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. 2011 May-Jun;5(3):205-9.
doi: 10.4161/chan.5.3.14856. Epub 2011 May 1.

Ethanol-mediated long-lasting adaptations of the NR2B-containing NMDA receptors in the dorsomedial striatum

Jun Wang  1 , Maria Fe Lanfranco, Stuart L Gibb, Dorit Ron
Affiliations

Ethanol-mediated long-lasting adaptations of the NR2B-containing NMDA receptors in the dorsomedial striatum

Jun Wang et al. Channels (Austin). 2011 May-Jun.

Abstract

We recently found that ethanol-induced long-term facilitation (LTF) of NMDAR activity is mediated by NR2B-NMDARs and is observed in the dorsomedial striatum (DMS) but not in the dorsolateral striatum (DLS). We also showed that repeated administration of ethanol causes a long-lasting increase in NMDAR activity in the DMS, resulting from ethanol-mediated Fyn phosphorylation of NR2B subunits. In this addendum, we report that the different sensitivity of NMDARs to ethanol between the DMS and DLS is not attributed to the abundance of synaptic NR2B-NMDARs or differences in Fyn levels. We further show that LTF is specific for NR2B-, but not NR2A-NMDARs, and that the duration of the in vivo ethanol-mediated increase in NMDAR activity is associated with the period of ethanol exposure, but not with alteration in NR1 or NR2A protein levels. Together, these results suggest that upregulation of NR2B-NMDAR activity by ethanol is selective and that ethanol's effect on NMDAR activity is gradual and cumulative.

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Figures

Figure 1
Figure 1
The abundance of synaptic NR2B-NMDARs does not differ between the DMS and DLS in naïve rats. Striatal slices were prepared from 3- to 4-week-old Sprague Dawley rats. NMDAR-mediated EPSCs were measured in the presence of 0.05 mM Mg2+, 0.1 mM picrotoxin, 0.01 mM NBQX and with neurons clamped at −70 mV. Inhibition of the EPSCs in the presence of a NR 2B-NMDAR specific antagonist, Ro 25-6981, was similar in DMS and DLS neurons. (A) Representative traces of NMDAR-mediated EPSCs in the absence (Baseline) and presence (Ro) of Ro 25-6981 (0.25 µM) in DMS (left) and DLS (right) neurons. Scale bars: 100 ms, 20 pA. (B) Bar graph showing no difference in the extent of inhibition of EPSCs by Ro 25-6981 between DMS and DLS neurons. p > 0.05 by t-test, n = 6 for DMS and DLS.
Figure 2
Figure 2
Fyn protein levels do not differ between the DLS and DMS in naïve rats. Western blot analysis was used to examine the total protein levels of Fyn kinase in the DLS and DMS of juvenile (21 days) and adults (90 days) Sprague-Dawley rats. Actin was used as a loading control.
Figure 3
Figure 3
The NR2A subunit-containing NMDAR does not contribute to ethanol-mediated LTF in the DMS. (A) Time-course of NMDAR-EPSCs in DMS neurons from NR2A knockout (KO) and wild-type (WT) mice before, during and after bath application of ethanol (80 mM). n = 7 slices for each group. (B) Bar graph comparing the magnitude of LTF in the DMS from NR2A WT and NR2A KO mice. LTF is calculated as the magnitude of averaged EPSCs from 21–30 min after ethanol was washed out. *p < 0.05 vs. baseline, **p < 0.01 vs. baseline, ##p < 0.05 WT vs. KO. Two-way ANOVA with repeated measures. n = 7 (NR2A KO mice). n = 7 (WT mice).
Figure 4
Figure 4
Duration of the ethanol-mediated increase in NMDAR activity is associated the period of ethanol exposure. (A) Single administration of ethanol did not lead to a detectable change in NMDA-induced current. Rats were systemically administrated with 2 g/kg of ethanol or saline and striatal slices were prepared 16 hrs after the administration to measure NMDA-induced currents. Left, Changes in holding currents were measured after NMDA (10 µM, 30 s) was applied to the slices. n = 16 (saline) and 16 (EtOH). Right, Bar graph summarizing the peak amplitudes of NMDA-induced currents in DMS neurons from ethanol- and saline-treated rats. p > 0.05 by t-test. (B) Repeated ethanol administration does not alter presynaptic release of neurotransmitter. The frequency of miniature EPSCs (mEPSCs) were compared in DMS neurons from saline- and ethanol-treated animals. n = 25 (saline) and 29 (ethanol) slices. p > 0.05, Mann-Whitney Rank Sum test. (C) Seven administrations of ethanol did not alter NMDA-induced currents 40 hrs later after the last treatment. Rats were systemically administered with 2 g/kg of ethanol or saline daily for seven days and striatal slices were prepared 40 hrs after the last administration to measure NMDA-induced currents. Left, Changes in holding currents were measured after NMDA (10 µM, 30 s) was applied to the slices. n = 14 (saline) and 14 (EtOH). Right, Bar graph summarizing the peak amplitudes of NMDA-induced currents in DMS neurons from ethanol- and saline-treated rats. p > 0.05 by t-test.
Figure 5
Figure 5
Repeated ethanol administration did not alter the protein levels of NR1 or NR2A subunits in the DMS. (A and B) Sprague-Dawley rats were treated with saline or ethanol (2 g/kg, i.p.) once a day for seven days. DMS was dissected 16 hrs after the last injection. (A) Repeated ethanol administration did not alter the protein levels of NR1 or NR2A subunits in total DMS homogenates. Left, image is representative of n = 3 for NR1 (top) and n = 9 for NR2A (bottom). p > 0.05 vs. saline-treated rats (two-tailed t-test). (B) Repeated ethanol administration did not alter the protein levels of synaptosomal NR1 or NR2A subunits in the DMS. Left, image is representative of n = 6 (saline) and n = 6 (ethanol) for NR1 (top). n = 6 (saline) and n = 5 (ethanol) for NR2A (bottom). (A and B) Middle and right, bar graphs summarizing the averaged changes in protein levels of NR1 (middle) and NR2A subunits (right). Western blot data was normalized to GAPDH and plotted as percentage of saline treatment. p > 0.05 vs. saline-treated rats (two-tailed t-test). (C) Intermittent access to ethanol did not alter the protein levels of NR1 or NR2A in total DMS homogenates. Long Evans rats were exposed to 18 ethanol-drinking sessions under an intermittent access to 20% ethanol in a two-bottle-choice paradigm. One day after the last drinking session, DMS tissue was dissected out and protein levels of NR1 and NR2A subunits in homogenates were measured. Left, Image is representative of n = 7 (water). n = 8 (ethanol). Middle and right, bar graphs summarizing the averaged changes in protein levels of NR1 (middle) and NR2A subunits (right). Western blot data was normalized to GAPDH and plotted as percentage of water treatment. p > 0.05 vs. control (two-tailed t-test).
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