Download 3. Results

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
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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
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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
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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%)
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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 GABR3 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).
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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
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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).
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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
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(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.
Therefore we thought of great importance (Chapter 7) to investigate whether activation
of brain NOP receptors could attenuate overall withdrawal signs in ethanol intoxicated
rats, after cessation of ethanol exposure. For this reason, the severity of withdrawal
symptoms was measured in rats after treatment with the endogenous N/OFQ or the
newly synthesized selective NOP receptor agonist, W-212393 (Chapter 7).
The main findings and possible implications of this thesis are summarized and
discussed in Chapter 8.
38
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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).
On the basis of the results so far obtained, it may be interesting to extend these
observations to other animal model of alcohol abuse, and possibly to develop metabolic
stable brain penetrating agents that can be administered peripherally.
Acknowledgements
The work was supported by Grant (COFIN 2002) from MIUR, Rome, Italy.
The financial support of NIAAA in occasion of the symposium on ‘‘Peptides: Their Role
in Excess Alcohol Drinking and Their Promise as a Therapeutic Tool’’ held at the SSIB
meeting in Santa Cruz, CA, 7–11 August 2002, is gratefully acknowledged.
85
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Chapter 3
Effect of novel NOP receptor ligands
on ethanol drinking in the alcoholpreferring msP rats
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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
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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.
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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. 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).
110
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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
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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.
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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.
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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
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150
agonist
into
the
nucleus
accumbens.
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
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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
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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.
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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.
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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.
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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.
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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.
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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
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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
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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)
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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
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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. 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).
174
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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
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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
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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.
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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 1g 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
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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
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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
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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