Zolpidem generalization and antagonism in male and female cynomolgus monkeys trained to discriminate 1.0 or 2.0 g/kg ethanol

DRUG DISCRIMINATION HAS been used extensively to identify the receptors mediating the discriminative stimulus effects of drugs of abuse including ethanol (Colpaert, 1986; Solinas et al., 2006). Drug discrimination is pharmacologically specific. For example, in rats, mice, gerbils, pigeons, and monkeys, systemically administered positive modulators of γ-aminobutyric acid (GABA)A receptors including benzodiazepines, barbiturates, and neuroactive steroids, generalize from ethanol in drug discrimination protocols, whereas direct GABAA agonists (e.g., muscimol) and μ-opioid receptor agonists (e.g., morphine) do not produce ethanol-appropriate responding (for a review, see Grant, 1999; Grant et al., 1996, 2000). Identification of the types of receptors mediating the discriminative stimulus effects of ethanol could contribute to the development of novel alcoholism pharmacotherapies. The GABAA receptors are pentameric assemblies of subunits forming a Cl- channel, typically including 2 α,2 β, and 1 γ subunit (McKernan and Whiting, 1996). Subunit composition influences the pharmacological effect of receptor ligands (Sieghart, 1995). Responses to GABA at GABAA receptors that include α1, α2, α3, or α5 subunits are positively modulated by benzodiazepines, resulting in sedation, anxiolysis, anterograde amnesia, and anticonvulsant effects (Mohler et al., 2002). The GABAA receptor subtypes that mediate the discriminative stimulus effects of ethanol, particularly in nonhuman primates, require further characterization. Zolpidem is a positive modulator with 5- to 10-fold selectivity for GABAA receptors containing α1 subunits compared with receptors containing α2 or α3 subunits (Luddens et al., 1994; Sieghart, 1995; Smith et al., 2001). The activity of zolpidem at receptors containing α5 subunits is in general lower than at α1-3 subunits but appears to vary depending on the receptor subunit composition. In 1 study, zolpidem slightly enhanced (∼15%) responses to GABA at α5-containing receptors coexpressed with β2γ2 subunits (Sanna et al., 2002). Zolpidem is reported to be inactive at receptors containing α5 subunits coupled with β3 subunits (Luddens et al., 1994), or slightly efficacious (19%) at α5β3γ2S receptors (Smith et al., 2001). Zolpidem appears to be inactive at α4-(Sanna etal., 2002) and α6-(Hadingham et al., 1996) containing receptors. Studies in which squirrel monkeys are trained to discriminate zolpidem from saline suggest that the GABAA receptor subtypes mediating the discriminative stimulus effects of zolpidem depend on the training dose (Rowlett et al., 1999, 2000, 2003), similar to ethanol (Grant, 1999). Benzodiazepines that are not selective among the α subunit types, such as diazepam, generalize from the discriminative stimulus effects of low (1.0 mg/kg, i.v.), but not intermediate or high (3.0 or 5.6 mg/kg, i.v.), doses of zolpidem (Rowlett et al., 1999, 2000). The discriminative stimulus effects of higher doses of zolpidem, in contrast, may be mediated by selective and high-efficacy positive modulation of GABAA receptors containing α1 subunits (Rowlett et al., 2003). In general, zolpidem partially generalizes from the discriminative stimulus effects of ethanol. For example, zolpidem partially generalized from 1.0 g/kg ethanol (i.p.) in rats (Bienkowski et al., 1997; Sanger, 1997) and from 1.0 g/kg ethanol (i.v.) in squirrel monkeys (Platt et al., 2005), but did not generalize from 1.5 g/kg ethanol (i.p.) in mice (Shannon et al., 2004) or 1.0 g/kg ethanol (i.p.) in rats (Jankowska et al., 1996). Likewise, only 47% of the human participants in a study by Evans et al. (1990) indicated that the subjective effects of zolpidem resembled benzodiazepines, barbiturates, or ethanol. The GABAA receptor subtypes by which zolpidem produces ethanol-like discriminative stimulus effects are not entirely clear. According to in vitro studies, zolpidem may generalize from ethanol by acting at GABAA receptors containing α1, α2, α3, or possibly α5 subunits. In vivo studies of squirrel monkeys found that the α1-preferring antagonist β-carboline-t-butyl ester (β-CCt) did not block the generalization of 1 mg/kg zolpidem (i.v.) from 1.0 g/kg ethanol (i.v.) (Platt et al., 2005). These findings suggest that zolpidem generalizes from 1.0 g/kg ethanol not by acting at α1-containing receptors, but at receptors containing α2, α3, or perhaps α5 subunits. The latter may be discounted because α5-preferring inverse agonists (L-655,708, RY-23) did not block the partial generalization of zolpidem from ethanol in squirrel monkeys (Platt et al., 2005). The imidazobenzodiazepine Ro15-4513 has been called a specific ethanol antagonist because it antagonizes a range of ethanol effects including discriminative stimulus effects of 1.0 and 1.5, but not 2.0 g/kg ethanol (i.g.) in rats (Gatto and Grant, 1997), ethanol self-administration in rats (Petry, 1995; but see Shelton and Grant, 2001; cynomolgus monkeys), ethanol-induced incoordination in mice (Hoffman et al., 1987), sedation in rats (Suzdak et al., 1986), and anticonvulsant effects in mice (Nutt and Lister, 1987). Ro15-4513 has affinity for GABAA receptor subtypes with α4 or α6 subunits that are insensitive to classical benzodiazepines (Hanchar et al., 2006). In addition, Ro15-4513 has approximately equal affinity for GABAA receptors containing α1, α2, or α3 subunits. At α5-containing receptors, however, Ro15-4513 has approximately 10-fold greater affinity compared with α1, α2 or α3-containing receptors (coexpressed with β1γ2 subunits; Hadingham et al., 1993). The current study assessed antagonism of the generalization of zolpidem from ethanol by Ro15-4513 to gain information about the GABAA receptors mediating the discriminative stimulus effects of ethanol. Zolpidem and Ro15-4513 are useful for this purpose because they vary in selectivity for GABAA receptors depending on the α subunit. Whereas both drugs act at receptors containing α1, α2, α3, and α5 subunits, zolpidem is most potent at α1-containing receptors (Sanna et al., 2002) and Ro15-4513 has the greater affinity for α5-containing receptors (Luddens et al., 1994). The potency with which zolpidem generalizes from the discriminative stimulus effects of ethanol, and the potency with which Ro15-4513 antagonizes this generalization, may suggest hypotheses about the receptor population at which zolpidem acts to produce ethanol-like discriminative stimulus effects.