Cell bodies of the neurons forming the major ascending dopaminergic pathways are located in the brainstem, in the substantia nigra pars compacta (SNc, A9) and ventral tegmental area (VTA, A10) (Björklund and Lindvall 1984). The A9 neurones project mainly to the caudate-putamen or to the dorsal striatum forming the nigrostriatal dopamine system (Björklund and Lindvall 1984; Fuxe et al. 1985) (Fig. 2.1.). A minor component of the nigrostriatal system projects from the A8 area to the ventral putamen. The mesolimbic dopaminergic pathway is formed by neurons that project from the A10 area to the ventral striatum, i.e. the nucleus accumbens, olfactory tubercle and other limbic regions such as amygdala and hippocampus and septum. In addition, the A10 area sends axons to the cortical areas, e.g. to the medial, prefrontal, entorhinal and cingulate cortex, which system is called the mesocortical dopaminergic pathway (Björklund and Lindvall 1984; Fuxe et al. 1985).
In these brain regions, dopamine binds to two main dopamine receptor subtypes, namely D1- and D2-like receptors, both groups having subtypes, i.e. D2, D3 and D4 belonging to the D2 family and D1 and D5 belonging the D1 family (Jackson and Westlund-Danielsson 1994). Both receptor families are G-protein-coupled, seven times cell membrane-spanning receptors, D1-type receptors being positively and D2-type receptors negatively coupled to adenylyl cyclase. The distribution of the subtypes within the striatum and also throughout the projection areas of dopaminergic pathways differs (Jaber et al. 1996).
Figure 2.1. Simplified presentation of the ascending dopaminergic pathways originating from the substantia nigra pars compacta (SNc, A9) and the ventral tegmental area (VTA, A10) and some of their main projection areas.
The D1 receptor is the most ubiquitous dopamine receptor. In addition to dorsal and ventral striatum, it is expressed in several limbic regions, hypothalamus and thalamus (Jaber et al. 1996). In the striatum D1 receptors are expressed mainly in the GABAergic medium-size spiney neurons projecting to the SNr. In contrast the D5 receptor is expressed at a lower level and its mRNA is detected in the hippocampus and some thalamic nuclei.
D2 receptors are expressed in the dorsal and ventral striatum in the GABAergic neurons together with enkephalins. D3 receptors, on the other hand are found mainly in the ventral striatum, i.e. nucleus accumbens and olfactory tubercle. The expression of D3 receptors is low in the dorsal striatum.. The expression of D4 receptor is low in the basal ganglia but high in the areas such as frontal cortex, amygdala and hypothalamus (Jaber et al. 1996).
Since dopamine was found to be concentrated especially into the striatum, a brain area known to participate in the extrapyramidal motor activity, it was suggested that dopamine would be involved in the regulation of motor functions (Carlsson 1959). Furthermore, it was suggested and later confirmed that deficient dopaminergic function would underlie the symptoms of Parkinson's disease.
By means of pharmacological and lesion studies it has been found that blockade and activation of nigrostriatal, mesolimbic and mesocortical projections, produce differential changes in spontaneous and drug-induced motor activity (Jackson et al. 1975b; Kelly et al. 1975; Pijnenburg and Rossum 1973). Activation of nigrostriatal dopaminergic transmission by locally applied dopamine (Jackson et al. 1975b), amphetamine (indirect DA receptor agonist) or apomorphine (direct agonist) (Kelly et al. 1975) induce mainly stereotyped behaviours, e.g. sniffing, grooming, oral movements but not locomotion. In contrast, selective activation of the accumbal dopaminergic transmission enhances locomotor activity but does not cause stereotyped behaviour (Jackson et al. 1975a; Kelly et al. 1975; Pijnenburg and Rossum 1973). Furthermore, lesioning nigrostriatal pathway abolishes amphetamine-induced stereotyped behaviour, while it enhances amphetamine-induced locomotor stimulation. Selective lesions of mesolimbic system causes opposite changes in the effects of amphetamine (Kelly et al. 1975).
In the caudate-putamen, the dopaminergic neurons make synaptic contacts with medium-size spiny neurons that are GABAergic but use also enkephalin, substance P and dynorphin as neurotransmitters (Gerfen 1992; Graybiel 1990). Dopamine is thought to serve as a modulator of glutaminergic signalling from the cortical areas through the basal ganglia (Mogenson 1987). Critical efferents from dorsal striatum involved in the regulation of motor activity are the GABAergic neurons projecting directly or indirectly via globus pallidus and subthalamic nucleus to the SNr (Figure 2.2.). The SNr, in turn, sends afferents to the thalamus. These project to the cortical areas innervating the dorsal striatum, closing the cortico-striato-pallido-thalamic loop (Alexander and Crutcher 1990; Gerfen 1992). This circuitry is believed to be central in the modulation of extrapyramidal motor processes and it is disturbed in Parkinson's disease, symptoms of which include tremor, rigidity and difficulties in the initiation of motor actions (Carlsson and Carlsson 1990; Fuxe et al. 1985; Gerfen 1992).
Figure 2.2. Schematic presentation of the brain structures and their connections between them involved in the regulation of psychomotor and reinforcement processes (modified from Gerfen 1992; Pulvirenti et al. 1991; Robbins and Everitt 1996)). CPu = caudate-putamen, GP = globus pallidus, Th = thalamus, STh = subthalamic nucleus, PPTg = Pedunculopontine Tegmental nucleus, SNc = substantia nigra pars compacta, VTA = ventral tegmental area, PCF = prefrontal cortex, VP = ventral pallidum, B = nucleus basalis of Meynert, LH = lateral hypothalamus, AcbSh = nucleus accumbens, shell; AcbC = nucleus accumbens core.
Similarly to the caudate-putamen, the nucleus accumbens receives glutamatergic afferents from some cortical areas and also from the amygdala and hippocampus (Heimer et al. 1995). Stimulation of inputs from the amygdala and hippocampus induces locomotor stimulation, which can be blocked with dopamine receptor antagonists (Mogenson 1987). This effect of glutamatergic activation can be enhanced by direct application of dopamine into the nucleus accumbens.
Similarly to the dorsal striatum, accumbal dopamine is believed to have a gating function, i.e. it regulates the information flow from the limbic structures such as amygdala and hippocampus to the motor nuclei (Mogenson 1987). The nucleus accumbens sends afferents to the VP, which, in turn projects, to the medial prefrontal cortex (MPC), thalamus (Th), pedunculopontine tegmental nucleus (PPTg), which is a part of the mesencephalic locomotor region (Schaefer and Michael 1987), the medial subthalamic nucleus and SN. In the nucleus accumbens, dopamine-induced hyperactivity is thought to be caused by its inhibitory effect on GABAergic neurons projecting to the VP, so that the release of GABA is decreased following activation of dopamine receptors in the nucleus accumbens (Bourdelais and Kalivas 1990; Inglis et al. 1994; Mogenson 1987). This is thought to result in the disinhibition neurons projecting to the mediodoral thalamus to enhance locomotor activity (Pulvirenti et al. 1991). Another output from the VP is a pathway to the PPTg, a brain area which could further mediate the information from forebrain structures to the spinal cord and modulate the motor behavior (Mogenson 1987).
The prefrontal cortex (PFC) is involved in the various cognitive processes such as regulation of working memory and focused attentione and these functions are modulated by mesocortical dopamine pathway (Barkley 1998; Le Moal and Simon 1991). However, dopamine in the PFC plays a role in locomotor activity and reinforcement as well. For instance, attenuation of dopaminergic tone in the PFC by 6-OHDA lesion increases the stimulatory effects of amphetamine (Carter and Pycock 1980). These effects have been suggested to result from disinhibition of the excitatory glutamatergic afferents projecting from the PFC to the ventral tegmental area and nucleus accumbens (Taber and Fibiger 1995). Furthermore, rats self-administer cocaine into the PFC indicating that this brain area could be an important part of the brain reward circuitry (Goeders and Smith 1983) .
Dopamine, like other catecholamine neurotransmitters, is synthesized from the amino acid precursor, tyrosine, which has to be taken up through the blood brain barrier by a transporter into the dopaminergic cells (Cooper et al. 1986). The first step in the synthesis of catecholamines is the hydroxylation of tyrosine to DOPA, by tyrosine hydroxylase, which is also the rate limiting enzyme in the synthetic cascade (Fig. 2.3.).
In the cytoplasm of cells, DOPA decarboxylase transforms DOPA to dopamine, which is then carried by another active transporter to synaptic vesicles, where the molecules are protected from catabolizing enzymes. The synthesis rate of the dopamine is dependent on the activity of the tyrosine hydroxylase, an enzyme which is under the control of many and complex mechanisms (Feldman et al. 1997). The main short-term regulatory factors are end-product inhibition, firing rate of the neuron and autoreceptors located in the nerve-endings. The end-product of the dopaminergic neurons, dopamine, decreases the affinity of the enzyme's pteridine co-factor for tyrosine hydroxylase, which results in a decrease of enzyme activity. All of the above mentioned mechanisms, as well as many other factors (Feldman et al. 1997), regulate the phosphorylation state of tyrosine hydroxylase, which is the major factor in controlling its activity.
Figure 2.3. Simplified presentation of the synthesis of dopamine and its major metabolic routes. COMT = catechol-O-methyltransferase, MAO = monoamine oxidase, 3-MT = 3-methoxytyramine, HVA = homovanillic acid, DOPAC = 3,4-dihydroxyphenylacetic acid.
Inside the dopaminergic cells the cytosolic dopamine is metabolized mainly by two successive reactions (Fig. 2.3. and Fig. 2.4.). First, monoamine oxidase (MAO) transforms dopamine to a corresponding aldehyde which then can serve as a substrate for aldehyde dehydrogenase to produce 3,4-dihydroxyphenylacetic acid (DOPAC). DOPAC then diffuses out of the cells and can be either conjugated to glucuronides or transformed to homovanillic acid (HVA) by catechol-O-methyltransferase (COMT) (Männistö et al. 1992; Westerink 1979; Westerink 1985). A portion of MAO is located outside of the dopaminergic neurons, i.e. in the glial cells, while COMT is found only outside dopaminergic neurons. A fraction of the released dopamine, the size of which varies in different brain areas (Karoum et al. 1994) is first O-methylated by COMT to 3-methoxytyramine (3-MT) and then oxidized by MAO to form HVA (Männistö et al. 1992; Westerink 1985; Wood and Altar 1988).
Since COMT does not exist inside the dopaminergic neurons (Kaakkola et al. 1987; Karhunen et al. 1995; Rivett et al. 1983; Roth 1992), all 3-MT found in the brain should be derived from dopamine that is released from the nerve endings (fig 2.4.).
Based on this scheme, it has been suggested that the 3-MT concentration in the brain tissue (Di Giulio et al. 1978; Kehr 1976; Kehr 1981; Wood and Altar 1988) or interstitial fluid (Brown et al. 1991; Kuczensky and Segal 1992) would be an indicator of dopamine release. Indeed, it has been shown that 3-MT levels in brain are elevated during electrical stimulation of the dopaminergic neurons and after drug treatments that increase dopamine release (Wood and Altar 1988).
Fig 2.4. Metabolism of dopamine in the dopaminergic nerve terminal and synapse. DA = dopamine, COMT = catechol-O-methyltransferase, MAO = monoamine oxidase, 3-MT = 3-methoxytyramine, HVA = homovanillic acid, DOPAC = 3,4-dihydroxyphenylacetic acid.
Methods such as in vivo microdialysis and voltammetry have enabled the measurement of concentrations of extracellular neurotransmitters, such as dopamine, in awake, freely moving animals (Lane and Blaha 1986, Di Chiara 1990). One major benefit of these methods is that they allow comparison of drug-induced changes in dopamine release as a function of time in the same animal, which is not possible when post mortem tissues are studied. However, utilization of post mortem metabolite analysis from brain samples has some advantages over the methods mentioned above. For instance, measurement of dopamine metabolites in post mortem tissues permits the examination of dopaminergic activity simultaneously in several brain regions.
Measurement of DOPAC and HVA concentrations have been also used to assess the activity of dopaminergic neurons (Di Giulio et al. 1978; Roffler-Tarlov et al. 1971; Westerink 1985; Wood and Altar 1988), but it is evident that after certain drug treatments dopamine release and formation of these acidic metabolites become dissociated (Miu et al. 1992). Since MAO exist also inside the dopaminergic cells, a large portion of DOPAC is formed from newly synthesised dopamine without release of dopamine to the synaptic cleft. This may occur especially during elevated dopamine synthesis. For instance, decreasing the impulse flow in the dopaminergic neurons by infusion of tetrodotoxin into the medial forebrain bundle elevates DOPAC and HVA concentrations due to increased dopamine synthesis, although it decreases dopamine release (Miu et al. 1992). On the contrary, amphetamine, cocaine and other dopamine reuptake inhibitors induce a robust increase of extracellular dopamine levels but decrease concentrations of DOPAC and HVA (Brown et al. 1991; Hurd et al. 1989; Westerink and Spaan 1982).
Utilisation of tissue 3-MT in measurement of dopamine release is complicated by the rapid and massive accumulation of 3-MT in brain after death (Carlsson et al. 1974; Damsma et al. 1990; Gonzales-Mora et al. 1989; Phebus et al. 1986), which may interfere with drug-induced changes. In order to prevent the effect of post mortem changes, experimental animals have been sacrificed with brain-focused microwave irradiation, which rapidly denaturate brain enzymes and prevents the formation of 3-MT by inactivating COMT (Blank et al. 1983; Ikarashi et al. 1985; Lenox et al. 1979). Another approach has been rapid freezing of brain tissue, which, however, is not so suitable at least when rats are being used as experimental animals (Carlsson et al. 1974; Di Giulio et al. 1978; Ikarashi et al. 1984; Westerink 1979).
However, despite the use of the microwave technique, the validity of measurement of steady-state 3-MT, especially in the assessment of increased dopamine release, has been questioned in some situations. In studies on the effects of neuroleptics on dopamine metabolism, drugs such as haloperidol, which blocks the pre- and postsynaptic dopamine receptors and thus increase dopamine release, have sometimes clearly increased 3-MT concentrations (Gropetti et al. 1978; Karoum and Egan 1992), induced only transient increase (Ponzio et al. 1981; Westerink and Spaan 1982) or even decreased 3-MT levels (Waldmeier et al. 1981). Similarly, µ-opioid receptor agonists have caused variable effects on 3-MT levels in rat striatum (Wood and Rao 1991; Yonehara and Clouet 1984). Drug-induced changes in extracellular 3-MT levels collected by microdialysis, however, are qualitatively similar to changes in extracellular dopamine indicating that 3-MT levels in brain do reflect dopamine release (Brown et al. 1991; Kuczensky and Segal 1992; Wood et al. 1988; Yonehara and Clouet 1984).
Extensive research during recent decades has revealed that dopaminergic an mechanisms are important part of the brain reward circuitry (Wise and Rompré 1989). These neuronal pathways are believed to mediate the reinforcing effects of different stimuli which exert control over behaviour. In operant psychology, a reinforcer is defined as a stimulus that increases the probability of operant or instrumental responses that precede or are contingent upon the stimulus (Robbins and Everitt 1996; Stolerman 1992). One example is animals which can learn to press a lever in order to stimulate certain areas of their brain in operant chambers (Olds and Milner 1954). The neuronal substrates of stimulation are believed to be, at least partly the neuronal circuitry which mediates reinforcement (Olds and Milner 1954; Wise and Rompré 1989). In the "Biological theory of reinforcement" Glickmann and Schiff (1967) suggested that the same brain structures e.g. medial forebrain bundle (mfb) that were found to be substrates for brain stimulation reward also mediate the species specific sequences of approach behaviour. According to this idea, the positive reinforcement process is reflected as forward locomotion or as some other approach response towards a stimulus.
Research also revealed that several drugs of abuse can serve as reinforcers in an operant situation, i.e. experimental animals learned to press a lever in order to get an intravenous infusion of heroin or amphetamine (Ettenberg et al. 1982; Pickens and Harris 1968; Pickens and Thompson 1968). The major neuronal systems that form the medial forebrain bundle are dopaminergic and noradrenergic pathways (Björklund and Lindvall 1984), emphasizing the involvement of these neurotransmitter systems in the intracranial self-stimulation (ICSS) reward. The role of these neurotransmitters both in drug reward and ICSS has also been supported by the observation that psychostimulants increased release of these substances in brain, while treatments that decrease noradrenergic and dopaminergic activity also attenuated ICSS (Fibiger 1978). However, studies, where the function of certain dopaminergic or noradrenergic pathways were blocked pharmacologically or with selective lesions, strongly supported the role of dopaminergic but not that of noradrenergic system as a substrate for the reinforcing effects of drugs of abuse (Fibiger 1978; Wise and Rompré 1989).
As reviewed above, there are several dopaminergic systems in the brain and also in the medial forebrain bundle (Björklund and Lindvall 1984). It is known that the role of these pathways in the regulation of behaviour is different and that each subsystem also has several functions, at least partly depending on the functional role of the neuronal systems they interact with (Amalric and Koob 1993; Le Moal and Simon 1991; Robbins and Everitt 1996; White 1989).
If one considers the mesolimbic dopamine system's projection areas, the nucleus accumbens has been shown to be critically involved in mediation of reinforcing properties of both drugs of abuse and of natural rewards such as feeding, drinking and sexual behaviour. Many different kinds of rewards increase dopamine release in that brain area (Di Chiara and Imperato 1988a; Kiyatkin 1995; Rosaria and Argiolas 1995), and attenuation of dopaminergic signalling in the nucleus accumbens decreases the reinforcing properties of these stimuli in different animal models (Ettenberg 1989; Rosaria and Argiolas 1995; Wise and Bozarth 1987). Based on these findings it has been suggested that accumbal dopaminergic neurons would be the final common pathway through which different reinforcers mediate their behavioural effects (Wise and Rompré 1989).
Although the mesolimbic dopamine system is now generally accepted to be central in the reinforcing effects of various rewards, during recent years its exact role has become the subject of intense debate (Bechara et al. 1998; Berridge and Robinson 1998; Di Chiara 1995; Salamone 1996; Schultz et al. 1997; Wise 1996). The psychomotor stimulant theory of addiction by Roy Wise and Michael Bozarth (1987), states that the nucleus accumbens dopamine mediates the rewarding, pleasurable or hedonistic effect of the reinforcer and blockade of dopamine receptors antagonizes the hedonistic effects of a given reinforcer. There is, however, also plenty of evidence in conflict with this idea. For instance, it seems that dopaminergic mechanisms are not critically involved in alcohol or opioid-induced reinforement, since reduction of dopaminergic tone does not always affect alcohol drinking, opioid self-administration or opioid-induced conditioned place preference (Ettenberg et al. 1982; Gerrits and Van Ree 1996; Kiianmaa et al. 1979; Linseman 1990; Mackey and Van Der Kooy 1985). Recent findings suggest that dopamine does not necessarily play a pivotal role in intracranial self-stimulation reward (Garris et al. 1999). Therefore, several other views about the role of dopamine in reinforcement processes have been put forward.
The theory formulated by Robinson and Berridge (Robinson and Berridge 1993) proposes that dopaminergic mechanisms in the nucleus accumbens would not be necessarily central to the subjective pleasurable effects of reinforcers. According to this idea, elevated dopaminergic tone "attributes incentive salience" to those stimuli that are associated with dopamine release, i.e. enhanced dopaminergic transmission make those events associated with elevated dopamine transmission salient and wanted. Strong activation and sensitization of the dopamine system induced by drugs would, therefore, make the drug effects pathologically attractive and wanted.
A modification from the theories involving dopamine holds that dopamine mediates incentive reward-dependent learning (Di Chiara 1995; Schultz et al. 1997). This idea is based, for instance, on electrophysiological experiments showing that dopaminergic neurons of the monkey will respond to a novel, rewarding stimulus, e.g. delivery of juice. However, the activation of the neurones fades when the animal learns to expect the reward, but subsequently, after the learning, the environmental cues predicting the reward are able to activate nigral dopamine neurons (Mirenowicz and Schultz 1996; Schultz et al. 1997). Thus, in the learning phase, dopamine may serve as signal about the significance of the event.
Furthermore, van der Kooy and his associates have suggested that brain dopamine would mediate reinforcement only in the withdrawal state, i.e. after cessation of repeated opioid treatment or in food-deprived animals (Bechara et al. 1992; Bechara and van der Kooy 1992). Normally other, non-dopamine mechanisms mediate positive reinforcing effects of opioids or food etc.
In addition to its role in the control of motor behaviour, also the nigrostriatal system is known to be involved in certain memory and learning processes. Dorsal striatum and its dopaminergic systems have been suggested to be central in the forming of habits or stimulus-response association, i.e. certain sequences of motor actions are "built in" the striatal circuitry, which then mediates learned responses to the appropriate environmental stimuli. This means that the dorsal striatum mediates "less cognitive" but more "rigid" learning (White 1997), which is also "impervious to fluctuations in the value of reinforcer" (Robbins and Everitt 1996). Therefore, also nigrostriatal dopamine system may have a role in drug reinforcement and the development of addiction.
It is, thus, evident that dopaminergic pathways in brain are important in the regulation of certain learning, motivational and reward processes. However, their exact role is not known, and it is likely that ascending dopaminergic pathways have multiple roles in these functions.
Several families of opioid peptides have been identified in the mammalian central nervous system, all of them being derived from precursors encoded by distinct genes (Darland et al. 1998; Loughlin et al. 1995; Zadina et al. 1997). Most of these peptides are known to regulate brain dopaminergic systems. Pro-opiomelanocortin's (POMC) postranslational processing generate an opioid peptide b-endorphin and also adrenocorticotrophic hormone (ACTH). The most important site for biosynthesis of POMC (Li and Chung 1976) is the pituitary and in the brain, POMC is synthesised in the arcuate nucleus and in the nucleus tractus solitarius. Although there is very little POMC containing innervation to the dopaminergic neurons, some fibers have been found in the SNc and the ventral striatum, including the nucleus accumbens and olfactory tubercle (Khachaturian et al. 1985).
The proenkephalin (PENK) gene encodes a protein, processing of which yields four copies of methionine-enkephalin (M-Enk) and one copy of leucine-enkephalin (L-Enk) that are the main products of this precursor molecule (Hughes et al. 1975). With respect to dopaminergic brain regions, enkephalinergic cell bodies and fibers have been found in the dorsal and ventral striatum as well as in the SN (Höllt 1993; Loughlin et al. 1995).
Dynorphin A, dynorphin B and leucine-enkephalin are derived from prodynorphin (PDYN) Localisation of PDYN synthesising neurons and peptides derived from it is fairly parallel with that of enkephalinergic system. PDYN synthesising cell bodies are found in the amygdala, hippocampus, cerebral cortex, hypothalamus and striatum, while nerve terminals and fibers containing these peptides are found in the SN, globus pallidus and raphe nuclei etc. (Loughlin et al. 1995).
Orphanin FQ or nociceptin was isolated independently by two groups in 1995 (see for review: Darland et al. 1998) and this peptide is widely expressed in brain including dopaminergic brain regions (Darland et al. 1998). Two novel opioid peptides, endomorphin-1 and endomorphin-2, were isolated from bovine brain in 1997 (Zadina et al. 1997) and found to show high affinity and selectivity for µ-opioid receptors (see below). Endomorphin-2 is expressed in several brain regions with known dopaminergic innervation, e.g. nucleus accumbens shell (Schreff et al. 1998).
Opioid binding sites in brain were first found by several groups independently in 1973 using ligand binding methodology (Pert and Snyder 1973; Simon et al. 1973; Terenius 1973). At present, four distinct subtypes of opioid receptors have been found, i.e. µ-, d - and k-, and orphan receptors. All four subtypes were cloned in the beginning of the 1990's (Chen et al. 1993; Darland et al. 1998; Evans et al. 1992; Kieffer et al. 1992). All of these receptors are negatively coupled to adenylyl cyclase via Gi/o protein, so the binding of agonists leads to a decrease of cAMP concentration in target cells. However, other effects on cellular functions for opioids have been also described, such as modulation of cation channels (Ca2+, K+ ) and phospholipase C (Dhawan et al. 1996).
Table 2.1. Brain opioid peptides, their precursor molecules and preferential binding sites
* b-endorphin binds with rather similar affinity to both d- and µ-receptors and no clearly preferential binding site for it has been found in the brain (Corbett et al. 1993).
µ-Receptors are widely distributed in the rat brain, and especially high concentrations are found in the dorsal and ventral striatum, limbic structures (amygdala) and thalamic nuclei (Mansour and Watson 1993). µ-Receptor concentrations are also high in the SNc but very sparse in the VTA (German et al. 1993). In contrast, most of the d-binding is seen in the rostral forebrain regions, e.g. in the caudate-putamen, nucleus accumbens and amygdala (Mansour and Watson 1993). The density and distribution of k-receptors in rat brain differs from those in several other species, such as guinea pig, monkey and human. If one considers the dopaminergic regions, the k-sites are densely found in the caudate-putamen, nucleus accumbens and olfactory tubercle of the rat but in the monkey k-binding is low in these regions. In the SN, in the rat the k-binding is sparse but relatively high in the monkey.
The first evidence that cerebral dopamine systems are regulated by opioids came from pharmacological studies showing that relatively selective µ-opioid receptor agonists such as morphine and methadone increase turnover and metabolism of dopamine in brain (Ahtee and Kääriäinen 1973; Clouet and Ratner 1970; Gunne et al. 1969; Laverty and Sharman 1965). For example, morphine has been shown to increase concentrations dopamine metabolites (Ahtee and Kääriäinen 1973; Moleman et al. 1984; Papeschi et al. 1975), increase dopamine depletion after inhibition of tyrosine hydroxylase (Attila and Ahtee 1984a; Eidelberg and Schwartz 1970; Gunne et al. 1969; Moleman and Bruinvels 1979) and increase the accumulation of dopa after inhibition of DOPA decarboxylase (Kalivas and Duffy 1987).
The effects of agonists for different opioid receptor subtypes on dopamine release differ. Studies conducted later by using in vivo methods, such as microdialysis and 3-MT accumulation, have confirmed that µ-opioids increase extracellular levels of dopamine both in the dorsal and ventral striatum as well as in the prefrontal cortex (Di Chiara and Imperato 1988b; Pei et al. 1993; Shoaib et al. 1995; Wood and Rao 1991). Furthermore, local application of an enkephalinase inhibitor into the VTA increases dopamine release in the nucleus accumbens and motor activity of the rats, suggesting that endogenous opioids activate dopaminergic neurons (Dauge et al. 1992). Electrophysiological evidence and results from local drug injections have shown that µ-opioids most likely cause their effects in the cell body regions, i.e. in the SN and VTA. If present at all, there are only very small amounts of µ-receptors in the dopaminergic neurons both in the VTA and SNc. Thus µ-opioid agonists are thought to activate dopaminergic neurons via inhibiting the GABAergic neurons that tonically control the firing rate of dopaminergic neurons (Gysling and Wang 1983; Henry et al. 1992; Johnson and North 1992; Trulson and Arasteh 1985). However, the evidence from the SN is somewhat indirect (Hommer and Pert 1983; Iwatsubo and Clouet 1977; Walker et al. 1987), since the GABA system which is believed to cause this disinhibition of dopaminergic neurons has not been pharmacologically characterized in that brain region. When infused directly into the nucleus accumbens, µ-opioids increase or have no effect on dopamine release (Mulder et al. 1989; Yokoo et al. 1994). On the other hand, there is some evidence that activation of µ-receptors might have an inhibitory effect on dopamine release in the caudate-putamen (Piepponen et al. 1999; Rossetti et al. 1990; Widdowson and Holman 1992, see also: Dourmap et al. 1992).
Although the µ-opioids are mainly sedative in humans, in rats and mice they have also dose-dependent stimulatory effects. Low doses of morphine < 2-3 mg/kg (s.c.) produce stimulation in rats without initial sedation, while higher doses produce first sedation and even a cataleptic state. After a certain period (depending on dose) also higher doses induce locomotor stimulation (Babbini and Davis 1972; Oliverio and Castellano 1974; Pert and Sivit 1977). In rats, morphine-induced acute psychomotor stimulation is seen mainly as horizontal locomotion (ambulatory activity) though there may be some stereotyped gnawing in the later phase (Cowan 1993).
µ-opioid-induced locomotor stimulation is thought to be mediated by dopamine, since blockade of dopamine receptors attenuates the stimulatory effect of systemically administered morphine (Di Chiara 1995; Di Chiara and North 1992). However, there are several other brain regions, such as the PPTg, VP and nucleus accumbens (Austin and Kalivas 1990; Kalivas et al. 1983; Klitenick and Kalivas 1994; Klitenick and Wirtshafter 1995), in which µ-opioids increase locomotor activity following local infusion. At least in the nucleus accumbens, this effect of µ-opioids is not dependent on dopamine since it is not affected by systemic administration of neuroleptics. It has been suggested that stimulatory action of intra-accumbal opioids is mediated via inhibition of the GABAergic pathway projecting from the nucleus accumbens to the VP (Pulvirenti et al. 1991; Swerdlow et al. 1986). Therefore, the critical pathway underlying locomotor stimulation induced by intra-accumbens opioids could be at least partly the same as that involved in dopamine-mediated activation (Wise and Bozarth 1987).
There is some evidence that µ-receptor agonists may alter dopamine receptors or pathways downstream from these receptors even after a single treatment. For instance, acute treatment with morphine (1.0 and 10 mg/kg) sensitizes mice to the stimulatory effects of apomorphine 3 h later (Martin and Takemori 1985). In that study, the enhanced response to apomorphine following morphine treatment (only 10 mg/kg) was associated with increased number D2 receptor binding sites. However, sensitivity of D2 receptors may first transiently decrease after µ-opioid treatment as indicated by attenuated D2-agonist-induced yawning behaviour in rats following morphine treatment (Zharkovsky et al. 1993). Thus these results indicate that µ-opioids may modulate behaviour also via postsynaptic dopaminergic mechanisms in addition to stimulation of dopaminergic neurons.
Also d-opioid receptor agonists increase dopamine metabolism and release in the dorsal and ventral striatum (Spanagel et al. 1990; Yonehara and Clouet 1984), but their mechanisms of action are different from those of µ-agonists. d-Agonists can activate dopaminergic neurons in the VTA as reflected in the increased dopamine release in the nucleus accumbens (Devine et al. 1993a). In contrast to µ-opioids, d-agonists elevate dopamine release locally in the nucleus accumbens and caudate-putamen (Dourmap and Costentin 1994; Dourmap et al. 1992; Lubetzki et al. 1982). However, this effect appears to be indirect as there is no evidence that d-receptors would be located on the dopaminergic neurons (Le Moine et al. 1994; Pasquini et al. 1992). d-Agonists induce locomotor stimulation in rats and mice (Cowan 1993) and this stimulation seems to be mediated via the dopaminergic system, since it can be antagonised by dopamine receptor blockers (Longoni et al. 1991). However, recent evidence from experiments with non-peptide agonists, SNC 80 and BW373U86, suggest that the stimulation induced by d-receptor agonists is not due to enhancement of dopamine release but is caused by the amplification of dopaminergic transmission at the postsynaptic messenger level (Longoni et al. 1998; Pinna and Di Chiara 1998).
k-Receptor agonists decrease dopamine release both in the dorsal and ventral striatum (Di Chiara and Imperato 1988b; Mulder et al. 1989). This effect is probably mediated by k-receptors located in the nerve terminals of the dopaminergic neurons, while in the cell body regions, k-opioids do not affect dopaminergic neurons (Devine et al. 1993b; Spanagel et al. 1990). In line with their effect on dopamine release, k-agonists decrease locomotor activity of rats (Di Chiara and Imperato 1988b), and they induce aversion both in the experimental animals and humans (Bals-Kubik et al. 1993; Pfeiffer et al. 1986).
Alcohol (ethanol) differs from many other psychoactive and abused drugs in that no specific receptor through which it would cause its effects has been found. In addition, compared with opioids and psychostimulants, much larger doses of alcohol are needed before any behavioural and neurochemical effects emerge (Deitrich et al. 1989; Tabakoff et al. 1996). It was proposed that cell plasma membrane lipids could be a target of action of alcohol (Deitrich et al. 1989; Tabakoff et al. 1996). However, recently it has become evident that ethanol may directly affect the function of the proteins, e.g., by binding to certain pockets in proteins containing both hydrophobic and hydrophilic components (Tabakoff et al. 1996).
Dopamine is one of the most thoroughly studied of the neurotransmitters affected by alcohol. Early evidence for the possible importance of dopaminergic systems in ethanol reward was provided by a study which showed that inhibition of dopamine synthesis with a -methyl-p-tyrosine abolished ethanol-induced euphoria in humans (Ahlenius et al. 1973). Previously Carlsson et al. (1972) had demonstrated that a-methyl-p-tyrosine could suppress alcohol-induced stimulation. Furthermore Engel et al. (1974) showed that the effect of a-methyl-p-tyrosine on alcohol-induced stimulation could be reversed with L-dopa, suggesting that catecholamines were central to this effect of alcohol. Subsequently, it was shown that acute administration of ethanol (1-4 g/kg, i.p. or i.g.) accelerated dopamine metabolism and synthesis in the brains of experimental animals such as rats and mice (Bustos and Roth 1976; Carlsson et al. 1973; Karoum et al. 1976; Khatib et al. 1988). In a microdialysis study, Imperato and Di Chiara (1986) found that alcohol increased extracellular dopamine levels in the caudate-putamen and nucleus accumbens.
Data on the effects on alcohol on dopamine mechanisms in humans is scarce. A recent PET study demonstrated that alcohol has little, if any, effect on binding of a D2 receptor ligand, raclopride, in striatum, suggesting that alcohol (1 g/kg) does not change synaptic levels of dopamine in man (Salonen et al. 1997). However, in that study the PET scanning was started one hour after the last alcohol-drinking bout and during scanning the blood alcohol concentrations (BAC) remained relatively stable. If alcohol induces its rewarding effect (dopamine release) during the increase in the BAC (Lewis and June 1990; Risinger and Cunningham 1992), it is likely that this phase was missed in that study.
As suggested already by Carlsson and Lindquist (1973), Imperato and Di Chiara (1986) also demonstrated that ethanol increased dopamine release in a impulse-dependent manner as the effect of ethanol was antagonised by g-butyrolactone, a drug which blocks the impulse flow in the dopaminergic neurons. This finding is consistent with earlier results showing that behaviourally relevant alcohol doses could increase the firing rates of dopaminergic neurons both in the SNc and VTA in vitro and in vivo (Brodie et al. 1990; Gessa et al. 1985; Mereu and Gessa 1985). Mereu and Gessa (1985) also showed that alcohol inhibited non-dopaminergic neurons in the SNr by stimulating GABAergic transmission. Since these SNr neurons are thought to exert inhibitory effect on SNc dopaminergic neurons, the authors suggested that alcohol-induced excitation of dopaminergic neurons and subsequent elevation of dopamine release could occur through disinhibition, i.e. an analogous mechanism to that observed with µ-opioid receptor agonists in the VTA.
The function of several ion channel-gated receptors is sensitive to ethanol, e.g. alcohol increases the Cl- flux through the GABAA anion channel and K+ and Na+ flux through 5-HT3-receptor-gated cation channels and decreases Na+ and Ca2+ influx through the NMDA-channel (Deitrich et al. 1989; Lovinger 1991; Lovinger et al. 1989; Tabakoff et al. 1996). All these effects of alcohol can lead to activation of dopaminergic neurons. Acute i.p. alcohol elevates also extracellular levels of 5-HT (Yoshimoto et al. 1991) suggesting that elevated dopamine release after alcohol might emerge indirectly via the 5-HT system. 5-HT3-receptor agonists have been shown to activate dopaminergic neurons in the VTA (Gillies et al. 1996; Mylecherane 1996) and antagonists of this receptor attenuate the alcohol-induced elevation of dopamine release (Wozniak et al. 1990; Yoshimoto et al. 1991).
Furthermore, mecamylamine, an antagonist of nicotinic acetylcholine receptors, has been shown to block alcohol-induced locomotor stimulation in mice, and increase of dopamine release induced by intraperitoneal injection of alcohol in rats. (Blomqvist et al. 1993; Blomqvist et al. 1992). Interestingly, Ericson et al (1998) reported that infusion of mecamylamine through a microdialysis probe into the VTA decreased alcohol drinking of rats and also prevented the increase of dopamine release normally associated with alcohol drinking behaviour. These findings strongly suggest that nicotinic acetylcholine receptors, especially those located in the VTA (Blomqvist et al. 1997; Ericson et al. 1998), are involved in regulation of alcohol drinking.
There is an abundance of evidence to suggest that some of the effects of alcohol emerge via activation of the endogenous opioid system (Herz 1997; O'Brien et al. 1996; Reid et al. 1991). For instance alcohol-induced antinociception (Boada et al. 1981; Pohorecky and Shah 1987) and hypothermia (Yirmiya and Taylor 1989) can be attenuated by opioid receptor antagonists, suggesting that these effects at least partly result from the interaction of alcohol with endogenous opioid mechanisms.
According to the current working hypothesis, alcohol stimulates the release of opioid peptides that, in turn could stimulate those opioid receptors that control dopaminergic neurons (Herz 1997). Increased opioidergic transmission could, thus, be responsible for alcohol's reinforcing effects. Evidence for the involvement of opioids in the regulation of alcohol intake has been provided by a number of studies showing that opioid receptor antagonists reduce alcohol drinking in both experimental animals and in humans (Altshuler et al. 1980; Hubbel et al. 1986; O'Brien et al. 1996; Volpicelli et al. 1995). In contrast, small doses of opioid receptor agonists, such as morphine, increase alcohol consumption in rats (Hubbel et al. 1986; Reid et al. 1991). Furthermore, an increase in accumbal dopamine release, following intraperitoneal (Acquas et al. 1993) or local (Benjamin et al. 1993) alcohol-administration and voluntary alcohol drinking (Gonzales and Weiss 1998), can be antagonized with opioid receptor antagonists.
Biochemical research has revealed that alcohol may affect brain endogenous opioid systems at several levels (Charness 1989; Gianoulakis 1989; Tabakoff et al. 1996). Several mechanisms for the mediation of alcohol-opioid peptide interactions have been suggested. For instance, alcohol may affect the enzymes involved in synthesis or processing of opioid peptide precursor and change the activity of enkephalinases, i.e. the enzymes which metabolise opioid peptides (Gianoulakis 1989).
With respect to the acute reinforcing effects of alcohol, the most relevant alcohol-opioid interactions sites are likely to be the hypothalamic POMC systems and the enkephalinergic systems associated with limbic and mesolimbic dopaminergic structures. For instance, there is evidence that alcohol increases the release of b-endorphin (or b-endorphin-like immunoreactivity) from rat hypothalamus slices in vitro (de Waele et al. 1994; Gianoulakis 1990). Acute alcohol administration also increases plasma b-endorphin levels in mice (Gianoulakis and Gupta 1986) and at least in human populations with a high risk for development of alcoholism (Aguirre et al. 1995; Gianoulakis et al. 1989; Gianoulakis et al. 1996). The source of the b-endorphin measured from plasma is the pituitary. Therefore it is not clear whether changes in this measure reflects changes in brain opioidergic function. The data from effects of acute and chronic alcohol on enkephalins and dynorphins is limited. Both decreases and increases of brain enkephalin levels after acute alcohol administration have been found. In contrast, it seems that chronic alcohol administration more consistently produces decreased met-enkephalin and dynorphin concentrations in several brain areas (Tabakoff et al. 1996).
Although moderate doses of alcohol consistently increase dopamine metabolism in the caudate putamen and nucleus accumbens of rats and mice, marked species and strain differences have been found in the behavioural effects of alcohol. Several mouse strains show locomotor stimulation following administration of a relatively large dose of ethanol (> 2 g/kg, i.p.), although sometimes after initial sedative phase (Frye and Breese 1981; Masur and Boerngen 1980; Phillips et al. 1995; Phillips et al. 1997). Ethanol-induced stimulation in mice appears to depend on the dopaminergic system since in those strains in which ethanol induces the largest increase of locomotor activity (DBA and BALB) it induces also the largest elevation of dopamine metabolism (Kiianmaa and Tabakoff 1983; Tabakoff and Kiianmaa 1982). Furthermore, as noted above alcohol-induced stimulation is attenuated by decreasing the brain catecholamine content (Carlsson et al. 1972). In rats, small doses of ethanol (< 1.0 g/kg, i.p.), have rarely induced stimulation (Carlsson et al. 1972; Waller et al. 1986), and usually no effects on motor activity or performance have been seen (Frye and Breese 1981; Lewis and June 1990; Päivärinta 1990). Large doses induce sedation and loss of the righting reflex.
When an organism is exposed repeatedly to dependence-producing pharmacologically active agents a common response to this is development of tolerance to a the drug effect. This phenomenon can be interpreted to be due to the response of the organism in order to oppose the effect of the drug and maintain the homeostatic state. However, in some situations, the opposite phenomenon, sensitization of the response to a drug, is observed during and after repeated drug administration.
The groups of which are able to induce sensitization are summarized in Table 2.2. and they include psychostimulants, amphetamine and cocaine (Kalivas and Stewart 1991; Wise and Leeb 1993), µ-opioids (Ahtee and Attila 1987; Bartoletti et al. 1983; Kalivas and Stewart 1991), nicotine (Johnson et al. 1995; Reid et al. 1996), NMDA-receptor antagonists (Wolf et al. 1993a; Xu and Domino 1994), D2 dopamine receptor agonists (Hoffman and Wise 1992; Mattingly et al. 1993; Traub et al. 1985; Wise and Carlezon 1994) and alcohol (Masur and Boerngen 1980; Phillips et al. 1994; Phillips et al. 1997).
The most commonly studied drug responses showing sensitization are different forms of motor activation, i.e. locomotor activity, stereotyped behaviour and rotational behaviour (Kalivas and Stewart 1991; Robinson and Becker 1986; Wise and Leeb 1993). Behavioural sensitization was first described for psychostimulants (Downs and Eddy 1932). Another widely studied group of drugs in sensitization research are the µ-opioids of which morphine and methadone were the first shown to have the ability to induce sensitization (Ahtee 1974; Babbini and Davis 1972; Bartoletti et al. 1983). There is evidence from different behavioural models that sensitization is really an opposite phenomenon to tolerance. For instance, in rats with unilateral 6-OHDA lesion of the ascending dopamine pathways, the dose-response curve of the amphetamine-induced circling behaviour has been shifted to the left (Badiani et al. 1997).
Table 2.2. Drugs of abuse (and some other compounds) that induce sensitization in different behavioural models in experimental animals. Model indicates the parameter on which the drug effect is increased following sensitization.
Studies by Babbini and Davis (1972) and Kumar et al. (1971) demonstrated that when morphine was given daily, over a wide dose range to rats, its locomotor activity increasing effect was enhanced and tolerance developed to the initial sedative and cataleptic effects of the larger doses. Although µ-opioids induce acutely very little stereotyped behaviour, after repeated and chronic treatment intense stereotypies emerge after subseqent acute treatment (Ahtee 1974; Cervo et al. 1981).
However, it is clear that although sensitization develops especially to some drug effects (motor activity, reinforcement), tolerance can also develop to these same responses when the doses or treatment schedule is different. For instance, when cocaine, amphetamine or opioids are administered in continuous i.v. infusion, with subcutaneous pellets or in large doses with frequent administrationd, tolerance emerges to the stimulant effects of the drugs as well (Stewart and Badiani 1993). It has been also shown that when morphine is given at large doses over short period, tolerance to the motor-stimulant effect develops, but when the same amount of the drug is given in smaller doses and over longer period, then sensitization is observed (Stewart and Badiani 1993). As a rule of thumb, it can be concluded that large doses and continuous administration of the drug induce initial tolerance. However, also after a tolerance-inducing treatment schedule, sensitization can develop after a long enough treatment-free withdrawal period (Acquas and Di Chiara 1992; Ahtee and Attila 1987).
All of the sensitizing drugs and other treatments share an ability to induce motor stimulation acutely, at least in certain conditions, and when given repeatedly, they induce sensitization. However, the expression of the acute stimulatory effect of the drug is not a prerequisite for its ability to induce behavioural sensitization. For instance, repeated local infusion of amphetamine or a D1 dopamine receptor agonist SKF 38393 into the VTA induces sensitization to subsequent systemic psychostimulant administration although these treatments do not induce acute motor effects (Pierce et al. 1996b; Vezina 1996). In addition to the drugs, certain types of stressful stimuli are able to sensitize experimental animals to subsequent stimulatory effects of the drugs mentioned above (Deroche et al. 1995; Kalivas and Stewart 1991; Roberts et al. 1995; Shaham et al. 1995).
Although drugs which are capable of inducing behavioural sensitization (e.g. table 2.2) have dissimilar behavioural actions especially in humans, like stress they are able to stimulate dopaminergic transmission in brain (Abercombie et al. 1989; Kalivas and Duffy 1995; Kalivas and Stewart 1991). Since dopamine, especially dopamine in the basal ganglia, is known to be involved in the control of motor activity, it was proposed that this brain dopaminergic system would be important also in sensitization (Ahtee 1974; Kalivas and Stewart 1991; Robinson and Becker 1986). The facts that repeated amphetamine may cause a psychosis very similar to schizophrenia in humans and that dopamine receptor antagonists are effective antipsychotics, also emphasized the importance of dopamine in sensitization. Indeed, amphetamine-induced sensitization has been widely used as an experimental model of schizophrenia in laboratory animals (Robinson and Becker 1986).
More recently, behavioural sensitization phenomenon has been linked to the development of drug dependence and it has been intensively used as a tool in research of chronic behavioural and neurochemical effects drugs of abuse (Ahtee and Attila 1987; Altman et al. 1996; Phillips 1997; Robinson and Berridge 1993). It has been found that following repeated treatment with sensitizing drugs, the reinforcing effects of these drugs are enhanced in experimental animals (Lett 1989; Robinson and Berridge 1993; Shippenberg et al. 1996). For instance, smaller doses of morphine are needed to induce conditioned placed preference in sensitized rats and the sensitized animals discriminate the effects of smaller doses of the drug than the control animals (Gaiardi et al. 1991; Gaiardi et al. 1986). Sensitized animals are more motivated to get i.v. amphetamine and they learn i.v. drug self-administration quicker than naïve animals in an operant paradigm (Piazza et al. 1989; Pierre and Vezina 1997).
In humans, the link between the development of drug dependence and sensitization has been more difficult to demonstrate. The fact that repeated administration of amphetamine may induce psychotic episodes very similar to schizophrenia suggest that dopaminergic systems may, indeed, sensitize the individual to the effects of amphetamine. Short-term controlled clinical trials have produced variable results showing either sensitization to behavioural effects of amphetamine (Strakowski et al. 1996) or, more frequently, no change in amphetamine or cocaine effects following repeated administration (Rothman et al. 1994; Wachtel and de Wit 1999). A study by Bartlett et al. (1997) provided some evidence that cocaine-abusers who had developed paranoid psychoses during repeated cocaine use (sensitized individuals) were also more vulnerable to relapse following abstinence, suggesting that sensitization might play a role in the maintenance of drug abuse.
Drug-induced sensitization is a long lasting phenomenon and may be linked to changes in mechanisms regulating motivation and reinforcement. Since the brain mechanisms mediating reinforcement are partially the same as those that are involved in sensitization, it is possible that sensitization of these brain circuitries play an important role in drug craving and relapse (Robinson and Berridge 1993).
In addition to the schedule of drug treatment and the doses used, another critical variable in development of behavioural sensitization is the environment where the repeated treatment is administered (Badiani et al. 1997; Hoffman and Wise 1992; Reid et al. 1996; Vezina et al. 1989). It has been hypothesised that two different forms of sensitization exist and different neuronal mechanisms may underlie these phenomena. Sensitization to µ-opioids and psychostimulants develops rapidly and with relatively small doses when the drugs are administered repeatedly in the same distinct environment (Babbini and Davis 1972; Badiani et al. 1997; Phillips et al. 1994; Reid et al. 1996; Vezina and Stewart 1984). This form of sensitization is called context-dependent sensitization. Thus, the most robust sensitization is seen when the experimental animals are removed from their home cage to distinct experimental cage where the drug treatment are given (Badiani et al. 1995a; Badiani et al. 1995b; Stewart and Vezina 1987; Vezina and Stewart 1984). When the same dose of the sensitizing drug is given unsignalled (e.g. via i.v. infusion) (Crombag et al. 1996) or injected in the home cage (Reid et al. 1996; Vezina et al. 1989; Vezina and Stewart 1984) sensitization to the subsequent drug challenge may not develop. It is, therefore, clear that environmental cues linked to drug effects have an important role in the expression of behavioural sensitization to acute drug effects (Robinson and Berridge 1993; Stewart 1992; Stewart et al. 1984).
The importance and role of context-dependency of sensitization can be explained by the classical Pavlovian conditioning associated with the drug effects (Stewart 1992; Stewart and Badiani 1993). According this idea, during a drug effect (unconditioned stimulus, UCS), normally neutral environmental stimuli associated with the drug effect acquire the ability to cause similar or partly similar activation of brain circuitries as the UCS, i.e. a neutral stimulus becomes a conditioned stimulus (CS). The results of this process can be seen, e.g. sometimes animals in a drug-free state show increased conditioned motor activity in the environment where they have been previously treated with sensitizing stimulant (Neisenwander and Bardo 1987; Walter and Kuschinsky 1989). However, this conditioned hyperactivity does not always occur and it is clear that it does not alone explain the development of sensitization. Thus, for expression of context-dependent sensitization, the interaction of UCS and CS are needed.
The initiation and expression of behavioural sensitization is thought to depend on the function of ascending dopamine pathways. In the case of µ-opioids, the link between behavioural sensitization and dopamine was revealed by studies showing that in rats withdrawn from chronic methadone treatment, acute methadone induced a larger increase in the striatal HVA concentration than in chronically saline-treated animals (Ahtee 1974). Since the methadone-withdrawn animals showed also intense stereotypies, a behaviour which psychostimulant studies have linked to striatal dopamine, it seemed evident that enhanced methadone-induced dopaminergic activity was the reason for the increased activity of the animals. Subsequently it has been shown that other µ-opioids share the same ability to induce sensitization of the dopaminergic system (Attila and Ahtee 1984b; Kalivas 1985; Kalivas and Duffy 1987; Spanagel et al. 1993).
In addition to stereotyped behaviour, the locomotor activity-increasing effects of morphine are enhanced following repeated µ-opioid treatment. As discussed above, these behaviours are thought to be mediated via separate dopaminergic pathways (Amalric and Koob 1993; Kelly et al. 1975). Indeed, this was confirmed in studies showing that the effects of acute of morphine on both mesolimbic and striatal dopamine turnover are sensitized in rats withdrawn long enough from chronic morphine (Attila and Ahtee 1984b). Similarly, behavioural sensitization to the psychostimulants, amphetamine and cocaine, has been shown to be associated with sensitization of ascending dopamine pathways (Heidbreder et al. 1996; Kiyatkin 1994; Robinson et al. 1988).
Although there is much of evidence indicating that changes in presynaptic dopamine release are important in the expression of behavioural sensitization to opioids and psychostimulants, it has been shown that this effect is not important in the initiation of sensitization. This is indicated by studies showing that although administration of amphetamine into the nucleus accumbens does not induce sensitization to itself, to systemic amphetamine or morphine, it increases dopamine release and the locomotor activity of the animals (Cador et al. 1995; Vezina 1993; Vezina and Stewart 1990). However, when amphetamine is administered repeatedly into the VTA it induces sensitization to both systemic amphetamine and morphine although intra-VTA administration does not affect either the motor activity or accumbal dopamine release (Cador et al. 1995; Perugni and Vezina 1994; Vezina 1993). Similarly, administration of µ-agonists into the nucleus accumbens induces acutely locomotor stimulation but not sensitization while intra-VTA µ-agonists induce both stimulation and sensitization (Vezina et al. 1987). Furthermore, local administration of naltrexone into the VTA but not into the nucleus accumbens prevents the sensitization by systemic morphine (Kalivas and Duffy 1987).
However dopaminergic mechanisms are not without a role in the initiation of sensitization, since the blockade of dopamine receptors during a sensitizing treatment can block the development of sensitization (Kalivas and Stewart 1991). Critical dopamine receptors are located in the VTA as indicated by the finding that blockade of D1 but not D2 receptors in this brain regions antagonizes amphetamine-induced sensitization (Kalivas and Stewart 1991; Stewart and Vezina 1989; Vezina 1996; Vezina and Stewart 1989). Furthermore, repeated infusion of D1 dopamine receptor agonists into the VTA induces long-lasting sensitization to the systemic administration of amphetamine and cocaine (Pierce et al. 1996b). These findings have not been convincingly repeated in studies with µ-opioids (Di Chiara 1995).
It has been suggested that a common factor in the initiation of behavioural sensitization by drugs with different primary mechanisms of action is that they all increase somatodendritic dopamine release (Kalivas and Stewart 1991) and, thus, activate D1 dopamine receptors in the cell body regions. Activation of the D1 receptors, therefore, could lead to molecular changes in the neurons in which they are expressed, which in turn, could lead to initiation of sensitization. Candidates for these cells are GABAergic and glutamatergic neurons projecting to the dopaminergic neurons (Kalivas et al. 1993). Although the importance of VTA dopamine receptors in the initiation is supported by several reports, these findings have not been replicated by other groups (Jeziorski and White 1995; Mattingly et al. 1994; Steketee 1998).
The role of glutamatergic mechanisms in the initiation of sensitization is supported by findings showing blockade of NMDA and AMPA receptors can antagonize the behavioural sensitization to amphetamine (Kalivas and Alesdatter 1993; Karler et al. 1991). However, there is evidence that the non-competitive NMDA receptor antagonist, MK-801, can block morphine-induced behavioural sensitization only at high doses (> 0.25 mg/kg) that together with morphine are lethal (Vanderschuren et al. 1997). Furthermore, repeated administration of morphine increases the expression of the GluR1 subunit of the AMPA receptors in the VTA, and the upregulation of this subunit in the VTA by virally mediated gene transfer enhances the effect of the stimulatory effects of morphine in rats (Carlezon et al. 1997). Thus these findings suggest that AMPA but not NMDA receptors are important in µ-opioid-induced sensitization.
A number of other alterations in the receptors, intracellular signalling systems, enzymes and cell structure have been reported to occur in the VTA during and after chronic treatment with psychostimulants, opioids and alcohol. One consistent finding is that early after discontinuation of repeated cocaine treatment, the number of D2 dopamine autoreceptors and the efficacy of D2 agonists is decreased in the VTA (Kalivas and Stewart 1991; Wolf et al. 1993b). This might lead to diminished autoregulation of dopaminergic neurons and enhanced efficacy of the stimulatory afferents to increase the firing rate of dopaminergic neurons. This change is transient, however, and not observed weeks after withdrawal although the sensitization may still be present. Alterations in autoreceptor functions may, therefore, play a role only in the initiation of events that lead to the development of sensitization (Wolf et al. 1993b).
Chronic treatment with morphine, cocaine and alcohol as well i.v. self-administration of heroin increase levels of tyrosine hydroxylase and glial fibrillary acidic proteins, and decrease the amount of neurofilament proteins in the VTA (Beitner-Johnson et al. 1992; Beitner-Johnson and Nestler 1991; Bonci and Williams 1996; Ortiz et al. 1995; Self et al. 1995). The latter finding, together with decrease of the size and number of dopaminergic neurons in the VTA (Beitner-Johnson et al. 1992; Beitner-Johnson and Nestler 1993), suggest that some cell damage takes place either during repeated drug exposure to drugs or withdrawal (Sklair Tavron et al. 1996). Similar changes have not been observed in the SN.
Even though there is evidence to support the involvement of presynaptic dopamine release as an important mechanism in the expression of behavioural sensitization, there are also several conflicting findings. Early after withdrawal from repeated cocaine or amphetamine treatment (< 5 days), behavioural sensitization is already present but the effect of an acute drug challenge on dopamine release is not enhanced (Heidbreder et al. 1996; Segal and Kuczenski 1992; Wolf et al. 1993b). Sensitization of dopamine release apparently develops later, and is maximal about three weeks after the end of the repeated treatment (Heidbreder et al. 1996; Wolf et al. 1993b). The fact that D2 dopamine receptor agonists, i.e. drugs that decrease rather than increase presynaptic dopamine release, also induce behavioural sensitization, indicates that dopamine-release independent mechanisms of sensitization must exist (Hoffman and Wise 1992; Rowlett et al. 1995; Szechtman et al. 1994).
Several intracellular molecular changes in both in the caudate-putamen and nucleus accumbens have been found following repeated psychostimulant and µ-opioid treatments (Beitner-Johnson et al. 1992; Hope et al. 1992; Miserendino and Nestler 1995). A consistent finding is that expression of Gs protein and cAMP levels are elevated in the nucleus accumbens (Nestler et al. 1993; Terwilliger et al. 1991). Furthermore, the efficacy of D1 receptor agonists to elevate of cAMP levels and to decrease the firing rate of the postsynaptic neurons is enhanced (Henry and White 1991; Unterwald et al. 1996). Repeated administration of µ-agonists into the nucleus accumbens sensitizes rats to the stimulant effect of systemically delivered amphetamine supporting the involvement of accumbal, possibly postsynaptic mechanisms in the sensitization (Cunningham et al. 1997). Changes in the functioning of accumbal excitatory amino acid systems have been also suggested to play a role in the sensitization to psychostimulants (Robinson et al. 1997). Pierce et al. (1996a) reported that only rats that expressed sensitization to systemic cocaine were sensitized to intra-accumbens AMPA and showed increased glutamate release following acute cocaine challenge.
Individuals within both animal and human populations differ substantially in their susceptibility to develop alcoholism and other addictions. Research during recent decades has confirmed that traits predisposing individuals to excessive and compulsive drug use are partially genetically regulated (Crabbe et al. 1994; Pickens et al. 1996). Of the different forms of addiction, experimental animals have been most widely used to study the genetic background of alcoholism (Crabbe et al. 1994; Samson et al. 1988). Furthermore, twin and adoption studies have revealed that a significant amount of the variability in human alcohol consumption can be explained by genetic factors (Ball and Murray 1994; Cloninger 1987; Pickens et al. 1996).
Evidence about the role of heredity in addiction for other drugs among humans is not as well understood as in the case for alcoholism, due to the paucity of studies carried out on this field (Pickens et al. 1996). However, it is very likely that genetic factors also play an important role in susceptibility to abuse of opioids and psychostimulants (Crabbe et al. 1994; Cunningham et al. 1992; Kosten et al. 1994; Shoaib et al. 1995).
Since drug-induced psychomotor stimulation and sensitization are thought to be related to reinforcement and development of addiction, differences in these responses have been sought in rodent lines differing in their voluntary drug intake (Phillips 1997). As reviewed below, some strain differences in these behavioural responses as well as in the sensitivity of the dopaminergic systems to the stimulatory effects of drugs have been found.
C57BL/6 (C57) inbred mice were the first to show a relatively high preference for 10 % alcohol-solution, while the DBA/2 (DBA) strain was found to avoid alcohol solution almost completely (McClearn and Rodgers 1959). Contrary to the prediction by psychostimulant theory of addiction, the DBA show greater locomotor stimulation as well as sensitization following acute and repeated i.p. alcohol treatment than C57 mice (Cunningham et al. 1992; Phillips et al. 1994).
C57 mice consume also large amounts of aqueous solutions morphine and cocaine (Belknap 1990; Eriksson and Kiianmaa 1971; George et al. 1991a; Horowitz et al. 1977) indicating that, in addition to alcohol, the reinforcing effects of other drugs of abuse are strong in C57 mice. Interestingly, C57 are more stimulated by acute morphine than the DBA mice and they show behavioural sensitization following repeated treatment, while the DBA mice do not (Cunningham et al. 1992; Oliverio and Castellano 1974; Phillips et al. 1994; Reggiani et al. 1980). In line with these results, morphine elevates dopamine metabolism more in the striatum of the C57 mice than in DBAs (Reggiani et al. 1980). The effects of acute amphetamine, cocaine and other dopamine reuptake inhibitors on locomotor activity do not differ between the C57 and DBA mice (Elmer et al. 1996; Phillips et al. 1994). There is evidence, however, that context-dependent sensitization of locomotor activity following repeated amphetamine treatment is greater in C57 mice than in DBA mice (Cabib 1993; Elmer et al. 1996; Tolliver and Carney 1994). These results may indicate that the C57 mice are more susceptible to conditioning effects of cocaine than the DBA mice (Cabib 1993).
Another pair of inbred rodent lines, frequently used in drug dependence research consists of Lewis and Fischer 344 rats. Lewis rats consume more aqueous solutions of alcohol (Suzuki et al. 1988), cocaine and morphine (George and Goldberg 1989) than the Fischer rats. In the conditioned place preference (CPP) paradigm, the reinforcement induced by cocaine and morphine is stronger in the animals of Lewis strain than in the Fischer strain (Beitner-Johnson et al. 1992; Kosten et al. 1994). Lewis rats also learn intravenous cocaine self-administration after fewer training sessions and with lower doses than Fischer rats (Kosten et al. 1997). These findings, therefore, suggest that the reinforcing effects of drugs of abuse from several classes are strong in the Lewis strain, making these animals susceptible to drug intake.
Stimulatory effects of cocaine and amphetamine are larger in the Lewis rats than in the Fischer rats (Camp et al. 1994; George et al. 1991b). Furthermore the Lewis strain is more susceptible to the development of behavioural sensitization than the animals of the Fischer strain (Kosten et al. 1994). These line differences in behavioural responses to the psychostimulants may be due to the larger amphetamine- and cocaine-induced increase of accumbal dopamine release in animals of the Lewis strain than in those of the Fischer strain (Camp et al. 1994). However, there is some evidence that these dissimilarities are at least partially due to differences in the bioavailability of these drugs (Camp et al. 1994).
Following the original finding by the McClearn and Rodgers that inbred mice differ in voluntary alcohol consumption (McClearn and Rodgers 1959), several rat strains for high or low voluntary alcohol consumption have been produced with selective bi-directional breeding from heterogenous strains. These kinds of strain pairs are effective tools in research into the biological mechanisms involved in regulation of alcohol drinking behaviour since all differences between the lines should be related to their differential alcohol intake and share at least partially the same genetic background (Crabbe et al. 1990). It is assumed that the selective breeding segregates the sets of genes contributing to the high and/or low drug-intake, and thus helps to identify physiological and behavioural correlates of differential drug preference (Crabbe et al. 1994).
The project, which produced the alcohol-preferring AA (Alko, Alcohol) and alcohol avoiding ANA (Alko, Non-Alcohol) rats was started by Kalervo Eriksson in the physiological laboratory of the Finnish state alcohol monopoly, ALKO, in Helsinki. The first report about these rat lines was published in 1968 (Eriksson 1968). Another breeding project was started already in the 1940īs in Chile, and it produced alcohol-preferring UChB and alcohol-avoiding UChA rats (Mardones and Segovia Riquelme 1983). Subsequently, several other similar rat line pairs have been produced, the most intensively studied being alcohol-preferring P and non-preferring NP rats (Li et al. 1993) bred in the Indiana University School of Medicine, Indianapolis, USA. In that institution, also another line pair has been produced, namely high-alcohol drinking HAD and low-alcohol drinking LAD rats (Li et al. 1993). Alcohol preferring SP (Sardinian Preferring) and alcohol-avoiding SNP (Sardinian Non-Preferring) have been bred in the University of Cagliari in Sardinia (Fadda et al. 1989).
2.11.2.1. Alko, Alcohol (AA) and Alko, Non-Alcohol (ANA) rats
Selection of the AAs and ANAs is based on the voluntary alcohol consumption under 24-h free choice situation between 10 % alcohol solution (in water) and plain water. The alcohol drinking behaviour of the AA and ANA rats has been described in several review articles (Eriksson and Rusi 1981; Hilakivi et al. 1984; Sinclair et al. 1989). For instance, alcohol consumption of male AA rats from generations F61-F77 has varied between 5 -7 g/kg of absolute ethanol/day, while the ANA rats consume less than 1 g/kg (Maija Sarviharju, personal communication). The level of alcohol drinking of the AA rats is comparable to that of other alcohol-preferring rat lines (McBride and Li 1998) (see below). The AA rats also learn to work for alcohol in an operant chamber without any previous alcohol experience (Hyytia and Sinclair 1989).
Several both behavioural and physiological differences as well differences in response to alcohol and other drugs and environmental stimuli (e.g. stress) have been found between the AA and ANA rats (Sinclair et al. 1989). With respect to possible roles of anxiety and stress in the etiology of alcoholism and drug dependence in general, it has been of great interest to explore different anxiety measures and responses to stressful situations in the AA and ANA rats. One could speculate that that the AA rats would drink alcohol in order to relieve anxiety. On the other hand, the AA rats could be sensitive to stress, a trait which could contribute to the sensitization of the brain mechanisms involved in drug reinforcement (see above).
However, in the tests for level of anxiety, such as elevated plus maze or conflict tests, either no differences between the strains have been found (Tuominen et al. 1990; Viglinskaya et al. 1995) or the ANA rats have shown greater anxiety than the AA rats (Knapp et al. 1997; Moller et al. 1997). Furthermore, the ANA rats show greater or faster response to different aversive stimuli, such as shock-prod in defensive burying paradigm (Sandbak et al. 1998), forced swimming test (Korpi et al. 1988; Sandbak et al. 1998) and less habituation to aversive stimuli during testing of nociception (Honkanen et al. 1995). These traits may play a role in the low alcohol drinking of the ANA rats and their lack of habituation to aversive properties of alcohol solution.
Also metabolic factors seem to be important in the differential alcohol drinking of AA and ANA rats. Following alcohol challenge, acetaldehyde accumulates in the blood of ANA rats probably causing aversion to alcohol (Eriksson 1973). The importance of the ethanol metabolism as the limiting factor of alcohol drinking by the ANAs is also suggested by the finding that AA rats do not develop alcohol preference when they are transplanted with livers from ANA rats (Eriksson et al. 1997). The ANA rats are also more sensitive to strong or aversive tastes, since they generally drink less of the bitter, salty, and sour solutions than the AAs or Wistars (Sinclair et al. 1992b). This finding suggests that the taste is an important factor in limiting the alcohol drinking of the ANA rats. However there is strong evidence for a post gustatory mechanism for differential alcohol drinking of these rats since i.v. alcohol maintains operant responding in the AAs but not in the ANAs (Hyytiä et al. 1996).
It was many years ago found that concentrations of the dopamine and 5-HT differed in the brains of AA and ANA rats, levels of both neurotransmitters being higher in the AA rats than in the ANA rats (Ahtee et al. 1980; Ahtee and Eriksson 1972; Ahtee and Eriksson 1975). Concentrations of the 5-HT metabolite, 5-HIAA, were also higher in the brains of the AA rats (Ahtee and Eriksson 1972; Korpi et al. 1988). The activities of tyrosine hydroxylase and dopa decarboxylase are markedly higher in the brains of AA rats than ANAs (Pispa et al. 1986). This difference may, at least partly, underly the greater concentrations of dopamine in the AAs than ANAs. However, no differences were found in the activities of dopamine-b-hydroxylase, MAO or COMT between the rat lines.
Dopamine-related behaviors have not been extensively studied in the AA and ANA rats. An acute intraperitoneal dose of alcohol (1 g/kg or lower) does not alter the locomotor activity of these animals, but the AA rats show significant stimulation after an alcohol drinking bout in a limited access paradigm (Päivärinta and Korpi 1993). This finding suggests that voluntary alcohol drinking activates dopaminergic mechanisms in the brains of AA rats. Intraperitoneal alcohol elevates extracellular dopamine levels in the nucleus accumbens of freely moving AA and ANA rats, but there is no difference in this response between the rat lines (Kiianmaa et al. 1995).
Given the possibility that alcohol can mediate its reinforcing effects via endogenous opioids, it is interesting that several differences in the brain opioid systems of the AA and ANA rats have been found. For instance b-endorphin-like immunoreactivity (b-EPLIR) is higher in the septum and lower in the amygdala and periaqueductal gray matter of the AA rats than ANA rats. However, there is no significant difference in the concentration of b-EPLIR in the nucleus accumbens, caudate-putamen or hypothalamus between the strains (Gianoulakis et al. 1992). Spontaneous release of b-EPLIR is larger in the ANA rats than in the AAs, but there is no difference in the alcohol-induced release between these strains (de Waele et al. 1994). Interestingly, most of the b-endorphin-like peptides released from the hypothalamus of the ANA rats were of acetylated forms that are devoid of opioid activity (de Waele et al. 1994). This may indicate a deficit of the reward system in the ANA rats that may contribute to their reduced level alcohol consumption.
Furthermore, basal levels of (Met)enkephalinArg6 Phe7, a marker of the proenkephalin system and levels of dynorphin A and dynorphin B, were are lower in the nucleus accumbens of the AA rats than ANA rats. The levels of (Leu)enkephalinArg6, another marker of the dynorphin system were also lower in the VTA of the AA rats than in ANAs. These results suggest that opioidergic regulation of mesolimbic the dopamine system differs in the AA and ANA rats.
de Waele et al. (1995) reported that d- and µ- opioid receptor densities are both markdly lower in the ANA rats than AAs in several brain areas, including caudate-putamen (µ and d) and VTA (µ). However, these results have not been replicated by others either with agonist (Soini et al. 1998) or antagonist (Soini et al. 1999) autoradiography. The reasons for these discrepancies are not clear. However, given the large within strain variability in these measures in the AAs and ANAs, one of the problems with the study by de Waele et al. is that most of the data was derived only from three animals/strain. There are inconsistencies also in the data from studies with agonist and antagonist binding (Soini et al. 1999; Soini et al. 1998), but one replicated result is that µ-receptor density is higher in the SNr of the AA rats than ANAs.
The AA rats consume more aqueous solutions of cocaine and etonitazene, a µ-opioid receptor agonist, than ANA rats or non-selected Wistar rats (Hyytiä and Sinclair 1993). Thus, the reinforcing effects of opioids may be stronger in the AA rats than in the ANA rats. Hence, the brain reward mechanisms of the AA rats may be also highly sensitive to activating stimuli other than alcohol. Together these data indicate that there are genetically regulated differences in the brain opioid systems between the AA and ANA rats and these traits may contribute to their differential alcohol preference
In conclusion, there are several physiological and behavioural differencies between the AA and ANA rats that may contribute to the differential alcohol preference of these animals. However, initial reason for the development of high alcohol preference and its maintenence in the AA rats is not known.
Table 2.3. Summary of major neurobiological and behavioural differences between the AA and ANA rats
Acb = nucleus accumbens, PAG = periaqueductal gray matter, SNr = substantia nigra pars reticulata, VTA = ventral tegmental area
2.11.2.2. P/NP, HAD/LAD and SP/SNP rats
The findings from several high or low alcohol-drinking rat strains have been recently reviewed (McBride and Li 1998) so these other strain pairs are only briefly dealt with here. In comparison with AA and ANA rats, different results have been obtained from studies with other rat lines pairs, i.e. both 5-HT and 5-HIAA concentration are lower in the brains of alcohol-preferring P and HAD rats than in their alcohol-avoiding counterparts (Gongwer et al. 1989; Murphy et al. 1982). Similarly, while no differences have been found in the levels of 5-HT receptors in the brains of AA and ANA rats (Ciccocioppo et al. 1997; Ciccocioppo et al. 1998; Korpi et al. 1992), the P rats have lower concentrations of 5HT2A and 5-HT3 receptors in some brain regions.
Concentrations of both dopamine and DOPAC are lower in the nucleus accumbens and dorsal striatum of the P and HAD rats than in the NP and LAD rats (Gongwer et al. 1989; McBride et al. 1995; Murphy et al. 1987). In the brains of the Sardinian rats, the levels dopamine and its metabolites do not differ (Fadda et al. 1990). However, the density of D2 dopamine receptors is lower in the striatum of the SP than in SNP rats, and this result has been replicated with P and NP rats (McBride et al. 1993; Stefanini et al. 1992). However, very small differences have been found in the D2 receptor binding or mRNA levels between the AA and ANA rats (Korpi et al. 1987; Syvalahti et al. 1994). In summary, these results suggest that compromised dopaminergic and 5-HTergic transmission may contribute to the high alcohol consumption of the P, SP and HAD rats but different mechanisms underlie the alcohol-preference of the AA rats.
No differences in the response of dopaminergic system to alcohol have been found in the case of HAD/LAD lines of rats (Yoshimoto et al. 1992). However, in the alcohol-preferring SP rats, i.p. alcohol induces a greater elevation of DOPAC and HVA concentrations in the several brain areas than in the SNP rats (Fadda et al. 1990). Small doses of i.p alcohol have been reported to increase locomotor activity of the P rats but not that of nP rats, but during 5-day repeated treatment the responses to alcohol did not change in either strain (Waller et al. 1986). However, in a work by Criswell et al. (1994) neither P nor nP rats were stimulated by alcohol. Weiss et al. (1993) provided evidence suggesting that the accumbal dopamine system in the P rats might be very sensitive to the activating effect of voluntary alcohol drinking.