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

Abstract

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 microM 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 approximately 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 microM 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. British Journal of Pharmacology (2000) 129, 87 - 94

Figure 1

Characterization of AMPA subtype glutamate receptors on MS/DB neurons. In (A), application of 3 s pulses of 100 μM AMPA activates inward currents in three MS/DB neurons. Note initial transient peak currents in cells 1 and 3, but not cell 2. Simultaneous tests of AMPA combined with GYKI 52466 (GYKI; 100 μM) blunts initial rising phase of AMPA currents and largely blocks the subsequent sustained current (cells 2 and 3). SYM 2801 (100 μM) tested alone for 3 s activates little or no current in cells 2 and 3, while AMPA applied in combination with cyclothiazide (Cyclo; 10 or 100 μM) causes currents to increase during the test in cells 1 and 2. In (B), are shown mean±s.e.mean results for GYKI 52466 (100 μM) inhibition of initial ‘transient' (tran.) and ‘sustained' (sus.) AMPA currents; of SYM 2801-activated (100 μM) current; and of cyclothiazide potentiation of AMPA currents for 5–9 neurons is shown. Paired t, two-tailed, ^**P<0.01; ^***P<0.001 when compared with 100 μM AMPA alone.

Figure 2

Ethanol inhibits AMPA and kainate-activated currents in MS/DB neurons. Traces in (A) show 100 μM AMPA current is reversibly inhibited by 100 mM ethanol applied in combination for 3 s. On the left, kainate (Ka; 100 μM) activates currents without initial transients. Co-application with 10–300 mM ethanol (top traces) for 3 s shows increasing, but reversible inhibition. Lower traces from another cell show that 3 s 100 mM ethanol also inhibits kainate current, and that sustained ethanol exposure (pair of traces representing 0.5 and 5 min in ethanol), causes slightly greater inhibition, but no acute tolerance. Removal of ethanol partially reverses the inhibition, although recovery is not complete even after 5 min (‘Wash' pair of traces represents 0.5 and 5 min after ethanol). In (B) are shown mean±s.e.mean results for 100 mM ethanol inhibition of initial transient (tran.) and subsequent sustained (sus.) AMPA 100 μM current as well as concentration-dependent inhibition of 100 μM kainate current by ethanol 10–300 mM. Mean results are shown in (C) for kainate (100 μM) current and brief (3 s) and sustained (5 min) ethanol 100 mM exposure and the 5 min wash out of ethanol. Data represent results from 6–9 neurons. Paired t, two-tailed, ^*P<0.05; ^**P<0.01; ^***P<0.001 compared with the appropriate initial 100 μM AMPA or kainate alone in (B); in (C) ^*P<0.05 for 3 s ethanol inhibition vs 0.5 min; ^**P<0.01 for ethanol+kainate at 0.5 or 5 min vs initial kainate alone; also for washout 5.5 or 10 min kainate alone vs earlier 5 min ethanol+kainate.

Figure 3

Effect of chronic in vivo ethanol treatment on the sensitivity of MS/DB neurons to kainate. (A) shows individual recordings from two neurons, from a Control and an Ethanol Dependent animal, where similar concentration-dependent activation of inward current was stimulated by kainate (Ka; 3–3000 μM). Note 30 μM DNQX completely blocks 30 μM kainate currents in both cells. Also currents from the Control neuron show some desensitization above 100 μM kainate, which is not seen in the Ethanol Dependent cell. (B) shows mean±s.e.mean results for 3–3000 μM kainate currents normalized to neuronal capacitance for ten Ethanol Dependent and 16 Control neurons and inhibition by 30 μM DNQX of 30 μM kainate currents. Smooth curves were fit with equation 1. Paired t, two-tailed, ^*P<0.05 when DNQX+kainate is compared to kainate alone for both groups.

Figure 4

Ethanol causes comparable inhibition of kainate currents in cells from Naive, Control and Ethanol Dependent treatments. Brief application (3 s) of ethanol (3–300 mM) in combination with kainate (Ka; 100 μM) caused consistent concentration-dependent inhibition in 8–9 neurons from each treatment group. All ethanol treatments except 3 mM caused significant inhibition with paired t, two-tailed, ^*P<0.05; ^**P<0.01 or ^***P<0.001 when compared with kainate alone; ‘ns' signifies no difference in the extent of inhibition with 300 mM ethanol across groups.