α2A‐Adrenergic Receptor Signaling Underlies Synergistic Enhancement of Ethanol‐Induced Behavioral Impairment by Clonidine

Interactions between alcohol and prescription drug medications, such as the antihypertensive drug clonidine, represent a serious concern for public health. The antagonistic hemodynamic interaction between ethanol and clonidine, which renders the antihypertensive effect of clonidine ineffective, has been documented (El-Mas and Abdel-Rahman, 1999, 2001; Mao and Abdel-Rahman, 1998); however, an equally important behavioral interaction between these two drugs also produces a clinically serious adverse effect. The synergistic behavioral impairment elicited by clonidine-ethanol interaction has been reported (Mao and Abdel-Rahman, 1996) and supported by findings from interactions with related drug classes (Czarnecka et al., 1986; Durcan et al., 1991; Idanpaan-Heikkila et al., 1995; Kushikata et al., 2002). Although the sedative effect of clonidine is utilized in some clinical applications, such as analgesia, anesthesia, or treatment of withdrawal symptoms (Fauler and Verner, 1993; Khan et al., 1999), the combined use of ethanol and clonidine and the resultant synergistic behavioral impairment is a greater concern for patients taking clonidine unsupervised as an antihypertensive agent. Little is known, however, about the mechanisms underlying this synergistic interaction. Furthermore, clonidine is a mixed α2/I1-receptor agonist that induces its hypotensive effect primarily via the I1-imidazoline receptor (El-Mas and Abdel-Rahman, 2001; Head and Mayorov, 2006) and its sedative effects primarily via the α2A-adrenergic receptor (AR) (Hunter et al., 1997; Lahdesmaki et al., 2003; Lakhlani et al., 1997). There is, nonetheless, some cross-over in this system, such that both receptor types also contribute to the nonprimary function. For example, hypotension is partly mediated by α2AR activation (Reis, 1996), while the rostral ventrolateral medulla (RVLM) sends I1-responsive projections to the locus coeruleus (LC), where clonidine is thought to produce its behavioral effects via α2AR (Meana et al., 1995; Ruiz-Durantez et al., 2002; Ruiz-Ortega and Ugedo, 1997). Therefore, while α2AAR signaling is likely implicated in clonidine–ethanol behavioral interaction, direct evidence in support of this notion is lacking. Further, the possibility has not been investigated that other molecular targets of clonidine (α2BAR or I1-receptor) might be implicated in the interaction. It is also important when undertaking mechanistic studies to investigate neurochemical responses that might identify the neuroanatomical substrate(s) implicated in the behavioral response. Induction of immediate early genes, such as c-fos and its protein c-Fos, can serve as a marker of neuronal activity (Dragunow and Faull, 1989). In many brain areas, induction or inhibition of c-Fos expression has been associated with ethanol-evoked behavioral responses (Ryabinin, 1998; Sharpe et al., 2005; Thiele et al., 2000). In the Edinger-Westphal nucleus (EW), ethanol-induced c-Fos expression was modulated by α2AR activation (Bachtell et al., 2002). In the few studies that described the effect of clonidine on c-Fos or its gene, clonidine was shown to decrease induced c-fos mRNA expression in the nucleus tractus solitarius (NTS) and RVLM (El-Mas and Abdel-Rahman, 2000). Importantly, the studies that have implicated the α2AR in the modulation of the neurochemical (c-Fos expression) effects of ethanol did not directly evaluate the effect of α2AR activation on c-Fos expression in the brainstem or investigate the associated behavioral effects of ethanol in the presence of α2AR blockade (Bachtell et al., 2002). Equally important, no studies have evaluated changes in c-Fos expression as a result of clonidine–ethanol combination, particularly in the LC, which is an obvious target for clonidine action due to its high density of α2AR binding sites (Aghajanian and VanderMaelen, 1982). Indeed, when injected into the LC, clonidine or the α2AR agonist dexmedetomidine produces α2AR mediated sedative effects by inhibition of LC firing, which likely disinhibits GABAergic neuronal activity downstream to cause behavioral impairment (Aghajanian and VanderMaelen, 1982; Correa-Sales et al., 1992; De Sarro et al., 1987; Guo et al., 1996; Pudovkina et al., 2001). Likewise, ethanol has also been shown to induce behavioral impairment by inhibition of LC firing, and it has been suggested that the pre-existing level of LC activity (level of arousal) may affect responsiveness to ethanol (Palmer and Granholm, 1992; Verbanck et al., 1991), possibly accounting for some inter-individual differences in observed behavioral responses to ethanol. Overall, clonidine and ethanol produce sedation independently by inhibiting LC firing; however, it is yet unclear whether the neurochemical effects of their combination in the LC could explain the additive/synergistic behavioral interaction between both drugs. Therefore, the major goal of the present studies was to test the hypothesis that central α2AAR signaling plays a key role in the synergistic enhancement of behavioral impairment caused by ethanol in the presence of clonidine and that the LC is implicated in the interaction. To test this hypothesis, we investigated the effect of clonidine pretreatment on the behavioral response (impaired rotorod performance and loss of righting reflex, LORR) elicited by moderate doses of ethanol. Pharmacological interventions were used to identify the α2AR subtype implicated in the interaction and whether the I1-receptor, which is also activated by clonidine, plays a role in the interaction. In these studies we utilized selective α2A or α2BAR antagonist and the I1-receptor agonist rilmenidine. Finally, we measured the changes in c-Fos in the LC elicited by a moderate dose of ethanol in the presence or absence of clonidine; whether similar changes in c-Fos occurred in other brain areas (e.g., cerebellum) was investigated.