Sustained ethanol inhibition of native AMPA receptors on medial septum/diagonal band (MS/DB) neurons.

The direct impact of ethanol on native, non-NMDA glutamate receptors was examined in acutely isolated MS/DB neurons from rat. The impact of ethanol functional tolerance and physical dependence on non-NMDA receptor function was also determined. Non-NMDA receptors were defined pharmacologically as predominantly the AMPA subtype, because both AMPA- or kainate-activated currents were blocked by GYKI 52466, a selective AMPA receptor antagonist. The relative magnitude of potentiation of AMPA-activated currents by 10 or 100 μM cyclothiazide was consistent with recombinant AMPA flop-subtype receptors. Finally, the selective kainate receptor agonist, SYM 8021, induced little current in MS/DB neurons. AMPA receptor currents when activated by kainate were sensitive to ethanol, showing inhibition of ∼5–50% when 10–300 mM ethanol and kainate were briefly co-applied (3 s). Ethanol (100 mM) also inhibited both the initial transient peak and sustained currents activated by AMPA. Inhibition was sustained during continuous ethanol superfusions of 5 min, suggesting a lack of acute tolerance to ethanol-induced AMPA receptor blockade. Rapid application of 3–3000 μM kainate activated concentration-dependent currents in MS/DB neurons from Control and Ethanol Dependent animals that were not significantly different. Also, direct ethanol inhibition (300 mM) of kainate-activated currents was not reduced by ethanol dependence, suggesting a lack of functional tolerance. These results suggest that native AMPA receptors on MS/DB neurons are inhibited by pharmacologically-relevant concentrations of ethanol. However, these receptors, unlike NMDA receptors, do not undergo adaptation with sustained ethanol exposure sufficient to induce physical dependence. Keywords: Medial septum/diagonal band, non-NMDA glutamate receptors, AMPA receptor, ethanol intoxication, tolerance and dependence, patch-clamp electrophysiology Introduction Glutamate serves as the principal excitatory neurotransmitter in the mammalian central nervous system (Watkins & Evans, 1981; Dingledine et al., 1999), generating fast excitatory postsynaptic potentials by activation of ionotropic receptors of the α-amino-3-hydroxy-5-methyl-4-isoxalonepropionic acid (AMPA; subunits GluR1-4), kainate (subunits GluR5-7, KA-1&2) and N-methyl-D-aspartate (NMDA; subunits NR1-3) subtypes and modulating neurotransmitter release through activation of metabotropic G-protein receptors (mGluR1-8) or kainate subtype receptors (Nakanishi et al., 1998; Dingledine et al., 1999). Glutamate neurotransmission generally appears to be a target for ethanol and likely plays important roles in aspects of ethanol intoxication such as cognitive impairment, blackouts and ethanol withdrawal excitability, as well as in excitotoxic mechanisms involved in longer-term brain damage in foetal alcohol syndrome and Wernicke-Korsakoffs syndrome (Tsai et al., 1995; Faingold et al., 1998). At a cellular level, ethanol exerts a well characterized acute inhibition of both native (Peoples et al., 1997; Thomas et al., 1998; Bhave et al., 1999) and recombinant (Dildy-Mayfield & Harris, 1992; Koltchine et al., 1993) NMDA receptors, an action which in some model systems may reverse within minutes of continuing exposure in a manner consistent with acute tolerance (Grover et al., 1994; Miyakawa et al., 1997). Under some conditions, ethanol induces functional receptor up-regulation during chronic treatment over several days (Trevisan et al., 1994; Chen et al., 1997; Grover et al., 1998; Kumari & Ticku, 1998; Thomas et al., 1998), although changes in binding sites for NMDA receptor ligands are not consistently robust (see for review Rudolph et al., 1997). Such ethanol-induced increases in NMDA receptor function likely make significant contributions to functional tolerance, withdrawal hyperexcitability, calcium-dependent excitotoxicity and long-term brain damage in chronic alcoholism (Tsai et al., 1995; Grover et al., 1998; Thomas et al., 1998). Interestingly, NMDA receptors containing certain subunits, show greater relative inhibition by ethanol (Yang et al., 1996; Bhave et al., 1999), and potentially may further increase the vulnerability of neurons expressing them to chronic ethanol toxicity. Ethanol, also inhibits, acutely, the activity of native and recombinant non-NMDA ionotropic receptors. Overall, a similar degree of inhibition is observed for both recombinant AMPA or kainate receptor subtypes in expression systems (Lovinger, 1993; Minami et al., 1998b; Valenzuela & Cardoso, 1999). Whether there are receptor subtype selective actions of ethanol for native neuronal AMPA/kainate receptors is less certain (Lovinger et al., 1990; Dildy-Mayfield & Harris, 1992; Lovinger, 1993; Martin et al., 1995; Yang et al., 1996), in part due to a lack until recently of selective pharmacological tools for distinguishing native AMPA and kainate subtypes (Johansen et al., 1995; Wilding & Huettner, 1995; Donevan et al., 1998; Dingledine et al., 1999; Savidge et al., 1999; Weiner et al., 1999). Evidence that in vitro or in vivo chronic ethanol exposure can up-regulate non-NMDA receptors is mixed (Trevisan et al., 1994; Molleman & Little, 1995; Chandler et al., 1999), and specific effects on pharmacologically characterized native AMPA or kainate receptor subtypes largely remains to be determined. Finally, the actions of acute or chronic ethanol on either native or recombinant metabotropic glutamate receptors have received even less attention than NMDA, AMPA or kainate receptors and their interactions with ethanol largely remain to be defined or confirmed (Minami et al., 1998a). The purpose of the present investigation was to define the impact of immediate in vitro ethanol application and in vivo tolerance and dependence-inducing chronic ethanol treatment on the function of native non-NMDA inotropic receptors found on medial septum/diagonal band (MS/DB) neurons. Previously, ethanol was shown, acutely to inhibit native NMDA receptors on MS/DB neurons from rat, while induction of physical dependence up-regulated peak NMDA receptor currents in these cells and induced resistance to inhibitory actions of immediate ethanol application (Grover et al., 1998). In the present study, currents activated by either AMPA or kainate in MS/DB neurons were studied and shown pharmacologically to be mediated by AMPA subtype glutamate receptors. AMPA receptor currents were inhibited by pharmacologically-relevant concentrations of ethanol (10–300 mM). However, unlike NMDA receptors, AMPA receptors on MS/DB neurons were not up-regulated following induction of physical ethanol dependence and did not show functional tolerance to immediate ethanol inhibition. Together, these findings are consistent with the idea that AMPA receptors, which are the primary carriers of fast excitatory neurotransmission, are relatively resistant to the sustained inhibitory influence of ethanol when compared with NMDA receptors, which have a prominent role in synaptic plasticity and thus undergo adaptive up-regulation in response to ethanol blockade.