Alcohol and Alcoholism Vol. 36, No. 4, pp. 318-322, 2001
© 2001 Medical Council on Alcoholism
Alcohol preference: association with reduced striatal nicotinic receptors
Department of Pharmacology, College of Medicine, Howard University, 520 W Street NW, Washington, DC 20059, USA
Received 14 January 2000; first review notified 14 February 2001; accepted 24 February 2001
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ABSTRACT |
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 TOPFOOTNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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This study was designed to determine whether inherent alcohol preference is associated with differential expression of central nicotinic receptors. [3H]Cytisine and [125I]
-bungarotoxin binding-site, ligands selective for 4ß2 and 7 nicotinic receptor subtypes, respectively, were determined in various brain regions of alcohol-preferring (P) and non-preferring (NP) rats. Only the striatum of P rats had a reduction in the number of binding sites for both ligands, compared to NP rats. The data suggest a link between striatal nicotinic receptors and alcohol preference. |
INTRODUCTION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Family, twin and adoption studies suggest that alcoholism is familial and that genetic factors play a significant role in its aetiology (Merikangas, 1990
; True et al., 1999). Indeed, through selective genetic breeding, distinct alcohol-preferring (P) and alcohol-non-preferring (NP) rat lines have been established (Li and McBride, 1995; McBride and Li, 1998). In contrast to many drugs of abuse, however, no specific receptors for alcohol have been identified.
The high incidence of cigarette smoking among alcoholics prompted the search for possible common neurochemical pathways mediating the effects of alcohol and nicotine (Blomqvist et al., 1992; Ericson et al., 1998). Central actions of nicotine are mediated by specific nicotinic cholinergic receptors. Various nicotinic receptor subtypes with distinct physiological and pharmacological properties have been identified (Olale et al., 1997; Changeux et al., 1998; Cordero-Erausquin et al., 2000). The most predominant and most extensively studied subtype in the brain has a high affinity for cytisine, nicotine or acetylcholine and is formed from the 4 and ß2 subunits (Clarke et al., 1985; Pabreza et al., 1991; Flores et al., 1992). The other major class with a high affinity for -bungarotoxin (-BT) and low affinity for nicotine is formed from 7 subunits and can be labelled by [125I]-BT. [125I]-BT binding sites are most abundant in the hippocampus and colliculi (see Clarke et al., 1985 for detailed distribution) and are believed to have a prominent role in neuronal growth and survival (De Fiebre et al., 1995), and cognitive functions, particularly attentional processes (Freedman et al., 1997). On the other hand, 4ß2 receptors are most abundant in the striatum, midbrain and the cortex and are believed to be involved in antinociceptive, locomotor activating, discriminative stimulus, and addictive properties of nicotine (Olale et al., 1997; Stolerman et al., 1997; Changeux et al., 1998; Damaj et al., 1998). Recently, a role for 7 receptor subtype in central reward pathway has also been suggested (Schilstrom et al., 1998).
It has been postulated that the reinforcing effects of alcohol are at least partially mediated by central nicotinic receptors (Ericson et al., 1998). This postulate is based on the finding that administration of the nicotinic antagonist mecamylamine into the ventral tegmental area markedly reduced ethanol intake and preference in rats. Further support for this hypothesis is provided by the findings that the locomotor stimulant effects of ethanol and the associated increases in dopamine turnover can be manipulated by nicotinic agents (Blomqvist et al., 1992). Moreover, chronic alcohol administration in rats and mice can lead to changes in nicotinic receptor densities in discrete brain regions (Yoshida et al., 1982; Booker and Collins, 1997). In addition, both acute (Katner et al., 1997; Dyr et al., 1999; Lê et al., 2000) and chronic (Potthoff et al., 1983; Blomqvist et al., 1996; Lê et al., 2000) administration of nicotine can affect alcohol consumption in rats. More recently, evidence for common stimulus properties for alcohol and nicotine in a conditioned taste aversion paradigm was provided (Kunin et al., 1999). In this study, we sought to determine whether inherent alcohol preference may also be associated with differential expression of central nicotinic receptors.
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MATERIALS AND METHODS |
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Animals and tissue collection
Adult male (3–4 months old, 300–350 g), alcohol-naive, P and NP rats from colonies maintained at Indiana University (Indianapolis, IN, USA) were killed by decapitation and the brains were quickly removed, frozen on dry ice and stored at –80°C until dissected and assayed for receptor density and binding affinity. Brains from eight P and eight NP rats were dissected into various regions as described in detail previously (Tizabi et al., 1999
). Briefly, the brains were partially thawed, maintained on ice, and frontal cortex excluding the olfactory bulb and olfactory tubercle and up to the genu of corpus callosum, the remaining cortical area, hippocampus (bilateral), striatum (bilateral and including nucleus accumbens), midbrain (including the thalamus), hypothalamus, colliculi (superior and inferior), and cerebellum were removed under a magnifying lens. Tissues were stored at –80°C before measuring nicotinic receptor binding. Preliminary studies established that this procedure does not affect the measurements of the receptor density or affinity.
Determination of nicotinic receptors
Tissue was homogenized in ice-cold 50 mM Tris–HCl buffer (pH 7.0) at room temperature. The tissue homogenate was centrifuged at 38 000 g for 12 min at 4°C. The pellet was washed twice by suspension in fresh buffer and centrifuged again. Aliquots of homogenate equivalent to ~10–20 mg tissue were divided into two sets of tubes for determination of [3H]cytisine and [125I]-BT binding. These ligands bind specifically to the 4ß2 and 7 nicotinic receptor subtypes, respectively (Flores et al., 1992; Barrantes et al., 1995). For [3H]cytisine binding, an assay procedure based on that by Pabreza et al. (1991) was used. For total binding, ~4 nM [3H]cytisine (38.2 Ci/mmol; Dupont/NEN, Boston, MA, USA) was incubated in a final volume of 0.25 ml at 2°C for 75 min. Non-specific binding was obtained in the presence of 100 µM (–)-nicotine bitartrate. For [125I]-BT binding, ~2 nM [125I]-BT (120 Ci/mmol; Dupont/NEN) was incubated in a final volume of 0.25 ml at 37°C for 2 h for total binding. Non-specific binding was obtained in the presence of 200 µM (–)-nicotine bitartrate. Membrane-bound [3H]cytisine, or [125I]-BT were separated from free ligand by filtration using Brandel GF/B filter paper (soaked in 0.5% polyethylenimine to reduce non-specific binding), and a Brandel cell harvester. Samples were run in triplicate for both total and non-specific binding. Protein concentration in the final homogenate was determined by the method of Bradford (1976).
Saturation studies were initially carried out in the cerebral cortex only where adequate tissue was available. In this case, six concentrations of [3H]cytisine (0.3–12 nM) or six concentrations of [125I]-BT (0.1–10 nM) were utilized. Scatchard plots (for determination of Bmax and Kd) were generated by Radioligand Binding Analysis Program. Because striatum was the only region that showed differences in the binding of the ligands when using a single ligand concentration, separate groups of P and NP rats (five rats/group) were utilized to determine the binding affinities in the striatum. In this case, ten concentrations of [3H]cytisine (0.1–14 nM) and ten concentrations of [125I]-BT (0.1–12 nM) were used.
Statistical analysis
Data were analysed by one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc tests when significant main effects were indicated. All analyses were two-tailed and used an alpha of 
0.05 to determine significance.
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RESULTS |
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[3H]Cytisine binding
Figure 1
illustrates the 4 nM [3H]cytisine binding in discrete brain regions of P and NP rats. Binding density was significantly lower (by ~22%, P < 0.05) only in the striatum of P, compared to NP, rats. Scatchard analysis of [3H]cytisine binding in the striatum confirmed a decrease (22%) in the Bmax of P compared to NP (Table 1). There were no significant differences in the binding affintity between the two groups (Table 1). Scatchard analysis of [3H]cytisine binding in the cortex did not reveal any significant difference in the Bmax or Kd values between P and NP rats (Table 2).
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[125I]
-BT bindingFigure 2
illustrates the 2 nM [125I]-BT binding in discrete brain regions of P and NP rats. Binding density was significantly lower (66%, P < 0.01) only in the striatum of P compared to NP rats. Scatchard analysis of [125I]-BT binding in the striatum confirmed a decrease (~55%) in the Bmax of P compared to NP (Table 1
). There were no significant differences in the binding affinity between the two groups (Table 1). Scatchard analysis of the [125I]-BT binding in the cortex did not reveal any significant difference in the Bmax or Kd values between P and NP rats (Table 2).
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DISCUSSION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The results of this study indicate a reduction in binding to nicotinic receptors in the striatum of P, compared to NP, rats. This reduction was evident in both
4ß2 and 7 nicotinic receptor subtypes, suggesting a possible role for the striatal nicotinic receptors in propensity for alcohol preference. The striatal region in this study included the nucleus accumbens (NACC). Although the significance of NACC in reward and addictive effects of substances of abuse, including alcohol, is well established (Koob et al., 1998), recent reports also support a role for the dorsal striatum (caudate putamen) in unrestricted free-choice ethanol consumption in rats (Nestby et al., 1999; Yoshimoto et al., 1999).
It should be noted, however, that, in addition to preference for alcohol, P rats may differ from NP rats in other behavioural parameters, such as approach and avoidance learning (Blankenship et al., 1998) and associative learning (Slawecki et al., 1999). Moreover, it has been demonstrated that P and NP rats exhibit differential sensitivity to locomotor effects of peripherally (Gordon et al., 1993a), or centrally administered (Katner et al., 1996) nicotine. Thus, the extent to which striatal nicotinic receptors may be responsible for alcohol preference in P rats is yet to be determined. Hence, the functional status of these receptors and their modulatory role on alcohol consumption and/or preference need to be elucidated.
Both in vitro and in vivo interactions between alcohol and nicotinic receptors are amply documented. Recent electrophysiological studies in cultured cortical neurons suggest that nicotinic receptors are sensitive conduits for mediating the central actions of ethanol (Aistrup et al., 1999). Long-term ethanol treatment may affect nicotinic receptor densities in selective brain regions of rats and mice (Yoshida et al., 1982; Booker and Collins, 1997). Moreover, alterations in nicotinic receptors can affect ethanol self-administration in rats (Nadal and Samson, 1999). Our findings suggest that inherent preference for alcohol may also be influenced by differential expression of nicotinic receptors in discrete brain regions. This contention is supported by studies demonstrating that, in drug discrimination paradigms, nicotine substituted for ethanol in P, but not in NP, rats (Gordon et al., 1993b; Mcmillan et al., 1999).
The majority of central nicotinic receptors are located presynaptically (Wonnacott, 1997; Vizi and Lendvai, 1999). It has been reported that the striatum of P rats receives less serotonergic and dopaminergic innervation compared to NP rats (Zhou et al., 1994, 1995). Thus, the observed reductions in neuronal nicotinic receptors in the striatum of P rats may be related to the reduced neuronal arborization of this region in P rats.
Stimulation of different nicotinic receptor subtypes can differentially influence the release or activity of other neurotransmitters (Wonnacott, 1997; Vizi and Lendvai, 1999). In the striatum, for example, stimulation of the 4ß2 subtype may lead to an increase in dopamine release, whereas stimulation of 7 receptors may lead to an increase in glutamate release. It will be of considerable interest to determine whether striatal dopaminergic, serotonergic or other neurotransmission (e.g. glutamatergic) in the P rats may be normalized by exogenous administration of nicotinic analogues.
In summary, P rats exhibit a reduction in the striatal nicotinic receptors. This reduction, consistent with reduced neuronal innervation in this region of the P rats, might play a role in propensity for alcohol preference.
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ACKNOWLEDGEMENTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The authors thank Drs W. J. McBride and T.-K. Li, and the Indiana Alcohol Research Center (AA07611) for generously providing the rat brains. This work was supported by NIAAA 1U24AA11898–01 and ORMH.
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FOOTNOTES |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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* Author to whom correspondence should be addressed.
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