We found robustly induced posphorylation of AKT at Thr-308 and of DARPP-32 at Thr-34 in the mouse striatum. The delayed onset of the ethanol-evoked phosphorylation observed here is in line with the concept that the acute behavioral effects of ethanol result from indirect effects on various neurotransmitter systems rather than from ethanol’s actions on its primary targets (Spanagel 2009
). Phosphorylation of DARPP-32 following an ethanol challenge is in agreement with previous observation of similar effects following administration of several other addictive drugs (Svenningsson et al. 2005
). For AKT on the other hand, drug responses seems to be more diverse. In contrast to psychostimulants, which induce a robust dephosphorylation of AKT, ethanol leads to a robust phosphorylation of AKT (Beaulieu et al. 2005
, Neznanova et al. 2009
). While distinct from psychostimulants, this profile is similar to that previously described for morphine, a prototypical mu-opioid receptor agonist (Muller & Unterwald 2004
). These observations suggest that ethanol mimics the actions of direct mu-opioid agonists by inducing transient AKT phosphorylation, presumably through mu-opioid receptor activation.
We next asked whether ethanol-induced AKT and DARPP-32 phosphorylation is downstream of opioid or dopamine neurotransmission. To this end, we pre-treated mice with either the opioid antagonist naltrexone (1mg/kg i.p.) or the dopamine D2 receptor antagonist sulpiride (20 mg/kg), 30 min before ethanol administration (1.5 g/kg, i.p.). Ethanol-induced striatal AKT Thr-308 phosphorylation was abolished by naltrexone but not sulpiride (). In contrast, phosphorylation of DARPP-32 at Thr-34 was blocked by both antagonists ().
The finding that a D2 antagonist failed to block ethanol-evoked AKT phosphorylation in the striatum suggests a dopamine independent mechanism. In fact, release of endogenous opioids in response to ethanol has previously also been shown directly within the striatum, and it has long been noted that opioid receptor activation can produce psychostimulant and reinforcing effects in a direct, dopamine independent manner (Marinelli et al. 2003
; Vaccarino et al. 1986
). The picture that emerges is thus that ethanol can activate opioid mechanisms at two levels. One of these actions is upstream of the established DA activation in response to ethanol, which ultimately leads to induction of striatal DARPP-32 phosphorylation through activation of D2 receptors. The second, DA-independent component may be induced through direct actions within the striatum, and result in phosphorylation of AKT. Blockade of opioid receptors by naltrexone may act synergistically to prevent both these ethanol actions.
The canonical model for DARPP-32 phosphorylation at Thr-34 posits that D1 activation and subsequent protein kinase A (PKA) activation increase phosphorylation, whereas D2 activation inhibits it (Svenningsson et al. 2005
). Clearly, this model cannot explain the ability of the D2 antagonist sulpiride to block ethanol induced DARPP-32 phosphorylation observed here. However, ethanol has been shown to facilitate interactions between adenosine A2 and D2 receptors on medium spiny neurons that robustly activate PKA. This would in turn be expected to lead to increased phosphorylation of DARPP-32 (Yao et al. 2002
). It is possible that D2 antagonism affects phosphorylation through this mechanism. That naltrexone is similarly able to abolish striatal DARPP-32 phosphorylation is likely due to blockade of opioid receptors within the mesencephalon, thus interfering with the ability of endogenous opioids released in response to ethanol to disinhibit dopaminergic neurons.
In conclusion, our results show that reinforcing doses of ethanol increase both DARPP-32 and AKT phosphorylation in the mouse striatum. The differential sensitivity of these effects to naltrexone and sulpiride suggests of two distinct but potentially synergistic striatal signalling cascades that are initiated by actions of ethanol on endogenous opioid systems. One of these is D2-dependent, while the other is not.