the ratio was increased by 1; for all the following deliveries the ratio was increased by 2. Each ethanol-reinforced response resulted in a 1.0 s illumination of the “house light” while sessions were terminated when more than 30 min had elapsed since the last reinforced response. Baseline PR was established for two consecutive days, and the third day drug testing begun. Between experiments, the baseline under PR schedule of reinforcement was re-established for two consecutive days. Sucrose PR For the determination of the BP for sucrose, an experimental procedure identical to that used for ethanol was used except that every ratio completed was reinforced by the delivery of 0.1 ml of 10% sucrose. 158 Cue-induced reinstatement of alcohol-seeking behaviour This experimental procedure consisted of three phases. Conditioning phase The purpose of the conditioning phase was to train rats to discriminate the availability of ethanol (reward) versus water (nonreward). Conditioning sessions began immediately after termination of the saccharin-fading procedure and were composed of ten ethanol and ten water 30-min daily sessions, during which discriminative stimuli (SD) predictive of ethanol versus water availability were presented. The S D for ethanol consisted of the odour of an orange extract (S+), whereas water availability was signalled by an anise extract (S-). The olfactory stimuli were produced by depositing five to six drops of the respective extract into the bedding of the operant chamber immediately before extension of the levers and session initiation, and remained present throughout the 30-min sessions. At the end of each session, the bedding of the chamber was changed, and bedding trays were thoroughly cleaned. In addition, each lever press resulting in delivery of ethanol was paired with illumination of the chamber’s house light for 1.0 s (CS+), while lever presses resulting in water delivery were followed by a 1.0-s white noise (CS-). During the 1.0-s presentation of these contingent cues, responses at the active lever were recorded but not reinforced by ethanol or water infusions (time out). During the first 3 days of this phase, the rats were given ethanol sessions only. Subsequently, ethanol and water sessions were conducted in random order. Extinction of lever-pressing phase After completion of the conditioning phase, rats were subjected to 30-min extinction sessions, for 20 consecutive days. Extinction sessions began by extension of 159 the levers without presentation of the olfactory discriminative stimuli, while responses at the previously active lever activated the syringe pump but did not result in the delivery of either ethanol or water or the presentation of the response-contingent cues (house light or white noise). This phase was introduced to eliminate the capacity of the self-administration chamber to non-specifically motivate the animal’s behaviour by leaving unaltered the ability of the cues to predict ethanol availability. Reinstatement testing Reinstatement tests began the day after the last extinction session and were conducted over two consecutive days. In these tests, rats were exposed to the same conditions as those during the conditioning phase, except that liquids (alcohol or water) were not made available. Sessions were initiated by extension of both levers and presentation of either the ethanol S+ or water S-, that remained present during the entire 30-min session. Responses at the previously active lever were followed by activation of the syringe pump motor and presentation of the CS+ (“house light”) in the S+ condition or the CS- (“white noise”) in the S- condition. Half of the animals were tested under the S+/CS+ condition on day 1 and under the S-/CS- condition on day 2. The other half were first tested under S-/CS- and then under S+/CS+. Experiment 1. Effect of subchronic i.c.v. injections of N/OFQ on alcohol selfadministration under a FR 1 schedule of reinforcement After acquisition of a stable 10% ethanol self-administration baseline (9 days), rats (n=24) were separated into three groups with similar baseline levels of responding for 10% ethanol. During the last 4 days of training (pre-treatment), immediately prior to 160 the self-administration sessions, animals were given 1 l of saline i.c.v. to familiarise them with the injection procedure. At this point, for six consecutive days, the first group (n=9) was injected i.c.v. with isotonic saline (control), whereas the second (n=9) and the third (n=9) groups received 0.5 g and 1.0 g per rat of N/OFQ, respectively. Immediately after, animals were tested for 10% ethanol self-administration. At completion of drug testing (6 days), ethanol self-administration was monitored for an additional 4 days (post-treatment) during which all animals received only i.c.v. saline. The number of responses at both active and inactive levers was recorded for the entire period of the experiment. Experiment 2. Effect of i.c.v. injections of N/OFQ on alcohol self-administration under PR schedule of reinforcement Following acquisition of a stable baseline of responding for ethanol (15 days) under a FR 1 condition, a group of animals (n=8) was treated i.c.v. with N/OFQ 0.5 g and 1.0 g per rat or its vehicle. In a counterbalanced order (Latin square), animals received all drug doses and vehicle. An interval of 3 days was imposed between drug testing. Responding on both the active and the inactive lever was recorded for the entire period of the experiment. Experiment 3. Effect of i.c.v. injections of N/OFQ on cue-induced reinstatement of alcohol-seeking behaviour After completion of the discrimination and extinction phases, animals were tested for the ability of N/OFQ to prevent the reinstatement of alcohol-seeking elicited by 161 cues predictive of ethanol availability. For this purpose, during the last three extinction sessions msP rats were injected i.c.v. with 1 l saline in order to familiarise them with the administration procedures. Animals were then separated into three groups with a similar baseline number of responses during extinction. For the reinstatement test, one group (n=9) of rats was injected i.c.v. with isotonic saline (control), while the other two groups (n=7–8) received 2.0 g and 4.0 g per rat of N/OFQ. Animals were placed in the self administration chambers immediately after drug administration. In one-half of the rats, the effect of N/OFQ was tested the day after the last extinction session under the S+/CS+ condition and on the following day under the S-/CS- condition. In the other half, N/OFQ was first tested under S-/CS- and then under S+/CS+ condition. The number of responses on both the active and the inactive levers was recorded throughout the experiment. Experiment 4. Effect of subchronic i.c.v. injections of N/OFQ on sucrose selfadministration under FR 1 schedule of reinforcement After acquisition of a stable 10% sucrose self-administration baseline (8 days), rats (n=25) were separated into four groups with similar baseline levels of responding for 10% sucrose. During the last 4 days of training (pre-treatment), immediately prior to the self administration sessions, animals were given 1 l saline i.c.v. to familiarise them with the injection procedure. At this point, for six consecutive days, the first group of animals (n=7) received i.c.v. isotonic saline (control), while the second (n=6), the third (n=7) and the fourth (n=5) groups received 0.5, 1.0 and 4.0 g per rat of N/OFQ, respectively. Immediately after, animals were tested for 10% sucrose selfadministration. At completion of drug testing (6 days), sucrose responding was monitored for another 4 days (posttreatment), during which all animals received only 162 i.c.v. saline. The number of responses on both the active and inactive levers was recorded for the entire period of the experiment. Experiment 5. Effect of i.c.v. injections of N/OFQ on sucrose self-administration under a PR schedule of reinforcement Following acquisition of a stable baseline of responding for 10% sucrose (12 days) under a FR 1 condition, a group of animals (n=7) was treated i.c.v. with N/OFQ 0.5, 1.0 and 4.0 g per rat or its vehicle. In a counterbalanced order (Latin square), animals received all drug doses and vehicle. An interval of 3 days was imposed between drug testing. Operant responding at both the active and the inactive lever was recorded for the entire period of the experiment. Statistical analysis For the self-administration experiments under the FR 1 condition, data were analysed by means of two-way analysis of variance (ANOVA) with one within-subjects factor (time) and one between subjects factor (treatment). For the PR experiments, the number of rewards earned were analysed by one-way ANOVA with repeated means. For the reinstatement experiment, differences among responses during the training, extinction and reinstatement phases were analysed in the vehicle-treated group by oneway within subjects ANOVA, followed by Newman-Keuls post-hoc tests to identify differences between experimental phases and responses in the presence of the S +/CS+ versus S-/CS-. The effect of N/OFQ on reinstatement responses was analysed by two- 163 way ANOVA, one factor within (reinstatement condition) and one factor between (treatment), followed by Newman-Keuls post-hoc tests. Statistical significance was set at P<0.05. Results Experiment 1. Effect of subchronic i.c.v. injections of N/OFQ on alcohol selfadministration under a FR 1 schedule of reinforcement All rats acquired responding reinforced by 10% ethanol and developed stable levels of ethanol-maintained behaviour. During the initial 5-day training phase, in 30min sessions, animals responded to the active lever from 31 to 53 times to selfadminister approximately 0.6–1.1 g/kg ethanol. As shown in Fig. 1, during the following 4 days, while establishing pre-treatment baseline, responding ranged between 40 and 65 corresponding to 0.8–1.3 g/kg ethanol. The stability of responding, suggests, therefore, that manipulation due to i.c.v. injections did not influence animals’ operant behaviour. In a separate study, measures of blood alcohol levels (BAL) taken from another group of msP rats immediately after the operant session demonstrated that over similar range of 10% ethanol self-administration pharmacologically relevant ethanol concentrations of 40–70 mg/dl are achieved. Subchronic i.c.v. treatment with N/OFQ, 0.5 mg and 1.0 g per rat, markedly reduced responding on the active lever. The effect was significant from the first day of treatment. Compared with controls, ethanol self-administration was reduced by about 40–50%, and statistical analysis revealed a significant overall effect of treatment (F2,24=7.30, P<0.01). Moreover, the Newman-Keuls posthoc test showed a significant 164 difference between controls and animals treated with both 0.5 mg and 1.0 mg per rat of N/OFQ (P<0.01). Responses at the inactive lever were almost absent in all treatment conditions and were not affected by drug injection (F2,24=1.95, n.s.). At the end of N/OFQ treatment, the msP rats progressively recovered from the effect of treatment, and ethanol self-administration returned to pre-treatment levels within 4 days. Fig. 1 Fixed ratio 1 10% ethanol self-administration in Marchigian Sardinian alcohol-preferring rats (n=9/group) treated i.c.v. for six consecutive days with 0.5 mg and 1.0 g per rat of N/OFQ or its vehicle (Veh). *P<0.05 versus vehicle Experiment 2. Effect of i.c.v. injections of N/OFQ on alcohol self-administration under PR schedule of reinforcement PR baseline was established for two consecutive days before the experiment. For each of these 2 days, values of overall responding were 10.4±1.5 and 9.5±1.8. On the 165 third day, N/OFQ or its vehicle were given prior to ethanol access and, as shown by the analysis of variance, an overall drug effect (F2,14=3.93, P<0.05) was observed. As shown in Fig. 2A, the Newman Keuls test demonstrated a significant reduction in earned reinforcers in rats treated with 1.0 g per rat of N/OFQ compared with drug vehicle. This effect was confirmed by the reduction of the BP for ethanol observed following N/OFQ treatment (Fig. 2B). Responses at the inactive lever were almost absent in all treatment conditions and were not affected by drug injection (F2,14=1.48, n.s.). Fig. 2 Performance on a progressive ratio schedule of reinforcement of rats (n=8) treated i.c.v. with 0.5 g and 1.0 g per rat of N/OFQ or its vehicle (Veh) in a counterbalanced order (Latin square). A Mean (±SEM) number of rewards earned. B Mean (±SEM) number of responses emitted at breaking point. *P<0.05 versus vehicle 166 Experiment 3. Effect of i.c.v. injections of N/OFQ on cue-induced reinstatement of alcohol-seeking behaviour At the end of the conditioning phase, the number of ethanol-reinforced responses was significantly higher (F1,46=57.21, P<0.01) compared with water-reinforced responses (Fig. 3). Lever pressing progressively decreased throughout the 20-day extinction phase (Fig. 3). For the reinstatement test, analysis of variance revealed a nonsignificant overall effect of treatment (F2,21=1.96, n.s.), but a significant treatmentreinstatement condition interaction (F4,42=3.18, P<0.05). Specifically, further post-hoc tests demonstrated a significant reinstatement of ethanol seeking in the vehicle-treated group (Fig. 3) under S+/CS+ stimulus condition; whereas, under S-/CS- responding remained at extinction levels (F2,16=20.71, P<0.01). Moreover, as shown in Fig. 3, pretreatment with 2.0 g and 4.0 mg per rat of N/OFQ significantly (F2,21=3.73, P<0.05) attenuated recovery of responding elicited by ethanol-paired cues (S+/CS+), whereas drug treatment did not modify responding under the S-/CS- condition (F2,21=0.23, n.s.). This difference was confirmed by a Newman-Keuls test showing that under S+/CS+ condition revealed a significant effect of N/OFQ (4.0 g per rat) compared with vehicle-treated rats (P<0.05). Responses at the inactive lever were almost absent throughout all experimental phases and were not affected by N/OFQ treatment (F2,21=2.34, n.s.). 167 Experiment 4. Effect of subchronic i.c.v. injections of N/OFQ on sucrose selfadministration under a FR 1 schedule of reinforcement As shown by the analysis of variance, subchronic i.c.v. treatment with N/OFQ 0.5, 1.0 and 4.0 g per rat did not affect responding for sucrose self-administration (F3,21=0.37, n.s.; Fig. 4). Responses at the inactive lever were almost absent in all treatment conditions and were not affected by drug injection (F3,21=0.04, n.s.). Fig. 3 Training: responses on the last day of the discrimination training phase in the presence of distinct discriminative stimuli predictive of the availability of 10% (w/v) ethanol (S +/CS+) or water (S-/CS-). Extinction: extinction responses during the 20 days of this phase. Reinstatement: responses in rats exposed to the S+/CS+ and S-/CS- conditions (in the absence of ethanol or water) and treated i.c.v. with 2.0 g and 4.0 g per rat of N/OFQ or its vehicle (Veh). Responding in the S +/CS+ test differed from S/CS- responses (P<0.01). Treatment with N/OFQ 4.0 g per rat significantly reduced responses under S+/CS+ but not under S-/CS- conditions. Values represent the mean (±SEM) of 7-9 subjects per group. Difference from vehicle *P<0.05. 168 Experiment 5. Effect of i.c.v. injections of N/OFQ on sucrose self-administration under a PR schedule of reinforcement As shown in Fig. 5A, N/OFQ at the doses of 0.5, 1.0 and 4.0 g per rat did not modify the sucrose reward earned, and statistical analysis showed absence of significant treatment effect (F3,18=0.24, n.s.). Consistently, the BP for sucrose was not modified by drug treatment (Fig. 5B). Responses at the inactive lever were almost absent in all treatment conditions and were not affected by drug injection (F3,18=0.84, n.s.). Fig. 4 Fixed ratio 1 10% sucrose self-administration in Marchigian Sardinian alcohol-preferring rats (n=25) treated i.c.v. for six consecutive days with 0.5, 1.0 and 4.0 g per rat of N/OFQ or its vehicle (Veh). 169 Fig. 5 Performance on a progressive ratio schedule of reinforcement of rats (n=7) treated i.c.v. with 0.5, 1.0 and 4.0 g per rat of N/OFQ or its vehicle (Veh) in a counterbalanced order (Latin square). A Mean (±SEM) number of rewards earned. B Mean (±SEM) number of responses emitted at breaking point DISCUSSION The results show that subchronic i.c.v. administration of 0.5 g and 1.0 g per rat of N/OFQ significantly reduced ethanol self-administration under a FR 1 schedule of reinforcement. Moreover, in the PR experiment a reduction in the BP for ethanol selfadministration was observed following N/OFQ treatment. A limitation in the interpretation of the results of the PR experiment is that, using this schedule, even vehicle-treated animals obtained a limited number of reinforcers (10–14 ethanol doses) 170 which unlikely lead to pharmacologically relevant BAL. However, in our animals, under PR conditions, the substitution of ethanol with water results in a 40–50% drop of lever responding (data not shown). This, therefore, argues in favour of the fact thatunder this condition-the animals’ behaviour is driven by the motivational value of the reinforcer (i.e., ethanol). At present we cannot exclude that factors such as the taste or the odour of ethanol may have contributed to maintain the reinforced responding. However, it is interesting to note that the peptide did not alter 10% sucrose selfadministration under either a FR 1 or a PR schedule. It seems, therefore, that the effect of the peptide is selective for ethanol, whereas the reinforcing magnitude of sucrose (a natural reinforcer) is not apparently modified by drug administration. A possible explanation for this phenomenon is that the NOP receptor system may be recruited and, therefore, may play a functional role, when the brain reward system/s is activated by potent pharmacological stimuli; whereas, it plays only a marginal role in the regulation of brain reward processes under basal conditions. This hypothesis is supported by several pieces of evidence. For example, in place conditioning studies, it has been shown that N/OFQ inhibits the rewarding effects of ethanol, morphine and cocaine, while it is devoid of motivational effects per se (Devine et al., 1996; Murphy et al., 1999; Ciccocioppo et al., 1999, 2000; Kotlinska et al., 2002). Moreover, microdialysis studies demonstrated that the activation of the NOP receptor by relatively low doses of N/OFQ potently inhibits morphine-induced mesoaccumbal DA release (Di Giannuario et al., 1999), whereas higher doses are needed to modulate basal DA activity (Murphy et al., 1996, Murphy & Maidment, 1999). Alcoholism is a chronic relapsing disorder characterised by compulsive drugseeking behaviour and use (O’Brien et al., 1990, 1998; American Psychiatric Association, 1994; O’Brien & McLellan, 1996). A critical factor implicated in the relapsing nature of alcohol and other drugs of abuse as well is the conditioning of their rewarding effects with specific environmental stimuli (cues). Indeed, clinical and 171 preclinical studies demonstrated that exposure to alcohol cues increases the urge to drink and facilitates ethanol “relapse” even after protracted periods of abstinence (McCusker & Brown, 1990, 1991; Staiger & White, 1991; Monti et al., 1993; Katner et al., 1999; Weiss et al., 2001; Ciccocioppo et al., 2001a, 2002a). The present study, using msP rats as an animal model of relapse, confirmed these previous findings and demonstrated that presentation of ethanol paired cues elicits a robust reinstatement of extinguished ethanol responding in this rat line. In contrast, no reinstatement was observed following presentation of cues predictive of water availability. More importantly, the present study demonstrated that treatment with 2.0–4.0 g per rat of N/OFQ significantly reduces cue-induced resumption of responding on the previously ethanol-paired lever. This, however, indicates that higher doses of the peptide are needed to prevent cue-induced ethanol-seeking behaviour relative to ethanol intake. This suggests that different mechanisms may control these two behaviours. To confirm the selectivity of the effect of the peptide, responding at the inactive lever was unaltered by N/OFQ treatment. Research utilising reinstatement models of relapse predominantly points to roles for DA and opioid systems in regulating the motivating effects of ethanol-associated environmental stimuli. For example, in rats, exposure to environments associated with ethanol availability increases extracellular DA levels in the NAcc (Katner et al., 1996; Gonzales & Weiss, 1998), whereas blockade of either D1 or D2 receptors dose dependently reduced the cue-induced reinstatement of ethanol-seeking behaviour (Liu & Weiss, 2002). In addition, a role of opioid systems in relapse has been implicated by clinical findings that the opiate antagonist naltrexone attenuates craving associated with exposure to ethanol cues (Monti et al., 1999) and by experimental evidence that naltrexone, as well as - and delta-selective opiate receptor antagonists, reverse conditioned reinstatement of ethanol-seeking by ethanol-associated contextual stimuli (Katner et al., 1999; Ciccocioppo et al., 2002a). 172 There is also evidence that N/OFQ reverses several of the actions of opiate drugs, which has given rise to the hypothesis that N/OFQ may act as a functional “antiopioid” agent. Specifically, N/OFQ blocks the analgesic effects of morphine (Mogil et al., 1996; King et al., 1998; Mogil & Pasternak, 2001) prevents the development of morphine-induced conditioned place preference (Murphy et al., 1999; Ciccocioppo et al., 2000) and, as mentioned before, inhibits morphine-induced DA release in the NAcc (Di Giannuario & Pieretti, 2000). In addition, electrophysiological data has demonstrated that the N/OFQ system inhibits the firing of b-endorphin cells in the hypothalamic arcuate nucleus (Wagner et al., 1998). These arcuate neurons project, among other brain regions to the ventral tegmental area (VTA) and the NAcc, where they interact with mesolimbic DA transmission and influence motivated behaviour (Di Chiara & North, 1992; Johnson & North, 1992; Devine et al., 1993a, 1993b; Herz, 1997). Moreover, it has been shown that 91% of tyrosine hydroxylase-positive cells in the VTA co-express NOP receptors, and that N/OFQ can directly and indirectly (via GABA interneurons) modulate (inhibit) neural activity of VTA DA neurons (Maidment et al., 2002; Norton et al., 2002; Zheng et al., 2002). The exact mechanism by which N/OFQ acts in the brain to modulate ethanol intake and cue-induced reinstatement of drug-seeking behaviour is not yet clear. However, taking into consideration the important role of the DA-ergic and the opiodergic systems in the regulation of these ethanol-related behaviours, and considering the modulatory role that N/OFQ has on corticomesolimbic DA and opioid activity, it may be hypothesised that the N/OFQ system interacting with these two other systems may reduce the motivational value of alcohol as well as that of stimuli predictive of its availability. In conclusion, the present study demonstrates that stimulation of NOP receptors by N/OFQ reduces the reinforcing effects of ethanol and prevents relapse elicited by environmental stimuli predictive of drug availability. Therefore, agents targeting NOP 173 receptors may represent a promising treatment for alcohol-relapse prevention and abuse as an alternative to existing medications such as naltrexone. An important consideration is that naltrexone, which has been successfully employed for the treatment of alcohol craving and prevention of relapse, can produce aversive side effects that limit compliance (Kosten & Kleber, 1984; Rabinowitz et al., 1997, 2002). In contrast, at least in laboratory animals, NOP receptor activation does not appear to produce aversive effects (Devine et al., 1996). Furthermore, NOP agonists exert a marked anxiolytic and anti-stress actions (Jenck et al., 1999, 2000; Martin-Fardon et al., 2000; Ciccocioppo et al., 2001b, 2002c) that may provide additional advantages over opiate antagonist treatments which, in fact, can induce anxiety (Lee & Rodgers, 1990) and are ineffective in preventing stress-induced ethanol-seeking behaviour (Le et al., 1998). Acknowledgements The authors thank Mike Arends for his assistance with manuscript preparation and Marino Cucculelli for technical assistance and animal care. 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Br J Pharmacol 136:1065-71. 181 Chapter 6 The Central Nucleus of the Amygdala is the neuroanatomical site of action for the effects of N/oFQ on alcohol drinking 182 The Central Nucleus of the Amygdala is the neuroanatomical site of action for the effects of N/oFQ on alcohol drinking Abstract N/oFQ acts in the brain as a functional antiopioid peptide. In previous studies, it has been shown that treatment with N/oFQ abolishes ethanol-induced conditioned place preference, reduces voluntary, 2 bottle choice alcohol intake and ethanol selfadministration in genetically selected marchigian Sardinian alcohol-preferring (msP) rats. However, at present no studies have examined the involvement of the N/oFQ-NOP receptor system in alcohol related behaviours in the nonselected rats. The aim of the present set of experiments is twofold: 1st we aimed at evaluating and compare the effect of N/oFQ treatment on alcohol self-administration in the alcohol-preferring msP rats respect the nonselected Wistars and, 2nd we sought to identify the site of action for the effects of N/oFQ on operant alcohol intake. Results, showed that ICV treatment with N/oFQ significantly reduced ethanol self-administration in the alcohol-preferring msP rats (0.5 and 1.0 g/rat) but had no effect in the nonselected Wistars (1.0 and 2.0 g/rat). In addition, by employing in situ hybridization and microinjection techniques, we demonstrated that the CeA is the brain site of action for the inhibitory effect of N/OFQ on ethanol self-administration. Keywords: Nociceptin/orphanin FQ, NOP receptor, alcohol self-administration, alcohol-preferring rats 183 INTRODUCTION The N/oFQ-NOP receptor system, because of its intracellular actions and structural similarities with the opioid peptides and receptors, respectively, it is considered to be the fourth member of the opioidergic family. However, from a functional point of view, N/oFQ is known to possess antiopioid properties (Morgan et al., 1997; King et al., 1998). In previous studies conducted in our laboratory, we investigated the effects of N/oFQ treatment, on ethanols’ positive reinforcing properties, in the genetically selected marchigian Sardinian alcohol-preferring (msP) rats. The msP rats, originate from the 13th generation of the Sardinian alcoholpreferring (sP) rats, an animal line selectively bred for high ethanol preference and consumption (Fadda et al., 1990; Gessa et al., 1991), and are bred in the Department of Pharmacological Sciences and Experimental Medicine of the University of Camerino (Marche, Italy). Results, showed that intracerebroventricular (ICV) treatment with N/oFQ inhibited ethanol-induced conditioned place preference, and significantly reduced voluntary alcohol consumption under the 2 bottle choice paradigm, in the msP rats (Ciccocioppo et al., 1999, 2000). In addition, under operant self-administration conditions animals treated with N/oFQ reduced their lever presses for alcohol (Ciccocioppo et al., 2004a), whereas, under an operant extinction/reinstatement paradigm ICV treatment with the peptide significantly attenuated the reinstatement of alcohol-seeking behavior elicited by environmental conditioning factors in the msP rats (Ciccocioppo et al., 2004a). No studies, however, have until now examined the significance of manipulation of the N/oFQ-NOP receptor system on alcohol related behaviours in nonselected animals. Here, the effect of ICV injection of N/OFQ on ethanol self-administration in msP and in non selected Wistar rats was compared. Results showed that peptide administration reduces ethanol intake in msP rats but not in 184 Wistars. Therefore, an extensive in situ hybridization study was carried out to understand the reasons for the difference observed between the two rat lines. Interestingly the results showed that msP rats have a higher expression of the gene encoding for NOP receptors in the central amygdala (CeA) in the basolateral amygdala (BLA) and in the bed nucleus of the stria terminalis (BNST). Site specific microinjection studies have, then, been carried out to demonstrate that the CeA is the brain site of action for the inhibitory effect of N/oFQ on ethanol self-administration. Materials and Methods Subjects Male genetically selected Marchigian Sardinian alcohol-preferring (msP) and the nonseleted Wistar (Charles River, Calco, Italy) rats were employed. The msP rats were bred in the Department of Pharmacological Sciences and Experimental Medicine of the University of Camerino (Marche, Italy) for 60 generations from the Sardinian alcoholpreferring rats (sP) of the 13th generation, provided by the Department of Neurosciences of the University of Cagliari (Fadda et al., 1990; Gessa et al., 1991). At the beginning of the experiments, the animals’ body weight ranged between 250 g and 300 g. They were kept in a room with a reverse 12-h/12-h light/dark cycle (lights off at 0800 hours), temperature of 20–22°C and humidity of 45–55%. All animals were handled once daily for 5 min for 1 week before the beginning of the experiments. All procedures were conducted in adherence with the European Community Council Directive for Care and Use of Laboratory Animals. During the experiments, rats were offered free access to tap water and food pellets (4RF18, Mucedola, Settimo Milanese, 185 Italy) except during the first 3 days of training to establish operant responding (see below). Drugs Nociceptin/orphanin FQ (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-ArgLys-Leu-Ala-Asp-Glu) was synthesized at the Department of Pharmacological Sciences and Biotechnology Centre (University of Ferrara, Ferrara, Italy), and was a generous gift of Dr. G. Calò. N/oFQ was dissolved in sterile isotonic saline and injected in a volume of 1.0 l per rat or 0.5 l per site/rat into the lateral cerebroventricle or into the specific brain areas, respectively. Intracranial surgery For intracranial surgery, animals were anaesthetized by intramuscular injection of 100–150 l of a solution containing tiletamine cloridrate (58.17 mg/ml) and zolazepam cloridrate (57.5 mg/ml). A guide cannula (0.65 mm outside diameter) for injections of N/oFQ was stereotaxically implanted and cemented to the skull. For cannula placements, the coordinates in millimetres with reference to bregma were as follows: intracerebroventricular: anteroposterior (AP),-1.0; lateral (L), 1.8; ventral (V), 2.0; BNST: AP, -0.1; L, ±1.3; V, 6.0; CeA: AP, -1.5; L, ±4.3; V, 7.0; BLA: AP, -1.5; L, ±5.1; V, 7.0. For the BNST, the CeA, and the BLA, cannulas were implanted bilaterally. N/OFQ, was injected through a stainless-steel injector protruding beyond the cannula tip: 2.5 mm for the lateral ventricle; 1.2 mm for the BNST; and 1.5 mm for the CeA, and the BLA. Coordinates were taken from Paxinos and Watson (1998) and adjusted for the body weight of the animals. Experiments began 1 week after surgery. 186 After completion of the experiments, rats were killed and cannula placements were verified histologically. Self-administration apparatus The self-administration stations consisted of operant conditioning chambers (Med Associate, Inc) enclosed in sound-attenuating, ventilated environmental cubicles. Each chamber was equipped with a drinking reservoir (volume capacity: 0.2 ml) positioned 4 cm above the grid floor in the centre of the front panel of the chamber, and two retractable levers located 3 cm (one to the right and the other to the left) of the drinking receptacle. An infusion pump was activated by responses on the right, or active, lever, while responses on the left, or inactive, lever were recorded but did not result in activation of the pump. Activation of the pump resulted in a delivery of 0.1 ml fluid (either ethanol or saccharin). During the infusion of ethanol (10% w/v), a house light located on the front panel was turned on for 5.0 s (TimeOut period, TO). Lever presses during this period were counted but did not lead to further infusions. An IBMcompatible computer controlled the delivery of fluids, presentation of visual stimuli and recording of the behavioural data. Alcohol self-administration training procedures Animals were trained to self-administer 10% (w/v) ethanol in 30-min daily sessions under a fixed-ratio 1 (FR1) schedule of reinforcement where each response resulted in delivery of 0.1 ml fluid, as previously described (Weiss et al., 1993). During the first 3 days of training, the rats were placed under a restriction schedule limiting water availability to 2 h per day in order to facilitate acquisition of operant responding maintained by a liquid reinforcer. During this time, responses at the lever were 187 reinforced by delivery of a 0.2% (w/v) saccharin solution into the drinking receptacle on a FR1 schedule, throughout daily 30-min sessions. During all subsequent training and testing, water was freely available in the home cages. After successful acquisition of saccharin-reinforced responding, rats were trained to self-administer ethanol using a modification of the “sucrose-fading procedure” (Samson, 1986), which employed saccharin instead of sucrose (Weiss et al., 1993). During the first 2 days of training, responses at the lever were reinforced by a 0.2% saccharin solution containing 5.0% (w/v) ethanol. Beginning on day 3, the concentration of ethanol was gradually increased from 5.0% to 8.0% and finally 10% (w/v), while the concentration of saccharin was correspondingly decreased to 0%. From the first day, rats began to press for 10% ethanol, the house light located on the front panel was turned on for 5.0 s (TO period). Experiments Experiment 1: Effect of subchronic ICV injections of N/oFQ on alcohol selfadministration in the msP rats After acquisition of a stable 10% ethanol self-administration baseline (10 days), the msP rats (n=21) were separated into three groups with similar baseline levels of responding for 10% ethanol. During the last 4 days of training (pre-treatment), immediately prior to the self-administration sessions, animals were given 1 l of saline ICV to familiarise them with the injection procedure. At this point, for six consecutive days, the first group (n=7) was injected ICV with isotonic saline (control), whereas the second (n=7) and the third (n=7) groups received 0.5 and 1.0 g per rat of N/oFQ, 188 respectively. Immediately after, animals were tested for 10% ethanol selfadministration. At completion of drug testing (6 days), ethanol self-administration was monitored for an additional 4 days (post-treatment) during which all animals received only ICV saline. The number of responses at both active and inactive levers was recorded for the entire period of the experiment. Experiment 2: Effect of subchronic ICV injections of N/oFQ on alcohol selfadministration in the Wistar rats Following acquisition of a stable baseline of responding for ethanol 10% (10 days) under a FR1 condition, a group of Wistar rats (n=24) was divided into three groups with similar baseline levels of responding for 10% ethanol. During the last 4 days of training (pre-treatment), immediately prior to the self-administration sessions, animals were given 1 l of saline ICV to familiarise them with the injection procedure. At this point, for six consecutive days, the first group of animals (n=8) received ICV isotonic saline (control), while the second (n=8), and the fourth (n=8) groups received 1.0 and 2.0 g/rat of N/oFQ, respectively. Immediately after, animals were tested for 10% ethanol self-administration. At completion of drug testing (6 days), ethanol selfadministration was monitored for another 4 days (post-treatment) during which all animals received only ICV saline. Responding on both the active and the inactive lever was recorded for the entire period of the experiment. 189 Experiment 3: Differences on the N/OFQ and NOP receptor gene expression levels between naïve msP and Wistar rats Ethanol naive msP and Wistar rats were used (n=8/group). Rats were sacrificed by decapitation and brains were quickly removed, snap frozen in –40 °C isopentane and stored at –70 °C until use. 10 m brain sections were taken at bregma levels (1) +2.5 to +1.7 mm, (2) –0.3 to -0.4 mm, (3) -1.7 – 2.0 mm and (4) -2.3 to –3.3 mm according to the atlas of Paxinos and Watson (1994). The sections mounted on slides (Superfrost slides, VWR, USA) were brought to room temperature and fixed for 15 minutes in 4% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.0. After fixation the slides were washed in PBS pH 7.4 for 10 min and rinsed two times in sterilized water for 5 min, each followed by a deproteination of the tissue with 0.1 M HCl for 10 min. The slides were rinsed twice in PBS pH 7.4 for 5 min and were acetylated in 0.1 M triethanolamine pH 8.0/0.25% acetic anhydride for 20 min, washed again briefly twice in PBS pH 7.4 for 5 min, dehydrated in graded ethanol and air-dried. The slides were prehybridized in a humidified chamber with prehybridization buffer (50% deionized formamide, 50 mM Tris-HCl pH 7.6, 25 mM EDTA pH 8.0, 20 mM NaCl, 0.25 mg/ml yeast tRNA, 2.5 x Denhardt’s solution (0.05% Ficoll, 0.05% polyvinylpyrrolidone, 0.05% bovine serum albumin) at 37°C for 2-3 hours. After draining the prehybridization buffer off the slides, the sections were hybridized with 80 l hybridization buffer (50% deionized formamide, 20 mM Tris-HCl pH 7.6, 1 mM EDTA pH 8.0, 0.3 M NaCl, 0.2 M DTT, 0.5 mg/ml yeast tRNA, 0.1 mg/ml poly-ARNA, 1 x Denhardt’s solution, 10% dextransulfate) containing 1 x 106 cpm of either the labeled antisense RNA or sense RNA. The sections were covered with siliconized coverslips and were incubated at 55°C for 18 hours in a humidified chamber. After hybridization the coverslips were removed by washing with 1x standard saline citrate (SSC) at 48°C for 30 min, followed by washing twice in 0.5 x SSC/50% formamide at 190 48°C for 30 min. After two additional washings in 1 x SSC for 15 min at 48°C the sections were treated with 1 g/ml RNAaseA in 0.5 M NaCl; 10 mM Tris-HCl; 5 mM EDTA, pH 8.0 for 45 min at 37°C. The hybridized sections were exposed to Fuji BAS5000 Phosphorimager plates. Phosphorimager generated digital images were analyzed using AIS Image Analysis Software (Imaging Research Inc., St. Catharines, Ontario, Canada). Regions of interest were defined by anatomical landmarks as described in the atlas (Paxinos & Watson, 1994). Based on the known radioactivity in the 14C standards, image values were converted to nCi/g. Afterwards slides were exposed for 1 month to Kodak BioMax MR film (Eastman Kodak Company, UK). RNA probe synthesis: Antisense and sense RNA probes for each gene were synthesized from a 1g linearized DNA template, incubated with transcription buffer (40 mM Tris-HCl pH 7.5, 6 mM MgCl2, 2 mM spermidine) in the presence of 12.5 nmol ATP, CTP, GTP, 500 pmol UTP and 125 pmol [-35S]UTP (1350 Ci/mmol, NEN/DuPont, Boston, USA), 1 U RNase inhibitor and 1 U RNA polymerase (Roche Molecular Biochemicals, Sweden) at 37°C for 1 hour. The DNA was removed by digestion with RNase-free DNase at 37°C for 15 min. The transcripts were purified using spin columns (Amersham Pharmacia Biotech, UK) and the quality of the riboprobes analyzed on an 8 M urea/5% polyacrylamide gel (PAGE). Experiment 4: Effect of microinjections of N/oFQ into the CeA on alcohol selfadministration in the msP rats Following acquisition of a stable baseline of responding for ethanol under a FR1 condition, a group of msP rats were divided into three groups (n=7) with similar baseline levels of responding for 10% ethanol. All animals received on day 1 (pretreatment) a mock injection in each of the two cannulas aiming the CeA, to familiarise 191 them with the injection procedure. On day 2 (test day), the first group of animals received isotonic saline (control), while the second and the third groups were injected into the CeA with N/oFQ at the doses of 0.125 and 0.25 g per site/rat, respectively. On day 3, all animals received 0.5 l per site/rat of isotonic saline into the CeA (posttreatment). Immediately after N/oFQ treatments, animals were placed in the selfadministration chambers and tested for 10% ethanol self-administration. Responding on both the active and the inactive lever was recorded for the entire period of the experiment. Experiment 5: Effect of microinjections of N/oFQ into the BLA on alcohol selfadministration in the msP rats After acquisition of a stable baseline of responding for ethanol under a FR1 schedule of reinforcement, msP rats were divided into three groups (n=7-8) with similar baseline levels of responding for 10% ethanol. All groups of animals received on day 1 (pre-treatment) a mock injection in each of the two cannulas aiming the BLA, to familiarise them with the injection procedure. On test day (day 2), animals were treated into the BLA with N/oFQ (0.25 and 0.5 g per site/rat) or its vehicle, respectively. On day 3, all animals received 0.5 l per site/rat of isotonic saline into the BLA (posttreatment). Immediately after N/oFQ treatments, animals were placed in the selfadministration chambers and tested for 10% ethanol self-administration. Responding on both the active and the inactive lever was recorded for the entire period of the experiment. 192 Experiment 6: Effect of microinjections of N/oFQ into the BNST on alcohol selfadministration in the msP rats Following acquisition of a stable baseline of responding for ethanol under a FR1 condition, the msP rats were divided into three groups (n=6-7) with similar baseline levels of responding for 10% ethanol. All animals received on day 1 (pre-treatment) a mock injection in each of the two cannulas aiming the BNST, to familiarise them with the injection procedure. On test day (day 2), animals were treated into the BNST with N/oFQ (0.25 and 0.5 g per site/rat) or its vehicle, respectively. On day 3, all animals received 0.5 l per site/rat of isotonic saline into the BNST (post-treatment). Immediately after N/oFQ treatments animals were placed in the self-administration chambers and tested for 10% ethanol self-administration. Responding on both the active and the inactive lever was recorded for the entire period of the experiment. Statistical analysis Data from the subchronic (6 days) N/oFQ treatment experiments were analysed by means of two-way analysis of variance (ANOVA) with one within-subjects factor (time) and one between subjects factor (treatment). The effect of N/oFQ into the CeA, BNST and BLA was analysed by one-way between subject ANOVA (treatment). Posthoc comparisons were performed by Newman-Keuls tests. For statistical purposes, only data for which correct cannula placement was confirmed by histological analysis were used. For the in situ hybridization studies, statistical analysis was performed by oneway ANOVA followed by Fisher’s PLSD post-hoc test. Statistical significance was set at *P<0.05. **P<0.01 and ***P<0.001. 193 Fig. 1 Histological reconstruction showing correct (filled circles) and incorrect (filled triangles) placements of cannula tips. Data represented in the figure are representative of only some of the animals used in the study and are indicative of the criteria used for identification of the correct injection sites. Drawing is from the atlas of Paxinos and Watson (1998). Results Histology At the end of the experiments, rats were killed with CO2 and perfused transcardially with isotonic saline followed by 3% formalin. Brains were removed, stored in formalin, and subsequently sectioned into 30 m coronal sections using a cryostat. After staining with cresyl violet, sections were examined under a microscope for the location of injector tip placement. An expert observer blind to treatment conditions and behavioral data was used for the histological verification. The analysis revealed that 62 of the 84 rats had injector tips placed in the expected brain sites. Correct cannula placements were found in 21 of 30 rats in the CeA (Fig. 1), 22 of 30 rats in the BLA, and 19 of 24 rats in the BNST. Intracerebroventricular cannulas were in the correct position in all of the animals. 194 Experiment 1: Effect of subchronic ICV injections of N/oFQ on alcohol selfadministration in the msP rats All rats acquired responding reinforced by 10% ethanol and developed stable levels of ethanol-maintained behavior. As shown in Fig. 2A, subchronic (6 days) ICV treatment with N/oFQ (0.5 and 1.0 g/rat), markedly reduced responding on the active lever in the msP rats. Compared with controls ethanol self-administration was reduced by about 40-50%, and statistical analysis revealed a significant overall effect of treatment [F(2,18) = 6.662; P < 0.01]. Moreover, the Newman-Keuls post-hoc test showed a significant difference between controls and animals treated with both doses 0.5 and 1.0 g/rat of N/oFQ (P<0.01). Responses at the inactive lever were almost absent in all treatment conditions and were not affected by N/oFQ treatment [F(2,18) = 2.731; NS] (Fig. 2B). At the end of N/oFQ treatment, the msP rats progressively recovered from the effect of treatment, and ethanol self-administration returned to pretreatment levels within 4 days (Fig. 2A). Fig. 2 Effect intacerebroventricular of subchronic (6 days) treatment with 0.5 and 1.0 g per rat of N/OFQ in the alcoholpreferring msP rats. Data represent the mean (± SEM) number of responses A) at the active and B) at the inactive lever. Difference from vehicle *P<0.05, **P<0.01. 195 Experiment 2: Effect of subchronic ICV injections of N/oFQ on alcohol selfadministration in the Wistar rats All rats acquired responding reinforced by 10% ethanol and developed stable levels of ethanol-maintained behavior. As shown by the analysis of variance, subchronic (6 days) ICV treatment with N/oFQ never affected at none of the doses tested (1.0 and 2.0 g/rat) responding for 10% ethanol self-administration in Wistar rats [F(2,21) = 0.07; NS] (Fig. 3A). Responses at the inactive lever were almost absent in all treatment conditions and were never affected by N/oFQ treatment [F(2,21) = 1.17; NS] (Fig. 3B). Fig. 3 Effect of intacerebroventricular subchronic (6 days) treatment with N/OFQ at the doses of 1.0 and 2.0 g/rat in the nonselected Wistar rats. Data represent the mean (± SEM) number of responses A) at the active and B) at the inactive lever. 196 Experiment 3: Differences on the N/OFQ and NOP receptor gene expression levels between naïve msP and Wistar rats Results, showed a significant increase on the expression of both the N/OFQ and NOP receptor gene levels, in the alcohol-preferring msP rats respect the nonselected Wistars. In particular, as shown in Fig. 4A, an increased expression of the N/OFQ gene was seen in the BNST (P < 0.001) and CeA (P < 0.01). For the NOP receptor, an increase gene expression was found in the BLA (P < 0.001) and again in the CeA (P < 0.001) of the msP respect the nonselected Wistar rats (Fig. 4B). Experiment 4: Effect of microinjections of N/oFQ into the CeA on alcohol selfadministration in the msP rats All rats acquired responding reinforced by 10% ethanol and developed stable levels of ethanol-maintained behaviour. The analysis of variance revealed an overall effect of treatment [F(2,18) = 0.61; P < 0.05]. As shown in Fig. 5A, the Newman-Keuls post-hoc test showed that microinfusion of N/oFQ into the CeA at the dose of 0.25 g per site/rat induced a marked reduction of ethanol self-administration in the msP rats (P<0.05). No statistical difference was observed between control animals and rats injected with 0.125 g per site/rat of N/oFQ (Fig. 5A). Responses at the inactive lever were almost absent in all treatment conditions and were never affected by drug injection [F(2,18) = 0.63; NS] (Fig. 5B). 197 Fig. 4 Densitometric evaluation of in situ hybridization from phosphoimaging plates showing A) N/OFQ and B) NOP gene expression levels in different brain regions of Wistar, msP naive rats. Values are given in nCi/g (mean ± S.E.M.; n = 7-8). Cingulate cortex (cg ctx, Bregma +2.5 mm); frontal cortex (fr ctx, Bregma +2.5 mm); frontoparietal cortex (fp ctx, Bregma –3 mm); bed nucleus of the stria terminalis, lateral division (BSTl, Bregma –0.5 mm); central amygdaloid nucleus (CeA, Bregma -1.8 mm); medial amygdaloid nucleus (MeA, Bregma –3 mm); CA, Cornus Ammon areas (CA1, CA3, Bregma –3 mm); dentate gyrus (DG, Bregma –3 mm); medial preoptic area (MPA, Bregma –0.5 mm); pituitary, anterior part (PIT). Statistical significance was set at: *p<0.05; **p<0.01; ***p<0.001 vs msP rats. 198 Fig. 5 Effect of direct microinjections of N/OFQ (0.125 and 0.25 g per site/rat) into the CeA in the alcohol-preferring msP rats. Data represent the mean (± SEM) number of responses A) at the active and B) at the inactive lever. Difference from vehicle *P<0.05. Experiment 5: Effect of microinjections of N/oFQ into the BLA on alcohol selfadministration in the msP rats All rats acquired responding reinforced by 10% ethanol and developed stable levels of ethanol-maintained behaviour. The ANOVA showed that microinfusion of N/oFQ into the BLA did not affect responding for 10% ethanol in the msP rats, in none of the doses tested [F(2,19) = 0.02; NS] (Fig. 6A). Responses at the inactive lever were almost absent in all treatment conditions and were never affected by drug injection [F(2,19) = 0.09; NS] (Fig. 6B). 199 Experiment 6: Effect of microinjections of N/oFQ into the BNST on alcohol selfadministration in the msP rats All rats acquired responding reinforced by 10% ethanol and developed stable levels of ethanol-maintained behaviour. The ANOVA showed that microinfusion of N/oFQ (0.25 and 0.5 g per site/rat) in the BNST did not affect responding for 10% ethanol in the msP rats [F(2,16) = 0.21; NS] (Fig. 6C). Responses at the inactive lever were almost absent in all treatment conditions and were never affected by drug injection [F(2,16) = 0.83; NS] (Fig. 6D). Fig. 6 Effect of direct microinjections of N/OFQ (0.25 and 0.5 g per site/rat) into the BLA and BNST, in the alcohol-preferring msP rats. Data represent the mean (± SEM) number of responses A) at the active and B) at the inactive lever after microinfusion of N/OFQ into the BLA and, C) at the active and D) at the inactive lever after microinfusion of N/OFQ into the BNST. 200 Discussion In the present study, we showed that ICV treatment with N/oFQ significantly reduced ethanol self-administration in the alcohol-preferring msP rats (as also shown by our previous studies), but had no effect in the nonselected Wistars. An in situ hybridization study was, therefore, undertaken to understand the reasons for the different effect of N/oFQ-NOP receptor system in these two rat strains. The results showed higher level of expression of the N/oFQ gene in the CeA and the BNST, whereas, for the NOP receptor, higher gene expression was found in the CeA and in the BLA of msP rats compared to Wistars. Based on these data, using msP rats, we tested the effect of N/oFQ on alcohol self-administration following direct microinjection of the peptide directly into the CeA, the BLA and the BNST. Results, showed that microinfusion of N/oFQ into the CeA significantly reduced ethanol self-administration in the msP rats. In contrast, intra-BLA or intra-BNST administration of N/oFQ did not modify ethanol self-administration in the msP rats. Ethanol, is known to mediate its positive reinforcing effects by interacting with various neurocircuitries and in a widespread neuroanatomical sites in the brain (Koob et al., 1998). Interestingly, the CeA and in particular the GABAergic activity in this nucleus, plays an important role in modulating important alcohol effects (Koob, 2003, 2004; Koob & Le Moal, 2001). For instance, it has been shown that acute intraperitoneal injection of ethanol increased c-fos immunoreactivity in the CeA and that over 70% of these cells were GABAergic neurons (Morales et al., 1998). Moreover, electrophysiological studies showed an increased GABAergic transmission in the CeA after ethanol superfusion, an effect blocked by treatment with a GABA A receptor antagonist (Roberto et al., 2003). In addition, treatment with GABA A receptor antagonists directly into the CeA significantly reduced ethanol self-administration both in nondependent (Hyytia & Koob, 1995) and dependent rats (Roberts et al., 1996), 201 suggesting the important involvement of this receptor in mediating the reinforcing properties of ethanol within the CeA. Interestingly, previous studies showed that N/OFQ decreases by presynaptic mechanisms GABA release in different brain areas (Meis & Pape, 1998, 2001). More impoirtantly, however, in a very recent study, Roberto et al., (Roberto & Siggins, unpublished observations) by employing electrophysiological techniques, demonstrated that N/oFQ potently inhibits ethanol-induced GABA release, in the CeA. An effect, found to be due to the ability of N/oFQ to inhibit GABA release in this brain area. Therefore, taking into consideration the importance of the GABAergic system in mediating ethanols’ actions especially in the CeA (Roberto et al., 2003; Roberts et al., 1996), a possible mechanism by which N/oFQ may reduce the reinforcing properties of ethanol could be by an inhibition of ethanol-induced GABA release in the CeA. N/oFQ, is also known to act in the brain as functional anti-CRF peptide by blocking CRF-induced anorexia in rats (Ciccocioppo et al., 2001, 2003, 2004b). Interestingly, however, N/oFQ was not able to block Urocortin II, a selective CRF2 receptor agonist, induced anorexia in rats (Fedeli et al., unpublished observations), suggesting, that the anti-CRF effects of N/oFQ are mediated via the CRF1 receptors. In respect to this, of a great interest, are the data by Day et al., (1999) and by Veinante et al., (1997) showing that in the CeA CRF is localized and co-synthesized within GABAergic neurons (Day et al., 1999; Veinante et al., 1997) and, particularly intriguing is a recent paper by Nie et al., (2004) demonstrating that ethanol-induced increased GABAergic transmission in the CeA depends on the CRF1 receptors (Nie et al., 2004). In this study, in fact, ethanol enhanced GABA release in the CeA in the wild-type and CRF2 receptor knockout mice, but not in the in CRF1 knockout mice (Nie et al., 2004). 202 Taking into consideration the data of the present study and the above mentioned recent electrophysiological data, we speculate that N/oFQ reduces ethanol selfadministration by inhibiting ethanol-induced GABA release in the CeA possibly via a functional antagonism at the CeA CRF1 receptors. However, at this point, we cannot exclude interactions with other neurochemical systems or receptors in N/oFQs actions. Further studies are needed to completely clarify the mechanism by which N/oFQ reduces ethanol reinforcing and rewarding properties. In conclusion, the present study suggest an important N/OFQ-CRF-GABAergic interaction in the CeA in mediating ethanols’ positive motivational effects, which may be of fundamental importance in the transition from controlled to excessive, compulsive drinking that is directly linked with the development of alcohol abuse and dependence in vulnerable individuals. Acknowledgements The study was supported by the EU 5th Framework Programme, grant QLRT-2001– 01048 (to RC), the NIH/NIAAA grant AA 10531 (to FW) and by grant MIUR 2002 to (MM). The authors thank Marino Cucculelli for technical assistance and animal care. 203 References Ciccocioppo R, Panocka I, Polidori C, Regoli D, Massi M (1999) Effect of nociceptin on alcohol intake in alcohol-preferring rats. Psychopharmacology 141:220-4. Ciccocioppo R, Angeletti S, Panocka I, Massi M (2000) Nociceptin/orphanin FQ and drugs of abuse. Peptides 21:1071-80. 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Samson HS (1986) Initiation of ethanol reinforcement using a sucrose-substitution procedure in food- and water-sated rats. Alcohol Clin Exp Res 10:436-42. Veinante P, Stoeckel ME, Freund-Mercier MJ (1997) GABA- and peptideimmunoreactivities co-localize in the rat central extended amygdala. Neuroreport 8:2985-9. Weiss F, Lorang MT, Bloom FE, Koob GF (1993) Oral alcohol self-administration stimulates dopamine release in the rat nucleus accumbens: genetic and motivational determinants. J Pharmacol Exp Ther 267:250-8. 206 Chapter 7 Activation of brain NOP receptors attenuates alcohol withdrawal symptoms in rats 207 Activation of brain NOP receptors attenuates alcohol withdrawal symptoms in rats Abstract Alcohol withdrawal, refers to a group of symptoms that may occur from suddenly stopping the use of alcohol after chronic or prolonged ingestion. These symptoms make alcohol abstinence difficult and increase the risk of relapse in recovering alcoholics. In previous studies, we demonstrated that treatment with N/OFQ significantly reduces alcohol consumption and attenuates alcohol-seeking behaviour induced by environmental conditioning factors or by stress, in rats. In the present report, we evaluated whether activation of brain NOP receptors may also attenuate alcohol withdrawal signs in rats. For this reason, animals received an intoxication cycle and, following cessation of alcohol exposure, they were treated either with the endogenous N/OFQ (0.0, 1.0 and 3.0 g/rat, ICV) or with a newly synthesized brain-penetrating NOP receptor agonist, W-212393 (0.0, 0.3 and 1.0 mg/rat, per o.s.). Results, showed a highly significant reduction of withdrawal score in animals treated with both doses of N/OFQ. W-212393, exhibited the same effect but with a minor intensity. A possible explanation for this result, may be the poor oral bioavailability observed with W212393. At present, benzodiazepines represent the mainstay of treatment of alcohol withdrawal with highly positive clinical outcomes. However, some limitations in the efficacy and use of these agents have emerged over years. The discovery of new potential targets for the treatment of alcohol withdrawal syndrome is of considerable practical importance. In our studies, we introduce the N/OFQ-NOP receptor system as a potential target of interest for the treatment of alcohol withdrawal. Key words: NOP receptor, alcohol intoxication, alcohol withdrawal. 208 INTRODUCTION Alcohol withdrawal refers to a group of symptoms that may occur from suddenly stopping the use of alcohol after chronic or prolonged ingestion. Up to 71% of individuals presenting for alcohol detoxification, manifest significant symptoms of alcohol withdrawal (Myrick & Anton, 1998). The most common symptoms include tremor, anxiety, insomnia, agitation, hypervigilance, irritability, and sometimes seizures (Hunter et al., 1974; Saitz, 1998). These symptoms make alcohol abstinence difficult and increase the risk of relapse in recovering alcoholics (Anton, 1999; Spanagel, 2003). Brain hyperexcitability is a hallmark feature of the ethanol withdrawal syndrome (Victor, 1970; Saitz, 1998; Becker, 2000). Under basal conditions, the central nervous system (CNS) is known to maintain neurochemical balance through inhibitory and excitatory neurotransmission. The main inhibitory neurotransmitter in the brain is the aminobutyric acid (GABA), which acts through the GABAA receptor, while, one of the major excitatory neurotransmitters is glutamate, which acts through the NMDA receptor. Dynamic adaptive changes in these systems have been linked to ethanol withdrawal-related CNS hyperexcitability (Dodd et al., 2000; Hoffman, 2003; Krystal et al., 2003; Sanna et al., 2003; Kumar et al., 2004), and GABAA and NMDA receptors have been shown to play an interactive role in various forms of neuronal plasticity relevant to sensitization of ethanol withdrawal, such as kindling and epileptogenic activity (Burnham, 1989; Collingridge & Singer, 1990; Bradford, 1995). Basically, prolonged ethanol exposure, has been reported to result in a reduced density of the GABAA receptor in the brain, as well as in a significant decrease of the activity of these receptors (Kuriyama et al., 1993; Lovinger, 1993; Nevo & Hamon, 1995), leading to a reduced inhibitory efficacy. On the other hand, alcohol intoxication 209 results in up-regulation of different NMDA receptor subunits (Trevisan et al., 1994; Follesa & Ticku, 1996; Hoffman & Tabakoff, 1996; Chandler et al., 1999; Bao et al., 2001) and abrupt cessation of alcohol exposure results in brain hyperexcitability because the NMDA receptors, previously inhibited by alcohol, are no longer inhibited (Grant et al., 1990; Rossetti & Carboni, 1995; Whittington et al., 1995; Thomas & Morrisett, 2000). To this regard, for the treatment of alcohol withdrawal syndrome in humans benzodiazepines are the medication of choice (Saitz et al., 1994; Mayo-Smith, 1997) and they are able to prevent most of the clinical manifestations of this condition. In addition, NMDA receptor antagonists have been reported to markedly reduce ethanol withdrawal signs in rodents (Morriset et al., 1990; Liljequist, 1991; Thomas et al., 1997). Interestingly, N/OFQ has been found to possess (like benzodiazepines) anxiolytic properties and to reduce responsiveness to stress in rodents (Griebel et al., 1999; Jenck et al., 2000a, 2000b; Ciccocioppo et al., 2001). In addition, N/OFQ by acting in the brain as a presynaptic neuron inhibitor has been shown to control, among other, the glutamatergic neurotransmission in different brain areas (Schlicker & Morari, 2000). Therefore, taking into consideration above considerations, the present study was undertaken to evaluate whether activation of the NOP receptor may reduce alcohol withdrawal signs induced by chronic ethanol drinking, in rats. For this reason, the effect of the endogenous N/OFQ and of a newly synthesized brain-penetrating NOP receptor agonist, W-212393 (Teshima et al., 2005), on alcohol withdrawal was evaluated in rats. 210 MATERIALS AND METHODS Subjects Male Wistar rats (Charles River, Calco, Italy) that weighed 300-350 g at the beginning of the experiments were used. The animals were individually housed in a room on a 12 hr light/dark cycle (lights off at 8:00 A.M.) at constant temperature (20– 22°C) and humidity (45–55°). Rats were offered food pellets (4RF; Mucedola, Settimo Milanese, Italy) and tap water ad libitum and were handled once a day for 5 min during the first week after arrival. All procedures were conducted in adherence to the European Community Council Directive for Care and Use of Laboratory Animals. Intracranial surgery For intracranial surgery, animals were anaesthetized by i.m. injection of 100–150 l of a solution containing tiletamine cloridrate (58.17 mg/ml) and zolazepam cloridrate (57.5 mg/ml). A guide cannula for intracerebroventricular (ICV) injections aimed at the left lateral cerebroventricle was stereotaxically implanted and cemented to the skull. The following co-ordinates, taken from the atlas of Paxinos and Watson (1998), were used: antero-posterior = 0.8 mm behind the bregma, lateral = 1.8 mm from the sagittal suture, ventral = 2 mm from the surface of the skull. Drugs Nociceptin/orphanin FQ (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-ArgLys-Leu-Ala-Asp-Glu) was synthesized at the Department of Pharmacological Sciences and Biotechnology Centre (University of Ferrara, Ferrara, Italy), and was a generous gift of Dr. G. Calò. N/oFQ was dissolved in sterile isotonic saline and injected in a 211 volume of 1.0 l per rat into the lateral cerebroventricle (ICV) by means of a stainlesssteel injector 2.5 mm longer than the guide cannula, so that its tip protruded into the ventricle. To verify the cannula placement, immediately before the rat was sacrificed, 1 l of black India ink was injected ICV, and ink diffusion into the ventricles was evaluated using a histological method. W-212393 was provided by (Mitsubishi Pharma Corporation). It was dissolved in 0.3% Tween 80 and distilled water and was administered to the animals per o.s. in a volume of 1 ml/kg. For intoxication, ethanol was given orally (per o.s.) to the animals in a liquid diet containing 25% w/v ethanol, 6% w/v saccharin and powdered milk (Mellin 2, Mellin S.p.A). Ethanol Intoxication In order to maintain a continuous elevation of blood alcohol levels (BLA), animals received per o.s. the liquid diet containing 25% w/v ethanol (see above) as sequent: the first and second intoxication days (days 1 and 2), animals received a total of 21-22 g/kg of 25% w/v ethanol in fractional doses of 2-3 g/kg separated by a 4-h interval over a 24-h period, during following intoxication days (days 3-5) animals received a total of 9-10 g/kg per day in intoxication cycles of 12-h and in fractional doses of 2-3 g/kg per cycle separated by a 4-h interval. Each intoxication day (cycle) was initiated by administering a priming ethanol (25% w/v) dose of 3 g/kg. Withdrawal period was defined as the interval between the administration of the last dose of ethanol and the disappearance of the signs of the ethanol withdrawal syndrome. Starting 7-8 hs after the last dose of ethanol, animals were scored for signs of withdrawal according to the following rating scale (0 = not present; 1 = moderate; 2 212 = severe). Four signs were measured: vocalization, ventromedial limb retraction (VLR), tail rigidity and tail tremors. Withdrawal rating was repeated at intervals of 2 hours. Blood ethanol determination. Blood samples were taken at three time points of the ethanol intoxication phase; on days 2 and 5 (d2 and d5, respectively) one hour after the second dose of ethanol, and on day 4 (d4) immediately before the initiation of the ethanol intoxication cycle. Blood samples were taken from the dorsal tail vein. Experiments Experiment 1: Effect of ICV N/OFQ treatment on alcohol withdrawal signs in Wistar rats To evaluate the effect of N/OFQ treatment on alcohol withdrawal, at the end of the intoxication cycle animals (n=32) were divided into 3 groups (n=8) and received, 10 min before behavioural measures for ethanol withdrawal signs, N/OFQ at the doses of 0.0, 1.0 and 3.0 g/rat (ICV), respectively. Behavioural observations were carried out in all animals at 8, 10, 12 and 24 hours after the last ethanol dose. Experiment 2: Effect of W-212393 treatment per o.s. on alcohol withdrawal signs in Wistar rats To evaluate the effect of W-212393 on alcohol withdrawal the end of the intoxication cycle, animals (n=21) were divided into 3 groups (n=7) and received per 213 o.s. W-212393 at the doses of 0.0, 0.3 and 1.0 mg/kg, respectively. W-212393 was given twice, 2 and 7 hrs after the last ethanol dose. Behavioural observation of the animals was carried out at 8, 10, 12, 14, 16 and 24 hours after the last ethanol dose. The longer time period assumed for behavioural measures in this experiment was due to the longer life time of W-212393. Statistical analysis The effects of drug treatments (N/OFQ or W-212393) on individual withdrawal signs were analysed by a two-way ANOVA, with one within-subjects factor (withdrawal sign) and one between subjects factor (treatment). Post-hoc tests were performed by Tukey test. Statistical significance was set at *P<0.05, **P<0.01 and ***P<0.001. Table 1. Blood Alcohol Levels (BLAs) during alcohol intoxication period BLAs (g/l) Intoxication day N/OFQ group W-212393 group d2 1,82 ± 0,57 (7) 2,10 ± 0,29 (6) d4 0,20 ± 0,22 (6) 0,15 ± 0,12 (6) d5 2,36 ± 0,28 (5) 2,27 ± 0,24 (6) Blood samples from tail vein were obtained as follows: d2) one hour after the second dose of ethanol on intoxication day 2; d4) immediately before the first ethanol dose on day 4; d5) one hour after the first dose of ethanol on intoxication day 5. BLAs were determined by gas chromatography. Numbers in parentheses represent the number of animals employed for blood samples. Results are means ± SEM. 214 Results Experiment 1: Effect of ICV N/OFQ treatment on alcohol withdrawal signs in Wistar rats As shown in Table 1, blood alcohol levels (BAL) were maintained at intoxication levels (Majchrowicz, 1975) during alcohol administration cycles (d2 and d5), while they returned to low levels between intoxication cycles (d4). The analysis of variance showed a significant overall effect of N/OFQ treatment on withdrawal score [F(2,21) = 9.28; P < 0.01]. As shown by post-hoc tests for both N/OFQ doses employed (1.0 and 3.0 g/rat) the total withdrawal score was significantly lower in drug-treated animals respect to vehicles (P < 0.05; P < 0.01, respectively). As to the effects of the peptide on individual withdrawal signs, a significant reduction was seen for both N/OFQ doses tested (1.0 and 3.0 g/rat, ICV) on vocalization at 8 (P < 0.01; P < 0.001, respectively) and 10 hours (P < 0.05) and on tail rigidity at 10 (P < 0.05; P < 0.01, respectively) and 12 hours (P < 0.001) (Fig. 1A,C). On tail tremors, both doses of N/OFQ induced a significant reduction at 10 h (P < 0.05) whereas at 12 h only the higher dose of 3.0 g/rat of the peptide was effective (P < 0.01). No significant effect was found after N/OFQ treatment on withdrawal score for VLR. As shown in fig. 2, at 24 h after withdrawal of ethanol diet ICV treatment with N/OFQ induced a significant overall effect on withdrawal score [F(2,21) = 7.60; P < 0.01]. Post-hoc tests showed a significant effect of both doses, 1.0 and 3.0 g/rat, of N/OFQ treatment (P < 0.05; P < 0.01, respectively). As to the effects on individual withdrawal signs, a significant reduction was seen for both N/OFQ doses (1.0 and 3.0 215 g/rat, ICV) on tail tremors (P < 0.05; P < 0.01, respectively) and tail rigidity (P < 0.05; P < 0.001, respectively) (Fig. 2). Fig. 1. Effect of N/OFQ treatment at the doses of 1.0 and 3.0 g/rat or its vehicle (Veh), on ethanol withdrawal score of: A) Vocalization, B) Ventromedial Limb Retraction (VLR), C) Tail Tremors and, C) Tail Rigidity at 8, 10 and 12 hours after the last ethanol dose. N/OFQ was ICV injected 10 min before each observation time point. At each withdrawal sign was assigned a score 0-3. Values represent the mean (±SEM) of 7 subjects per group. Difference from vehicle *P<0.05, **P<0.01, ***P<0.001. 216 Fig. 2. Effect of N/OFQ treatment (1.0 and 3.0 g/rat, ICV) or its vehicle (Veh), on ethanol withdrawal score 24 h after the last ethanol dose. Values represent the mean (±SEM) of 7 subjects per group. Difference from vehicle *P<0.05, **P<0.01, ***P<0.001. Experiment 2: Effect of W-212393 treatment per o.s. on alcohol withdrawal signs in Wistar rats As shown in Table 1, during alcohol administration cycles (d2 and d5) BALs were maintained at intoxication levels (Majchrowicz, 1975), whereas they returned to low levels between intoxication cycles (d4). The analysis of variance showed a significant overall effect of W-212393 treatment on withdrawal score [F(2,18) = 6.29; P < 0.01]. More detailed post-hoc tests showed a significant effect of W-212393 treatment only with the higher dose of 1.0 mg/kg (P < 0.01). As to the individual withdrawal signs, a significant reduction of score was seen for VLR after treatment with 1.0 mg/kg of W-212393 (P < 0.05) at 10, 12, 14 217 and 16 hours (Fig. 3B). Treatment with 1.0 mg/kg of W-212393 also significantly reduced withdrawal score for tail tremors at 16 h (Fig. 3C). Fig. 3. Effect of treatment with 0.3 and 1.0 mg/kg of W-212393 (per o.s.) or its vehicle (Veh), on ethanol withdrawal score of: A) Vocalization, B) Ventromedial Limb Retraction (VLR), C) Tail Tremors and, C) Tail Rigidity at 8, 10, 12, 14, 16 and 24 hours after the last ethanol dose. W-122393 was administered per o.s. 2 and 7 hs after the last ethanol dose. At each withdrawal sign was assigned a score 0-3. Values represent the mean (±SEM) of 8 subjects per group. Difference from vehicle *P<0.05, **P<0.01. 218 Discussion In our studies we showed that activation of brain NOP receptors either by ICV treatment with the endogenous N/OFQ or by peripheral administration (per o.s.) of the selective brain-penetrating NOP receptor agonist W-212393 (Teshima et al., 2005), significantly attenuated overall withdrawal signs in ethanol intoxicated rats, after cessation of ethanol exposure. In fact, animals treated with N/OFQ immediately before (10 min) behavioural observations, exhibited a withdrawal score significantly lower respect vehicles in most of withdrawal signs measured and in most time points observed. On the other hand, treatment with W-122393 significantly reduced, at the dose of 1.0 mg/kg, overall withdrawal score but a potent and continuous effect of this drug was seen only on ventromedial limb retraction (Fig. 3). A possible explanation for the lower ability of W-212393 to effectively attenuate (like N/OFQ) alcohol withdrawal symptoms may be the poor oral bioavailability of this drug. In fact, in a study by Teshima et al., (2005) a very low occupancy of the brain NOP receptors following per o.s. administration of W-212393 was reported. In this study, in fact, intraperitoneal administration of 3.0 mg/kg of the drug resulted in 40.0 ± 10.5% brain NOP receptor occupancy, whereas, to obtain the same occupancy following oral (per o.s.) administration, doses as high as 10 times superior were required (Teshima et al., 2005). In our studies, however, treatment with 3.0 mg/kg of W-212393 (per o.s.) induced marked sedation to the animals (data not shown, personal observations). Different studies demonstrated an important role of the brain GABAergic and glutamatergic systems in mediating alcohol withdrawal symptoms. In fact, chronic alcohol exposure has been shown to result in a hypoactivity of the GABAergic system (Kuriyama et al., 1993; Lovinger, 1993; Nevo & Hamon, 1995) and to up-regulate the NMDA receptors (Trevisan et al., 1994; Follesa & Ticku, 1996; Hoffman & Tabakoff, 219 1996; Chandler et al., 1999; Bao et al., 2001). Therefore, what is considered to lead to the signs and symptoms of alcohol withdrawal following abrupt cessation of alcohol exposure, is that in the brain of the alcoholic patient, when alcohol is removed acutely, the increased number of excitatory glutamate receptors remains, but without the suppressive GABA effect (Saitz, 1998). Pharmacological data, employing GABAergic agonists (clonazepam, lorazepam etc) or NMDA receptor antagonists (MK-801, CGP 39551 etc) confirmed above considerations (Morriset et al., 1990; Liljequist, 1991; Thomas et al., 1997; Jung et al., 2000; Strzelec & Czarnecka, 2001). In fact, in these reports, pre-treatment with these compounds significantly attenuated ethanol withdrawal symptoms in different animal models of alcohol intoxication. An important role in the physiological and psychological manifestations of alcohol withdrawal is also attributed to the corticotrophin releasing factor (CRF). It has been shown, for instance, an increased neuronal CRF release in the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) during acute withdrawal from ethanol (Merlo-Pich et al., 1995; Olive et al., 2002). Furthermore, antagonism of CRF neurotransmission in the CeA significantly attenuated the behavioral signs of alcohol withdrawal (Rassnick et al., 1993). An important aspect, in regard to the ability of NOP receptor activation (reported in our studies) to attenuate overall withdrawal symptoms in rats, may be that (as mentioned above) a hallmark feature of the ethanol withdrawal syndrome is a general brain hyperexcitability mainly due to an increased glutamatergic neurotransmission (Victor, 1970; Saitz, 1998; Becker, 2000). Treatment with the endogenous N/OFQ, on the other hand, has been shown to result in the brain in a general reduction of presynaptic neuronal excitability and neurotransmitter secretion (Calo’ et al., 2000). Interestingly, one of the main excitatory neurotransmitters the release of which is inhibited by N/OFQ treatment is glutamate (Nicol et al., 1996; Schlicker & Morari, 220 2000; Meis & Pape, 2001). Therefore, a possible mechanism by which activation of brain NOP receptors reduces withdrawal score in rats, could be by an inhibitory action on glutamate neurotransmission. Another important property of the NOP receptor, in regard to withdrawal seizures, are the anxiolytic and anti-stress properties reported following treatment with the endogenous agonist, N/OFQ (Griebel et al., 1999; Jenck et al., 2000a, 2000b; Ciccocioppo et al., 2001). It has been shown, in fact, that ICV injections with N/OFQ increased the time spent in the open arms of an elevated plus maze, as well as, the time spent in the light compartment of a light-dark box, in mice (Griebel et al., 1999; Jenck et al., 1997, 2000a). In addition, knockout mice for the N/OFQ gene consistently show high vulnerability to social stress and impaired adaptation to repeated stress in comparison to wild-type mice (Koster et al., 1999). Moreover, in previous studies conducted in our laboratory we demonstrated that, in rats, ICV treatment with N/OFQ blocked restraint-, footshock stress- and CRF-induced anorexia and prevented stressinduced reinstatement of alcohol-seeking behaviour (Martin-Fardon et al., 2000; Ciccocioppo et al., 2001). Lastly, this peptide prevented the increase in plasma corticosterone induced by intracerebral injections (Le Cudennec et al., 2002). Therefore, another possible approach by which, in our study, activation of brain NOP receptor reduced overall alcohol withdrawal symptoms, may be by an anxiolytic and/or an anti-CRF mechanism. In fact benzodiazepines are, in recent days, the medication of choice for the treatment of alcohol withdrawal (Saitz et al., 1994; Mayo-Smith, 1997), whereas CRF antagonists have been reported to significantly attenuate alcohol withdrawal symptoms in rats (Rassnick et al., 1993). As mentioned above, at present, benzodiazepines represent the mainstay of treatment of alcohol withdrawal (Saitz et al., 1994; Mayo-Smith, 1997). However, even though positive clinical outcomes are reported, some problematic following treatment 221 with these agents should be mentioned. For instance, the use of benzodiazepines is associated with several side effects, such as risk of excess sedation, memory deficits and respiratory depression in patients with liver impairment, as is often the case in alcoholics (Mayo-Smith, 1997). Moreover, these agents act by increasing (like alcohol) the effects of GABAergic neurotransmission in the brain and may serve, therefore, as a substitute. In addition, benzodiazepines could have addictive properties, which constitutes a limitation to their use in subjects affected by substance abuse disorders (Ross, 1993; Mayo-Smith, 1997). Consequently, the discovery of new potentially useful drugs for the treatment of alcohol withdrawal syndrome is of considerable practical importance. In our studies, we showed that activation of brain NOP receptors significantly reduced overall withdrawal signs induced by cessation of alcohol exposure, in alcohol intoxicated rats. Yet more studies have to be performed in order to better understand the way of action for this effect. However, brain-penetrating peripherally administered selective NOP receptor agonists, may offer important advantages respect to classic treatments in alcohol withdrawal, such as benzodiazepines. For instance, treatment with N/OFQ has been reported to antagonize alcohol’s motivational properties (Ciccocioppo et al., 1999, 2004). Moreover, N/OFQ is devoid of intrinsic motivational properties per se, and therefore may possess low abuse potential (Devine et al., 1996; Ciccocioppo et al., 1999). Acknowledgments This study was supported by the European Union’s Fifth Framework Program, grant QLRT-2001-01048 (to MH and RC); National Institute on Alcohol Abuse and Alcoholism, grant AA01435 (to FW subcontract to RC); and by a Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) grant 2004 to (MM). We wish to thank Mr. Marino Cucculelli for his skilful technical assistance. 222 References Anton RF (1999) What is craving? Models and implications for treatment. Alcohol Res Health 23:165-73. Bao X, Hui D, Naassila M, Michaelis EK (2001) Chronic ethanol exposure increases gene transcription of subunits of an N-methyl-daspartate receptor-like complex in cortical neurons in culture. Neurosci Lett 315:5-8. Becker HC (2000) Animal models of alcohol withdrawal. Alcohol Res Health 24:10513. Bradford HF (1995) Glutamate, GABA and epilepsy. Progr Neurobiol 47(6):477-511. Burnham WM (1989) The GABA hypothesis of kindling: recent assay studies. Neurosci Biobehav Rev 13(4):281-8. 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Victor M (1970) The alcohol withdrawal syndrome: theory and practice. Postgrad Med 47:68-72. Whittington MA, Lambert JDC, Little HJ (1995) Increased NMDA receptor and calcium channel activity underlying ethanol withdrawal hyperexcitability. Alcohol Alcohol 30:105-14. 228 Chapter 8 Summary, conclusions and suggestions for further research 229 Summary, conclusions and suggestions for further research The work performed in the present thesis was focused on investigating the importance of the NOP receptor as a potential target in the treatment of alcohol abuse. For this reason, the effect of treatment with the endogenous N/OFQ, as well as, of different NOP receptor agonists, in various alcohol-abuse related behaviors was studied in rats. Currently, only three medications -disulfiram, naltrexone, and acamprosate- have been approved by the U.S. Food and Drug Administration (FDA) for use in the treatment of alcohol dependence. Disulfiram, however, has been shown to have significant adverse effects and compliance difficulties with no clear evidence to increase abstinence rates, decrease relapse rates, or reduce cravings (Kiefer & Mann, 2005; Williams, 2005). Naltrexone, has been found, when combined with psychosocial treatments, to decrease drinking days, and relapse rates in alcohol dependent patients (Kiefer & Mann, 2005; Williams, 2005). However, clinical data indicate that naltrexone may have adverse side effects and can give compliance problems (Kosten & Kleber, 1984; Rohsenow et al., 2000; Rabinowitz et al., 1997, 2002). Consistently, in rats, naltrexone administration results in development of conditioned place and taste aversion and in stress-like reactions. In addition, this drug did not block stress-induced alcohol-seeking behaviour in rats (Liu & Weiss, 2002). Treatment with acamprosate, on the other hand, was effective in maintaining abstinence in alcoholics and reduced shortterm and long-term (more than six months) relapse rates, when combined with psychosocial treatments (Johnson & Ait-Daoud, 2000; Kiefer & Mann, 2005). In 230 animal studies, acamprosate reduced ethanol consumption (Czachowski et al., 2001; Olive et al., 2002), blocked cue-induced alcohol-seeking (Batcheler et al., 2005) and attenuated some ethanol withdrawal symptoms in rats (Spanagel et al., 1996). To my knowledge, no studies have until now examined the effect of this drug on alcohol reinstatement induced by stress in rats. However, in a study by Spanagel et al., (1998) treatment with acamprosate did not modify stress-induced heroin-seeking behaviour (Spanagel et al., 1998). On the other hand, CRF receptor antagonists have been reported to reduce anxiety-like behaviour observed in ethanol-withdrawn rats (Rassnick et al., 1993; Koob et al., 1998) and to attenuate foot shock-induced reinstatement of ethanolseeking (Le et al., 2000). These agents, however, had no effect on cue-induced reinstatement (Liu & Weiss, 2002). The data, obtain in this thesis are of great importance because, we showed that activation of brain NOP receptors significantly reduces, in the msP rats, ethanol intake (under both voluntary and operant conditions), as well as, reinstatement of ethanolseeking behavior induced either by environmental conditioning factors or by stress. Moreover, activation of NOP receptors also significantly reduced alcohol withdrawal symptoms in rats. Overall data, could suggest that the activation of brain NOP receptors could reduce, in alcoholics, relapse risk induced by environmental factors or stress, but also relapse rates induced in the abstinent alcoholics, because of the aversive affective symptoms experienced during withdrawal. Moreover, because most situations encountered by alcoholics are likely to be both stressful and to have cues strongly associated with drinking, NOP receptor agonists might be expected to have greater therapeutic efficacy than either naltrexone or CRF antagonists alone. In fact, animal data, suggests that in situations in which both stress- and alcohol-paired cues are present, neither naltrexone nor the CRF receptor antagonist, D-Phe-CRF, are sufficient to prevent reinstatement when they are administered alone (Liu & Weiss, 2002). 231 N/OFQ, though, by having a modulatory (inhibitory) effect on the regulation of brain dopamine and opioid function, on the one hand, and of brain CRF activity, on the other hand, could be expected to have greater therapeutic efficacy than either naltrexone or CRF antagonists alone. Furthermore, in previous studies conducted in our laboratory we demonstrated, by employing the condition place preference as a model, that N/OFQ is devoid of motivational properties per se (Ciccocioppo et al, 1999; Devine et al., 1996). This property of N/OFQ is of great importance because naltrexone, that in patients can give compliance problems, induces conditioned place and taste aversion in rats (Kosten & Kleber 1984; Rohsenow et al., 2000; Rabinowitz et al. 1997, 2002). CLINICAL IMPLICATIONS The N/OFQ-NOP receptor system, may represent numerous advantages respect other targets currently employed for the treatment of alcohol abuse. In fact, activation of the NOP receptor by the endogenous N/OFQ, has been found to reduce, in rats, ethanol consumption (Chapter 3 and 5), cue- and stress-induced seeking behavior (Chapter 5), as well as to attenuate alcohol withdrawal symptoms (Chapter 7). Moreover, N/OFQ is devoid of motivational properties per se (Ciccocioppo et al, 1999; Devine et al., 1996). On the other hand, naltrexone does not block stress-induced relapse in rats. Moreover, this drug, by being an opioid antagonist, induces aversion and can therefore give compliance problems (Kosten & Kleber, 1984; Rohsenow et al., 2000; Rabinowitz et al., 1997, 2002). In addition, naltrexone treatment instead of ameliorate could further exacerbate ethanol withdrawal symptoms. Acamprosate, has never being reported, to my knowledge, to reduce stress-related behaviours. 232 Overall considerations, suggest that agents targeting the brain NOP receptor may not only have pharmacotherapeutic potential for treatment of alcoholism, but could also offer various advantages over other classes of drugs. On the basis of the results obtained in the present thesis, it may be interesting to extend these observations to other animal models of alcohol abuse, and to develop metabolic stable brain penetrating agents that can be administered peripherally. Interestingly, in regard to this, as shown in Chapter 4, the analgesic drug, buprenorphine, that has recently been approved in the maintenance treatment of opioid dependence, at higher doses reduced, in rats, voluntary alcohol intake. This effect of buprenorphine, as we demonstrated (Chapter 4), was due to its agonistic properties to the NOP receptor. FUTURE PERSPECTIVES We suggest that agents targeting brain NOP receptors may represent a promising potential for treatment of alcohol abuse, and we alert the necessity and exigency of developing brain-penetrating peripherally administered selective NOP receptor agonists. 233 References Bachteler D, Economidou D, Danysz W, Ciccocioppo R, Spanagel R (2005) The effects of acamprosate and neramexane on cue-induced reinstatement of ethanolseeking behavior in rat. Neuropsychopharmacology 30(6):1104-10. Ciccocioppo R, Panocka I, Polidori C, Regoli D, Massi M (1999) Effect of nociceptin on alcohol intake in alcohol-preferring rats. 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Le AD, Harding S, Juzytsch W, Watchus J, Shalev U, Shaham Y (2000) The role of corticotrophin-releasing factor in stress-induced relapse to alcohol-seeking behavior in rats. Psychopharmacology 150:317-24. Liu X, Weiss F (2002) Additive effect of stress and drug cues on reinstatement of ethanol seeking: exacerbation by history of dependence and role of concurrent 234 activation of corticotropin-releasing factor and opioid mechanisms. J Neurosci 22:7856-61. Olive MF, Nannini MA, Ou CJ, Koenig HN, Hodge CW (2002) Effects of acute acamprosate and homotaurine on ethanol intake and ethanol-stimulated mesolimbic dopamine release. Eur J Pharmacol 437(1-2):55-61. Rabinowitz J, Cohen H, Tarrasch R, Kotler M (1997) Compliance to naltrexone treatment after ultra-rapid opiate detoxification: an open label naturalistic study. Drug Alcohol Depend 47:77–-86. Rabinowitz J, Cohen H, Atias S (2002) Outcomes of naltrexone maintenance following ultra-rapid opiate detoxification versus intensive inpatient detoxification. Am J Addict 11:52-6. Rassnick S, D’Amico E, Riley E, Koob GF (1993) GABA antagonist and benzodiazepine partial inverse agonist reduce motivated responding for ethanol. Alcohol Clin Exp Res 17:124-30. Rohsenow DJ, Colby SM, Monti PM, Swift RM, Martin RA, Mueller TI, Gordon A, Eaton CA (2000) Predictors of compliance with naltrexone among alcoholics. Alcohol Clin Exp Res 24(10):1542-9. Spanagel R, Putzke J, Stefferl A, Schobitz B, Zieglgansberger W (1996) Acamprosate and alcohol: II. Effects on alcohol withdrawal in the rat. Eur J Pharmacol 305(13):45-50. Spanagel R, Sillaber I, Zieglgansberger W, Corrigall WA, Stewart J, Shaham Y (1998) Acamprosate suppresses the expression of morphine-induced sensitization in rats but does not affect heroin self-administration or relapse induced by heroin or stress. Psychopharmacology 139(4):391-401. Williams SH (2005) Medications for Treating Alcohol Dependence. American Family Physician 72(9):1775-80. 235 SCIENTIFIC PUBLICATIONS OF DAINA ECONOMIDOU 1. Economidou D, Hansson A, Fedeli A, Massi M, Heilig M, Ciccocioppo R. The Central Nucleus of the Amygdala is the neuroanatomical site of action for the effects of N/oFQ on alcohol drinking. J Neurosc Manuscript submitted for publication 2. Economidou D, Fedeli A, Massi M, Ciccocioppo R. Effect of novel NOP receptor ligands on ethanol drinking in the alcohol-preferring msP rats. Peptides Manuscript submitted for publication 3. Economidou D, Ubaldi M, Lourdusamy A, Massi M, Ciccocioppo R. Activation of brain NOP receptors attenuates alcohol withdrawal symptoms in rats. Manuscript 4. Ciccocioppo R, Economidou D, Rimondini R, Sommer W, Massi M, Heilig M. Buprenorphine reduces alcohol drinking through activation of the NOP receptors. Biol Psychiatry Mar 11, 2006 [Epub ahead of print] 5. Economidou D, Policani F, Angellotti T, Massi M, Terada T Ciccocioppo C. Effect of novel NOP receptor ligands on food intake in rats. Peptides Feb 14, 2006 [Epub ahead of print]. 6. Economidou D, Mattioli L, Cifani C, Perfumi M, Massi M, Cuomo V, Trabace L, Ciccocioppo R. Effect of the cannabinoid CB(1) receptor antagonist SR141716A on ethanol self-administration and ethanol-seeking behaviour in rats. Psychopharmacology 183(4):394-403, 2006. 7. Bachteler D*, Economidou D*, Danysz W, Ciccocioppo R, Spanagel R. The effects of Acamprosate and Neramexane on cue-induced reinstatement of ethanol-seeking behavior in rat. Neuropsychopharmacology 30(6):1104-10, 2005. 8. Ciccocioppo R, Cippitelli A, Economidou D, Fedeli A, Massi M. Nociceptin/OrphaninFQ acts as a functional antagonist of corticotropinreleasing factor to inhibit its anorectic effect. Physiol Behav 82: 63-8, 2004. 236 9. Ciccocioppo R, Economidou D, Fedeli A, Angeletti S, Massi M. Attenuation of Ethanol Self-Administration Reinstatement of Alcohol-Seeking Behaviour by the Nociceptin/OrphaninFQ in Alcohol-Preferring Rats. 172:170-8, 2004. 10. Ciccocioppo R, Fedeli A, Economidou D, Policani F, Weiss F, Massi M. The bed nucleus is a neuroanatomical substrate for the anorectic effect of corticotropin-releasing factor and for its reversal by nociceptin/orphanin FQ. J Neurosci 23: 9445-51, 2003. 11. Ciccocioppo R, Economidou D, Fedeli A, Massi M. The nociceptin/orphanin FQ/NOP receptor system as a target for treatment of alcohol abuse: a review of recent work in alcohol-preferring rats. Physiol Behav 79:121-8, 2003. 12. Fedeli A, Ciccocioppo R, Economidou D, Angeletti S, Massi M. Autoradiografic analysis of 5-hydroxytryptamine 5-HT2A binding sites in the rat brain after chronic intragastric ethanol treatments. Res Commun Mol Pathol Pharmacol 112:113-27, 2002. 237 Weiss F, Heilig, M, and of Conditioned Antiopioid Peptide Psycopharmacology Thank You! This work was carried out at the Department of Experimental Medicine and Public Health, University of Camerino, Camerino, Italy. I wish to express my sincere gratitude to all those who in different ways have contributed to the realization of this work. I would especially like to mention and thank: First of all, my supervisors Associate Professor Roberto Ciccocioppo and Professor Maurizio Massi. Thank you for introducing me into research! For guiding me into the field of neuroscience and animal behavior and for always providing me with distinct scientific support. Extra many thanks for your endless care and encouragement and not least for your trust in me. Roberto, you’ve been inspiring in many ways and you’ve generated in me enthusiasm and passion for research. I will always be grateful that I had you as a mentor. Thank you! Massimo Ubaldi and Anbarasu Lourdusamy, thank you for your generous statistical advises. Massimo, many many thanks for always being available and for stopping me -different times- from throwing my computer through the window. Marino Cuculleli, Mariangela Fiorelli and Rina Righi, the animal caretakers at the University of Camerino. Many thanks for all your help with the animals and for your contribution to a pleasant working atmosphere. Marino, I’m grateful I had the opportunity to work with you. 238 Marino Cuculleli and Alfredo Fiorelli, thank you for all the technical assistance and advices regarding animal experiments. All the members of the Dpt. of Experimental Medicine and Public Health. With the risk of forgetting someone, I don’t mention you specifically. Thank you for all your help. All the guys -from different laboratories- here in our Department for being nice fellows. Special thanks to Amalia Fedeli and Laura Mattioli for being (as they are always) helpful and good friends. All the students who have helped me during these years. Back to Cyprus… my best friends (my four high-school classmates!)!!Even though I haven’t always been the best friend, but in the contrary, I’ve mostly been away in the past years…thank you for always been there for me! You are the best!!! All my friends in Italy!!For reminded me that there is a life outside the lab. Gèza, for all your love and support through these years… I’m fortunate to have you… My family, for all the trust and care you have given me and for always being there for me. Thank you! 239