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. 2007 Mar 28;27(13):3593-602.
doi: 10.1523/JNEUROSCI.4749-06.2007.

Ethanol induces long-term facilitation of NR2B-NMDA receptor activity in the dorsal striatum: implications for alcohol drinking behavior

Jun Wang  1 , Sebastien Carnicella, Khanhky Phamluong, Jerome Jeanblanc, Jennifer A Ronesi, Nadia Chaudhri, Patricia H Janak, David M Lovinger, Dorit Ron
Affiliations

Ethanol induces long-term facilitation of NR2B-NMDA receptor activity in the dorsal striatum: implications for alcohol drinking behavior

Jun Wang et al. J Neurosci. .

Abstract

Addiction is characterized by compulsive alcohol or drug taking and seeking, and the dorsal striatum has been implicated in such maladaptive persistent habits. The NMDA receptor (NMDAR), which is a major target of alcohol, is implicated in striatal-based habit learning. We found that, in the dorsal striatum, alcohol (ethanol) exposure produced an increase in the phosphorylation of the NR2B subunit of the NMDAR, and a corresponding increase in the activity of Fyn kinase, which phosphorylates NR2B. We further observed an ethanol-mediated long-term facilitation (LTF) of the activity of NR2B-containing NMDARs (NR2B-NMDARs) in the dorsal striatum. This LTF is Fyn kinase dependent, because it was observed in Fyn wild-type but not in Fyn knock-out mice. Importantly, none of these biochemical and physiological changes was observed in the ventral striatum. Finally, dorsal but not ventral striatum infusion of a Fyn or NR2B-NMDAR inhibitor reduced rat operant self-administration of ethanol. Our results suggest that the Fyn-mediated phosphorylation and LTF of NR2B-NMDAR activity in the dorsal striatum after exposure to ethanol may underlie aberrant plasticity that contributes to mechanisms underlying alcohol drinking behavior.

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Figures

Figure 1.
Figure 1.
Ethanol treatment increases NR2B subunit phosphorylation and Fyn activation in the rat dorsal striatum. A, Left, Ethanol incubation increased the phosphorylation level of the NR2B subunit in the dorsal, but not the ventral, striatum. Dorsal or ventral striatal slices were treated with 100 mm ethanol for 30 min. Phosphorylation of the NR2B subunit was detected by anti-[pY1252]NR2B antibodies (pY; top). The membrane was stripped and reprobed with anti-NR2B antibodies (bottom). The image is representative of five experiments. Right, Bar graph showing ethanol-mediated phosphorylation of the NR2B subunit in the dorsal striatum. The level of [pY1252]NR2B (p-NR2B) was normalized to total NR2B. *p < 0.05 versus control; n = 5 experiments, two rats per experiment. B, Ethanol incubation did not alter the phosphorylation state of the NR2A subunit in the dorsal or the ventral striatum. Dorsal or ventral striatal slices were treated as in A. The NR2A subunit was immunoprecipitated with anti-NR2A antibodies, and phosphorylation of NR2A was detected with anti-phosphotyrosine antibodies (pY; top). The membrane was stripped and reprobed with anti-NR2A antibodies (bottom). The image is representative of three independent experiments. C, Left, Ethanol treatment increased the activity of Fyn kinase in the dorsal but not the ventral striatum. Dorsal or ventral striatal slices were treated with 100 mm ethanol for 20 min. Fyn was immunoprecipitated with anti-Fyn antibodies, and activation of Fyn was detected by [pY417]Fyn antibodies (pY; top). The blot was stripped and reprobed with anti-Fyn antibodies (bottom). The image is representative of four independent experiments. Right, Quantification of the level of [pY417]Fyn (p-Fyn) to total immunoprecipitated Fyn. *p < 0.05 versus control; n = 4 experiments, 2 rats per experiment. D, Left, Systemic administration of ethanol increased the activity of Fyn kinase in the dorsal striatum. C57BL/6 mice received three intraperitoneal injections of saline for habituation before test injection of saline or ethanol (2.5 g/kg). Fifteen minutes after test injection, the dorsal striatum was dissected and homogenized, and activation of Fyn was detected by [pY417]Fyn antibodies (pY; top). The blot was stripped and reprobed with anti-Fyn antibodies (bottom). The image is representative of six saline and six ethanol injections. Right, Quantification of the level of [pY417]Fyn (p-Fyn) to total immunoprecipitated Fyn. **p < 0.01 versus the saline group by Student's t test; n = 6 experiments. E, Ethanol does not affect the activity of Src kinase. Dorsal or ventral striatal slices were treated with ethanol as in C. The slices were homogenized, and Src was immunoprecipitated. Activation of Src was detected by [pY418]Src antibodies (pY; top). The blot was stripped and reprobed with anti-Src antibodies (bottom). The image is representative of four experiments. F, Ethanol modulates the association of RACK1, Fyn, and the NR2B subunit in the dorsal striatum. Dorsal or ventral striatal slices were treated with or without 100 mm ethanol for 30 min. The slices were homogenized and RACK1 was immunoprecipitated with anti-RACK1 antibodies (bottom), and coimmunoprecipitation was detected with anti-Fyn (middle) and anti-NR2B (top) antibodies. The input lane shows level of proteins in total lysates. The image is representative of three independent experiments.
Figure 2.
Figure 2.
LTF of NMDAR EPSCs in the rat dorsal but not ventral striatum. A, Ethanol-mediated induction of LTF in the dorsal striatum. Left, Time course of NMDAR EPSCs recorded before, during (15 min as indicated by the horizontal bar), and after 50 and 100 mm ethanol application. Note that the mean EPSC amplitude is facilitated after ethanol washout (n = 15 slices). The inset shows sample traces at times 1, 2, and 3. The stimulus artifacts have been omitted for clarity. Calibration: 100 ms, 100 pA. Middle, Same as left, but with 50 mm ethanol. Right, Bar graph comparing the direct (EtOH) and washout (post-EtOH) effects of different concentrations of ethanol on NMDAR EPSCs. EPSCs were averaged from 10–14 min after ethanol application (EtOH), and from 21–30 min after ethanol washout (post-EtOH). #p < 0.01, *p < 0.05 versus baseline (average of EPSCs over the 10 min before ethanol application) by paired Student's t test; ##p < 0.05. B, Ethanol failed to induce LTF in the ventral striatum. Left, Time course of NMDAR EPSCs recorded in the ventral striatum before, during, and after 100 mm ethanol application. Note that, on average, no LTF was observed (n = 14 slices). Right, Bar graph comparing the ethanol (100 mm) effect on mean NMDAR EPSCs in the dorsal (same data as A, left) and the ventral (same data as B, left) striatum. #p < 0.01, *p < 0.05 versus baseline; **p < 0.05. C, Sustained NR2B subunit phosphorylation after ethanol washout in the dorsal striatum. Dorsal or ventral striatal slices were treated with 100 mm ethanol for 20 min. The slices were homogenized and the NR2B subunit was immunoprecipitated. Phosphorylation of the NR2B subunit was detected by anti-[pY1252]NR2B antibodies (pY; top). The membrane was stripped and reprobed with anti-NR2B antibodies (bottom). The image is representative of three independent experiments. Quantification of the level of pYNR2B (p-NR2B) to total immunoprecipitated NR2B subunits is shown. *p < 0.05, **p < 0.01 versus control; n = 3 experiments, two rats per experiment. D, Sustained activation of Fyn activity after ethanol washout in the dorsal striatum. Dorsal or ventral striatal slices were treated with 100 mm ethanol for 15 min. Ethanol was removed, and fresh aCSF was added for 10 min. The slices were homogenized, and Fyn was immunoprecipitated with anti-Fyn antibodies. Activation of Fyn was detected by anti-[pY417]Fyn antibodies (pY; top). The blot was stripped and reprobed with anti-Fyn antibodies (bottom). The image is representative of four independent experiments. Quantification of the level of [pY417]Fyn (p-Fyn) to total immunoprecipitated Fyn is shown. *p < 0.05, **p < 0.01 versus control; n = 4 experiments, two rats per experiment.
Figure 3.
Figure 3.
LTF in the rat dorsal striatum is mediated by NR2B-NMDARs. A, Blockade of NR2B-NMDARs prevents LTF induction by ethanol. Left, Time course of NMDAR EPSCs in the absence and presence of the NR2B antagonist, Ro 25-6981 (0.5 μm), and ethanol (100 mm). Note that LTF was not observed after ethanol washout in the presence of Ro 25-6981 (n = 9 slices). Right, Bar graph showing the mean NMDAR EPSCs before (pre-EtOH), during (EtOH), and after (post-EtOH) 100 mm ethanol application in the presence of Ro 25-6981. EPSCs were averaged from 13–15 min after starting perfusion of Ro 25-6981 for “pre-EtOH,” from 10 to 14 min after starting perfusion of EtOH for “EtOH,” and from 21 to 30 min after ethanol washout for “post-EtOH.” *p < 0.05; n.s., not significant, p > 0.05 by paired Student's t test. Error bars indicate SEM. B, Ro 25-6981 blocks ethanol-induced LTF of NMDAR EPSCs. Left, time course. n = 10 slices. Right, Left, Bar graph comparison of EPSCs in the presence of Ro 25-6981 before ethanol application (“pre-EtOH”; quantification of the mean EPSCs 13–15 min after starting perfusion of Ro 25-6981 in A, left) and after LTF is established (“LTF”; quantification of the mean EPSCs 13–15 min after starting perfusion of Ro 25-6981 in B, left). Note that the EPSCs in the presence of Ro 25-6981 before ethanol application and after LTF is established are not significantly different (p > 0.05). Right, Right, Bar graph comparison of the ratios of NR2B to non-NR2B EPSCs before ethanol application and after LTF is established. The non-NR2B EPSC was defined as the EPSC in the presence of Ro 25-6981, whereas the NR2B EPSC was calculated as the difference between the EPSCs before and after Ro 25-6981 application. Note that the ratio of NR2B/non-NR2B EPSCs is increased in LTF compared with pre-EtOH. *p < 0.05.
Figure 4.
Figure 4.
Fyn kinase is required for ethanol-induced LTF in the dorsal striatum. A, The Src family antagonist, PP2, prevents LTF induction by ethanol in the rat dorsal striatum. PP2 (1 μm) was bath-applied and present throughout the recordings. Note that LTF was not detected after ethanol washout (n = 11 slices). B, Deletion of the Fyn kinase gene prevents ethanol-induced LTF in the mouse dorsal striatum. Note that the LTF induced by ethanol was observed in Fyn+/+ mice (n = 12 slices) but not in Fyn−/− mice (n = 16 slices). C, Summary of the mean NMDAR EPSCs during (EtOH) and after (post-EtOH) ethanol applications in A and B is shown. #p < 0.01, *p < 0.05 versus corresponding baselines; **p < 0.05. Error bars indicate SEM.
Figure 5.
Figure 5.
Probability of neurotransmitter release remains unchanged during ethanol-induced LTF. A, Sample traces of the paired-pulse NMDAR EPSCs obtained before ethanol application (3, solid line) and after LTF was established (4, solid line) and explanation of how the PPR of the EPSCs was calculated (1–3). 1, A single EPSC elicited by one stimulus; 2, paired-pulse EPSCs elicited by two stimuli with an interval of 100 ms. Single EPSCs and paired-pulse EPSCs were alternately induced at an interval of 20 s; 3, the true amplitude of the second EPSC (dashed line) can be seen after digital subtraction of the single EPSC (1) from the paired-pulse EPSCs (2). The PPR of NMDAR EPSCs was defined as the ratio of P2 over P1, where P2 and P1 are the amplitudes of the subtracted second EPSC (dashed line) and the first EPSC in the paired-pulse EPSCs (solid line), respectively. 3 also shows unsubtracted sample traces obtained before ethanol application and 4 after LTF was established. B, Line graph summarizing PPRs of NMDAR EPSCs obtained before ethanol application (pre-EtOH) and after LTF is established (post-EtOH). PPRs are not significantly different (p > 0.05 by paired Student's t test; n = 7 slices). Error bars indicate SEM. C, Sample traces of the paired-pulse AMPAR EPSCs obtained before ethanol application (1) and after LTF was established (2). D, Line graph summarizing the PPRs of AMPAR EPSCs obtained before and after ethanol application. The PPRs between pre- and post-EtOH applications are not significantly different (p > 0.05 by paired Student's t test; n = 6 slices).
Figure 6.
Figure 6.
A, Intradorsal striatal injections of PP2 and ifenprodil attenuate operant ethanol self-administration. PP2 (0.3, 1.5, and 3 ng; equivalent to 1, 5, and 10 μm, respectively), PP3 (1.5 ng), and ifenprodil (0.5 μg) were microinjected (1 μl/side) into the dorsal striatum 15 min before the beginning of the test session. Data are expressed as mean ± SEM of number of lever presses in 1 h (n = 11 rats). A two-way ANOVA with repeated measures (active vs inactive lever as the first factor and treatment as the second factor) found significant main effects and a significant interaction between both factors (F values > 4.55; p values < 0.01). Post hoc comparisons revealed significantly reduced active lever responding after 1.5 and 3 ng PP2 and ifenprodil, compared with those after vehicle, 0.3 ng PP2, and PP3 (p values < 0.01); PP2 and ifenprodil did not change inactive lever responding (p values > 0.89), *p < 0.01, **p < 0.001. Mean ethanol intakes were 0.48 ± 0.06 g/kg for vehicle, 0.46 ± 0.08, 0.31 ± 0.05 and 0.31 ± 0.05 g/kg, respectively, for the doses of 0.3, 1.5, and 3 ng of PP2, 0.46 ± 0.07 for PP3, and 0.34 ± 0.07 for ifenprodil. B, Intradorsal striatal injection of PP2 or ifenprodil does not change the distribution of interresponse intervals. Mean ± SEM of the relative number of interresponse intervals (IRIs) (expressed as the percentage of total IRIs) with all intervals ≤20 s presented in 1 s time bins, as well as the percentage of IRIs that were >20 s (n = 11 rats). A two-way ANOVA with repeated measures (time interval as the first factor and treatment as the second factor) found a significant effect of the time interval (F(60,599) = 48.61; p < 0.001), but no effect of treatment and no interaction (F values < 0.93; p values > 0.54). C, Intradorsal striatal injection of PP2 (1.5 ng) or ifenprodil (0.5 μg) does not attenuate operant sucrose self-administration. A two-way ANOVA with repeated measures found a significant effect of lever (F(1,26) = 82.41; p < 0.001), but no effect of treatment and no interaction (F values < 0.55; p > 0.59); n = 14 rats. D, Infusion of an effective dose of PP2 (1.5 ng) or ifenprodil (0.5 μg) into the ventral striatum does not reduce operant ethanol self-administration. A two-way ANOVA with repeated measures found a significant effect of lever (F(1,12) = 36.67; p < .001), but no effect of treatment and no interaction (F values < 0.46; p > 0.64); n = 7 rats.
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