Ethanol-mediated facilitation of AMPA receptor function in the dorsomedial striatum: implications for alcohol drinking behavior

Abstract

We found previously that acute ex vivo as well as repeated cycles of in vivo ethanol exposure and withdrawal, including excessive voluntary consumption of ethanol, produces a long-lasting increase in the activity of NR2B-containing NMDA receptors (NR2B-NMDARs) in the dorsomedial striatum (DMS) of rats (Wang et al., 2010a). Activation of NMDARs is required for the induction of long-term potentiation (LTP) of AMPA receptor (AMPAR)-mediated synaptic response. We therefore examined whether the ethanol-mediated upregulation of NMDAR activity alters the induction of LTP in the DMS. We found that ex vivo acute exposure of striatal slices to, and withdrawal from, ethanol facilitates the induction of LTP in DMS neurons, which is abolished by the inhibition of NR2B-NMDARs. We also report that repeated systemic administration of ethanol causes an NR2B-NMDAR-dependent facilitation of LTP in the DMS. LTP is mediated by the insertion of AMPAR subunits into the synaptic membrane, and we found that repeated systemic administration of ethanol, as well as cycles of excessive ethanol consumption and withdrawal, produced a long-lasting increase in synaptic localization of the GluR1 and GluR2 subunits of AMPARs in the DMS. Importantly, we report that inhibition of AMPARs in the DMS attenuates operant self-administration of ethanol, but not of sucrose. Together, our data suggest that aberrant synaptic plasticity in the DMS induced by repeated cycles of ethanol exposure and withdrawal contributes to the molecular mechanisms underlying the development and/or maintenance of excessive ethanol consumption.

Figure 1.

Ex vivo ethanol exposure and withdrawal upregulates NMDAR but not AMPAR activity. Striatal slices from Sprague Dawley rats were treated for 1 h with 40 mm ethanol, which was then washed out for 30 min before electrophysiological measurements. Control slices (Ctrl) were exposed to the same treatment but without ethanol (EtOH). A, Ex vivo ethanol treatment causes an increase in NMDA-induced currents. Changes in holding currents in DMS neurons were measured after NMDA (10 μm, 30 s) was bath applied to control and ethanol-treated slices. n = 8 per group. B, Ex vivo ethanol treatment does not alter AMPA-induced currents. Changes in holding currents were measured in DMS neurons after AMPA (0.2 μm, 30 s) was bath applied. n = 8 per group. C, Ex vivo ethanol treatment causes an increase in NMDA/AMPA ratio. Left, Sample traces of NMDAR-mediated and AMPAR-mediated EPSCs in control slices (top) and in ethanol-treated slices (bottom). Calibration: 30 ms, 30 pA. Right, Bar graph summarizing the mean of NMDA/AMPA ratios in control slices and ethanol-treated slices. **p < 0.01 (t test). n = 9 (Ctrl) and n = 10 (EtOH). D, Ex vivo ethanol treatment does not alter the amplitude or frequency of AMPAR-mediated mEPSCs (AMPAR-mEPSCs) in DMS neurons. Left, Sample traces of mEPSCs in control slices (top) and in ethanol-treated slices (bottom). Calibration: 0.2 s, 10 pA. Middle, right, Bar graph summarizing the mean amplitudes (middle) and frequencies (right) of AMPAR-mediated EPSCs in control and ethanol-treated slices. n = 10 per group. n.s., Not significant.

Figure 2.

Ex vivo ethanol exposure and withdrawal facilitates the induction of LTP in an NR2B-NMDAR-dependent manner. Striatal slices from Sprague Dawley rats were treated with 40 mm ethanol for 1 h, ethanol was washed out for 30 min, and HFS was delivered in the absence (EtOH) or presence (EtOH/Ro) of Ro 25-6981 (0.5 μm), in which Ro 25-6981 was present throughout the recording period. Control slices (Ctrl) were exposed to the same treatment but without ethanol or Ro 25-6981. A, Sample traces of fEPSP/PSs before (1, 1′) and after (2, 2′) HFS in Ctrl (top) as well as EtOH-treated (middle) and EtOH/Ro-treated (bottom) slices. Note that the peak of the fEPSP/PSs (the second downward waveform) increases after HFS in EtOH-treated slices, but not in Ctrl or EtOH/Ro-treated slices. The stimulus artifacts have been omitted for clarity. Calibration: 2 ms, 0.1 mV. B, Time course of fEPSP/PSs before and after HFS in Ctrl (white circles) as well as EtOH-treated (black circles) and EtOH/Ro-treated (triangles) slices. Note that HFS induced greater increases in fEPSP/PSs amplitude in EtOH-treated slices than in Ctrl and EtOH/Ro-treated slices. The numbers 1, 1′, 2, and 2′ indicate time points where the sample traces in A were selected. C, Bar graphs summarizing the mean amplitudes of fEPSP/PSs 20–30 min after HFS in control (white bar), EtOH-treated (black bar) and EtOH/Ro-treated (gray bar) slices. n.s., p > 0.05 for EPSCs at time point 2 versus time point 1 (SNK test); ***p < 0.001 for EPSCs at time point 2′ versus time point 1′ (SNK test); ^##p < 0.01 (SNK test); ^###p < 0.001 (SNK test). n = 11, 10, and 9 for Ctrl, EtOH, and EtOH/Ro, respectively.

Figure 3.

Repeated daily in vivo administration of ethanol facilitates the induction of LTP in an NR2B-NMDAR-dependent manner in the DMS. Sprague Dawley rats were systemically administered once daily with saline or ethanol (20%, 2 g/kg) for 7 d, and striatal slices were prepared 16 h after the seventh administration. HFS was delivered in slices from saline (Sal)- and ethanol-treated animals. In the latter case, Ro 25-6981 was absent (EtOH) or present (EtOH/Ro) throughout the recording period. A, Time course of fEPSP/PSs before and after HFS in Sal (white circles), EtOH (black circles), and EtOH/Ro (triangles) groups. Note that HFS induced greater increases in fEPSP/PSs amplitude in EtOH than Sal and EtOH/Ro conditions. B, Bar graphs comparing the mean amplitudes of fEPSP/PSs after HFS in Sal, EtOH, and EtOH/Ro groups. ***p < 0.001 for EPSCs after HFS versus baseline (SNK test); ^###p < 0.001 (SNK test). n = 11, 8, and 10 for Sal, EtOH, and EtOH/Ro, respectively. n.s., Not significant.

Figure 4.

Repeated systemic ethanol administration and excessive ethanol intake upregulate the protein levels of synaptic AMPAR subunits in the DMS. A, Repeated systemic administration of ethanol upregulates the protein levels of synaptic AMPARs. Sprague Dawley rats were systemically administered ethanol or saline once a day for 7 successive days. DMS tissues were dissected out 16 h after the seventh administration, and GluR1 and GluR2 levels in total homogenates (Total) and at synaptosomal membranes (Synaptic) were measured by Western blot. Left, Sample images of total and synaptosomal protein levels of GluR1 and GluR2. Right, Bar graph summarizing mean protein levels of GluR1 and GluR2 in saline- and ethanol-treated animals. n = 3 for each group. B, Excessive ethanol intake upregulates synaptic GluR1 and GluR2 subunits of AMPARs. Long–Evans rats underwent an intermittent-access to 20% ethanol in a two-bottle choice drinking procedure for 7–8 weeks, DMS tissues were dissected 1 d after the last ethanol drinking session [ethanol withdrawal (EW)], and protein levels of GluR1 and GluR2 in total homogenates and synaptosomal membranes were measured. Left, Sample images of total and synaptosomal protein levels of GluR1 and GluR2. Right, Average of protein levels of GluR1 and GluR2 subunits in water controls (Water) and ethanol-treated animals. n = 7 (water) and 6 (EW) for total GluR1; 7 (water) and 8 (EW) for total GluR2; 5 (water) and 5 (EW) for synaptic GluR1, and 7 (water) and 7 (EW) for synaptic GluR2. *p < 0.05.

Figure 5.

Inhibition of AMPARs reduces operant self-administration of ethanol, but not of sucrose in the DMS. Aa–Ac, Mean ± SEM of the number of lever presses for ethanol after intra-DMS infusion of vehicle or NBQX (Aa) and mean ± SEM of ethanol intake (Ab) in rats trained on a FR3 schedule to obtain 0.1 ml of a 20% ethanol solution per delivery during a 30 min session. Ac, Schematic representation of cannulae placements (gray circles) in coronal sections from the ethanol self-administration experiments. **p < 0.01 (SNK test). n = 7 for each group. Ba, Mean ± SEM of the number of lever presses for sucrose after intra-DMS infusion of vehicle or NBQX. Bb, Schematic representation of cannulae placements (gray circles) in coronal sections from the sucrose self-administration experiments. n = 9 for each group.