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, 108 (16), 6650-5

Alcohol Inhibition of the NMDA Receptor Function, Long-Term Potentiation, and Fear Learning Requires Striatal-Enriched Protein Tyrosine Phosphatase

Affiliations

Alcohol Inhibition of the NMDA Receptor Function, Long-Term Potentiation, and Fear Learning Requires Striatal-Enriched Protein Tyrosine Phosphatase

Tianna R Hicklin et al. Proc Natl Acad Sci U S A.

Abstract

Alcohol's deleterious effects on memory are well known. Acute alcohol-induced memory loss is thought to occur via inhibition of NMDA receptor (NMDAR)-dependent long-term potentiation in the hippocampus. We reported previously that ethanol inhibition of NMDAR function and long-term potentiation is correlated with a reduction in the phosphorylation of Tyr(1472) on the NR2B subunit and ethanol's inhibition of the NMDAR field excitatory postsynaptic potential was attenuated by a broad spectrum tyrosine phosphatase inhibitor. These data suggested that ethanol's inhibitory effect may involve protein tyrosine phosphatases. Here we demonstrate that the loss of striatal-enriched protein tyrosine phosphatase (STEP) renders NMDAR function, phosphorylation, and long-term potentiation, as well as fear conditioning, less sensitive to ethanol inhibition. Moreover, the ethanol inhibition was "rescued" when the active STEP protein was reintroduced into the cells. Taken together, our data suggest that STEP contributes to ethanol inhibition of NMDAR function via dephosphorylation of tyrosine sites on NR2B receptors and lend support to the hypothesis that STEP may be required for ethanol's amnesic effects.

Conflict of interest statement

Conflict of interest statement: M.D.B. has a financial interest in PhosphoSolutions, Inc., which provided several of the antibodies in this study.

Figures

Fig. 1.
Fig. 1.
Microinjection of STEP C/S into postsynaptic neurons blocks the inhibitory effects of ethanol on NMDAR EPSCs. (A) Time course of cumulative data shows ethanol effects on NMDAR whole-cell current recordings from male Sprague-Dawley rats for control (Con), TAT-Myc (Myc), and STEPC/S (C/S)-injected neurons. (B) Representative NMDAR whole-cell current traces for control, C-Myc, and C/S-injected neurons. BSL, baseline; EtOH, ethanol; and WASH, ethanol washout period. (C) Composite data show quantification of the percent change from baseline in NMDAR currents during ethanol treatment (E) and ethanol washout (W). One-way ANOVA analyses with post hoc Tukey test show that ethanol significantly inhibited NMDAR currents in Con (P < 0.001) and in Myc-treated (P < 0.001) neurons, but it had no significant effect on C/S treated neurons (P = 0.847). During washout, there were some residual effects of ethanol in Con (P < 0.005) and no effects in Myc-treated neurons (P = 0.150), but C/S-treated neurons showed a significant increase (P < 0.001). ***P < 0.001. Number of cells: Con (n = 10); Myc (n = 5); STEPC/S (n = 5). (Scale bar, 50 ms and 50 pA.)
Fig. 2.
Fig. 2.
Ethanol fails to inhibit NMDAR EPSCs but reintroduction of STEP can restore ethanol's inhibition in STEP KO mice. (A) Ethanol (40, 80, and 120 mM) show differential effects on NMDAR EPSCs. Among WT mice, one-way ANOVA indicated that ethanol dose-dependently inhibited NMDAR currents [F(3,21) = 24.976, P < 0.001]. Post hoc Tukey test analyses showed that 80 and 120 mM ethanol significantly affected NMDAR responses (P < 0.001). In KO mice, ethanol significantly enhanced NMDAR currents [F(3,28) = 3.017, P < 0.05]. Post hoc Tukey test analyses showed that 80 and 120 mM ethanol significantly increased NMDAR currents (P < 0.05). (B) Time course of ethanol (80 mM) effects on NMDAR EPSC amplitudes in WT (n = 8) and KO (n = 6) animals. Representative traces show whole-cell currents of a hippocampal CA1 neuron from a WT and KO mice. Dashes indicate baselines. (C) (Left) Composite data show that ethanol application (WT, E, n = 8) inhibited the NMDAR EPSCs in slices from WT mice but had no effect during the washout (WT, W, n = 8). In contrast, ethanol potentiated the NMDAR EPSCs both during ethanol application (KO, E, n = 10) and washout (KO, W, n = 8). (Right) Composite data show that, in neurons from WT mice, PP2 had no effect either during ethanol application (WT-PP2, E, n = 13) or during washout (WT-PP2, W, n = 10). However, in neurons from KO mice, PP2 blocked the NMDAR enhancement seen with ethanol application (KO-PP2, E, n = 10) and during washout (KO-PP2, W, n = 9). (D) Intracellular microinjection of the control peptide, TAT-Myc, (WT, Con, C, Clear bars) and TAT-wtSTEP (STEP, cross-hatched bar) do not alter ethanol effects on NMDAR EPSCs in neurons from WT mice. Intracellular microinjection of TAT-Myc (KO, Con, C, striped bar) does not alter the enhancing effects of ethanol on NMDAR EPSCs, but microinjection of TAT-wtSTEP (KO, STEP, cross-hatched bar) restores ethanol inhibition of NMDAR EPSCs. TAT-wtSTEP has no effect in WT neurons [Con (C), n = 8]; [wtSTEP (STEP), n = 9]. TAT-STEP restores ethanol inhibition in STEP KO neurons [Con (C), n = 6]; [wtSTEP (STEP), n = 5]. One-way ANOVA with a post hoc Tukey test analysis determined significance, *P < 0.05, **P < 0.01, ***P < 0.001. (Scale bar, 150 ms and 150 pA.)
Fig. 3.
Fig. 3.
Ethanol fails to inhibit LTP induction and fear learning in STEP KO mice. Cumulative time course shows ethanol significantly blocks LTP slope in WT (A) but not in STEP KO (B) hippocampal slices. Control responses (○) and ethanol- (●) (80 mM) treated responses. (C) Composite data show ethanol inhibits LTP slope from WT slices, control (Con, C, n = 11); ethanol (E, n = 5) and not in STEP KO slices (Con, C, n = 10); EtOH (E, n = 4). Solid bars: WT; striped Bars: KO. In addition, the amplitude of LTP did not differ between WT and STEP KO slices (t = 1.158, P > 0.261). (D) Quantification of freezing behavior during a 3-min interval testing cued fear conditioning. WT animals froze 80.6 ± 3.8% of the time with saline and 43.1 ± 8.6% with ethanol (P < 0.001). STEP KO animals froze 69.1 ± 5.3% and froze 56.2 ± 7.8% with saline and ethanol, respectively (P > 0.19). WT (n = 8); KO (n = 9). (E) Quantification of freezing behavior during a 5-min testing interval for contextual learning. WT treated with saline froze 50.4 ± 7.4% of the time- and ethanol-treated froze 15.8 ± 5.3% of the time (P < 0.002). Saline-treated STEP KO mice froze 38.9 ± 8.7%, and ethanol-treated STEP KO animals froze 22.2 ± 4.9% (P > 0.10). (F) Composite data for BEC (mg%) for WT and STEP KO animals at 15 and 60 min post ethanol administration. Two-way ANOVA determined significant time-dependent decrease in BEC levels but no genotypic differences of BEC. Solid bars, WT animals; striped bars, KO animals; S, saline (white); E, 1.5 g/kg ethanol (gray). *P < 0.05, **P < 0.002.
Fig. 4.
Fig. 4.
Ethanol treatment does not reduce NR2B tyrosine phosphorylation in STEP KO mice. (A) Representative Western blot and quantification of decreased tyrosine phosphorylation on immunoprecipitated NR2B subunits after 10-min 80 mM ethanol treatment. Tyr (P): WT (n = 13); KO (n = 10). (B) Tyrosine phosphorylation on NR2A subunits after ethanol treatment. No significant change on NR2A tyrosine phosphorylation was detected in either WT or STEP KO mice Tyr (P): WT (n = 15); KO (n = 11). (C) Significant decrease in Tyr1472 phosphorylation of NR2B subunits after 80-mM ethanol treatment in slices from WT (n = 13) (P < 0.05), but not KO (n = 10) mice. (D) NR2B from immunoprecipitated hippocampal homogenates WT (n = 15); KO (n = 12). *P < 0.05, **P < 0.01. Solid bars, WT; striped bars, KO; C, control aCSF-treated minislices; E, ethanol-treated minislices.

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