Neurons of the ventral tegmental area (VTA) project to several regions of the extended amygdala including the nucleus accumbens, prefrontal cortex, and amygdala (Koob, 2003). Dopaminergic (DAergic) projections from neurons of the VTA participate in the mediation of the rewarding/reinforcing properties of numerous drugs of abuse (Wise, 1996). The dopamine (DA) neurons themselves are regulated by numerous afferent connections, including glutamatergic and GABAergic projections, as well as local GABAergic neurons (Adell and Artigas, 2004). In addition, the firing rates of DAergic neurons of the VTA are regulated by autoreceptors that inhibit the firing of these neurons (Grace, 1987).
There are predominantly 5 subtypes of DA receptors. There is structural homology between the D1 and D5 receptors, and both of these receptors are generally linked to Gαs, and stimulation of these receptors increases cAMP formation. The other 3 DA receptor subtypes (D2, D3, and D4) are structurally homologous and have been linked to Gαi and to reduction in cAMP formation (Neve et al., 2004). The autoreceptor on DAergic VTA neurons is of the D2 subtype, and activation of this receptor in mesencephalic DA neurons reduces the firing frequency of these neurons through a direct interaction of its G-protein with somatic membrane potassium channels (Kim et al., 1995; Lacey et al., 1988).
Ethanol (EtOH) has numerous specific actions on DA VTA neurons. In the VTA, acute EtOH increases h-current (Brodie and Appel, 1998), reduces M-current (Koyama et al., 2007), and increases barium-sensitive potassium current (McDaid et al., 2008). In addition, EtOH increases glutamatergic (Deng et al., 2009) and increases GABAergic postsynaptic potentials (Theile et al., 2008). Some actions of EtOH may directly cause the phenotypic response to EtOH (e.g., increased firing; Brodie et al., 1990; Gessa et al., 1985) and other effects may not directly play a role (McDaid et al., 2008), but may be involved in modulating those direct effects; the direct and modulatory effects may play a role in adaptation to EtOH subsequent to chronic (intermittent, repeated, or continuous) administration (Okamoto et al., 2006). A careful assessment of the acute effects of EtOH on these important neurons is the first step in understanding how EtOH actions in the VTA are related to the development of alcoholism.
We recently reported a time-dependent and concentration-dependent desensitization of DA D2 receptors requiring concurrent activation of D1-like and D2-like receptors which we called DA inhibition reversal (DIR; Nimitvilai and Brodie, 2010), and this desensitization is mediated by phospholipase C and the protein kinase C (PKC) pathway (Nimitvilai et al., 2012). The present report describes experiments to determine whether EtOH interacts with DIR.
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- Materials and Methods
The results presented here indicate that a low concentration of EtOH can interfere with desensitization of pDAergic VTA neurons. Human blood alcohol concentrations at legal intoxication in the United States (0.08 mg%) is equivalent to about 17 mM, and rats appear to be less sensitive to the effects of EtOH than humans (Haggard et al., 1940; Majchrowicz and Hunt, 1976), so the concentrations at which EtOH reduces DIR are pharmacologically relevant. We have previously reported a phenomenon of DIR with extended periods of exposure to moderate concentrations of DA, which differs from homologous and heterologous desensitization as it requires the concurrent stimulation of D1/D5 and D2 DA receptors (Nimitvilai and Brodie, 2010). We have begun characterizing this phenomenon and have found that it is dependent on PKC, but not on cAMP or protein kinase A, is sensitive to external calcium concentration, and is dependent on intracellular calcium release (Nimitvilai et al., 2012). Pure DA D2 agonists like quinpirole do not exhibit DIR, but reversal of quinpirole inhibition can be observed in the presence of D1/D5 agonists (Nimitvilai and Brodie, 2010) or activators of PKC like PMA (Nimitvilai et al., 2012). Our observation that EtOH blocks the reversal of quinpirole inhibition in the presence of PMA indicates that EtOH interferes with DIR at a step at or beyond the activation of PKC.
The action of EtOH on PKC is a possible mechanism by which it blocks DIR. There are numerous reports examining the action of EtOH on PKC (for review, see Newton and Messing, 2006; Stubbs and Slater, 1999). Acute EtOH can inhibit activation of PKC by interfering with translocation to the plasma membrane (Slater et al., 1993; Steiner et al., 1997). The effect of EtOH on PKC differs by subtype of PKC, as PKCα (Reneau et al., 2011; Slater et al., 1997), PKCγ (Harris et al., 1995; Rex et al., 2008), and PKCδ (Rex et al., 2008) have been reported to be inhibited by EtOH, but other isoforms are resistant to EtOH inhibition, like PKCβ1 and PKCε, for example (Rex et al., 2008). Other laboratories have shown that EtOH can enhance the activity of some PKC isoforms, such as PKCε (Messing et al., 1991; Satoh et al., 2006). The effect of EtOH on PKC isoforms can be tissue specific, as EtOH decreases the activity of PKCδ in HEK293 cells (Rex et al., 2008) but increased PKCδ activity in neural PC12 cells (Messing et al., 1991). One study found that the function of highly purified PKC was not reduced by EtOH (Machu et al., 1991). Additional studies will be necessary to ascertain whether EtOH acts on PKC to disrupt DIR in pDA VTA neurons and whether other actions of EtOH contribute to this disruption.
Once DIR is activated, it is not reversed (i.e., sensitivity to DA-induced inhibition is not returned to initial levels) by application of D1/D5 antagonists (Nimitvilai and Brodie, 2010). This indicates a long-lasting change in processes that regulate the sensitivity of the response to D2 agonists, such as a sustained change in the phosphorylation state of the D2 receptor or in the surface expression of that receptor. Alternatively, it could be due to other intracellular processes (like PKC activation state) that are initiated by concurrent D1/D5 and D2 stimulation and that persist after those processes have been triggered. As EtOH application following DIR induction did not restore sensitivity to DA-induced inhibition (Fig. 3), the mechanisms by which EtOH disrupts DIR does not influence expression of DIR once it is induced. As additional studies elucidate the molecular pathways involved in the mechanism of DIR, the points at which EtOH interferes with those pathways may become clearer.
As the reduction in desensitization to DA-induced inhibition can be blocked by low-to-moderate concentrations of EtOH, acute administration of EtOH may cause a functional enhancement of inhibition by endogenous DA in the presence of EtOH. The net effect of this action of EtOH would be to decrease the excitability of pDAergic VTA neurons, making them less sensitive to excitatory neurotransmitters or other factors. The primary effect of EtOH on DA VTA neurons is excitation, whether it is measured in vivo (Gessa et al., 1985) or in vitro (Brodie et al., 1990). Excitation is due to a direct action on the DA neurons (Brodie et al., 1999b) and may be the result of EtOH blockade of some potassium channels, including M-current (Koyama et al., 2007). The antagonism by acute EtOH of DIR would act to undermine that direct excitation. EtOH itself increases the concentration of DA in the VTA over time (Kohl et al., 1998), and the inhibitory effect of that DA would be maintained (would not exhibit desensitization) in the presence of EtOH, as we show here. Like the effect of EtOH on barium-sensitive potassium channels (McDaid et al., 2008), this would tend to decrease the excitatory effects of acutely administered EtOH in naïve subjects.
The acute and chronic actions of EtOH on regulation of autoreceptor sensitivity of pDAergic VTA neurons may be important for determining initial sensitivity as well as chronic response of these neurons to EtOH. As noted previously, this action of EtOH would tend to decrease the acute excitatory response to EtOH. Initial low sensitivity to the effects of EtOH has been correlated with a higher risk of the development of alcoholism (Schuckit, 1994). The reduction of DIR induced by acute EtOH would reduce the activity of DAergic neurons of the VTA and might have a variety of effects on motor activity. It may be that reduced sensitivity to EtOH blockade of DIR may be related to increased risk of alcoholism. Extensive additional studies would be needed to determine whether the variant of the D2 receptor (Blum et al., 1991; Curtis et al., 1999) or associated gene products (Dick et al., 2007) that are correlated with risk to alcoholism differ in sensitivity to desensitization or to EtOH action on the processes related to desensitization, respectively. As genetic variants in D2 receptors are linked to other psychiatric diseases (Noble, 2003), these variants may also differ in their sensitivity to DIR.
As DIR appears to be an expression of desensitization, our results show a very potent effect of EtOH on that desensitization. During withdrawal from chronic EtOH exposure, the activity of DA VTA neurons has been reported to be reduced (Diana et al., 1992); others have reported an increased number of silent DA neurons in the VTA (Shen and Chiodo, 1993). Desensitization of D2 receptors in the VTA has been reported to be reduced in mice after 7 days of repeated intraperitoneal injection of EtOH, which resulted in greater autoinhibition and reduced DA tone (Perra et al., 2011). In contrast, an increase in DA release in nucleus accumbens and caudate/putamen was observed in rats after exposure to EtOH vapor for 5 to 10 days (Budygin et al., 2007); in this study, there was a lack of effect of quinpirole on DA efflux in the nucleus accumbens, suggesting a lack of change in autoreceptor regulation of DA release. As we show here that acute EtOH potently reduces desensitization of D2 autoreceptors, chronic or repeated EtOH may induce adaptive changes in regulation of DA neuronal activity to compensate for the lack of desensitization of D2 receptors in the persistent presence of EtOH. As the studies noted previously use different species as well as different methods of EtOH exposure, additional studies will be necessary to examine the mechanisms of adaptation to persistent block of DIR by EtOH and to determine whether the mode of EtOH administration influences how the physiology of DA VTA neurons adapts to the persistent presence of EtOH.
In VTA neurons from mice, autoreceptor inhibition is attenuated after chronic EtOH (Perra et al., 2011). In that study, the desensitization appeared to be linked to calmodulin kinase II and not to PKC; desensitization to DA in rat pDA VTA neurons is clearly dependent on PKC (Nimitvilai et al., 2012). We show here that the processes that result in the production of DIR by DA do not result in the reduction of EtOH excitation, but activation of PKC by PMA did in fact result in a reduction of sensitivity to EtOH excitation. This paradox may be due to a more selective activation of PKC by DA or by a more potent activation of PKC by PMA than by DA. The calcium dependence of DIR, and blockade of DIR by the conventional PKC antagonist Gö6976, suggests the involvement of a conventional PKC (Nimitvilai et al., 2012), whereas acute or chronic EtOH can affect some isoforms of novel PKC, including PKCε (Jiang and Ye, 2003) and PKCδ (Gerstin et al., 1998) (for review, please see Newton and Messing, 2006). Many additional studies would be necessary to identify the cellular elements altered by PKC that result in the decrease in EtOH excitation in DA VTA neurons. The role of EtOH to block DIR may reveal important actions of acute EtOH to disrupt information processing in the VTA. Examination of this action of EtOH on pDAergic VTA neurons may permit a better understanding of the array of actions of EtOH on these neurons, especially when the acute actions of EtOH are compared with the effects of EtOH after chronic alcohol administration.