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Fyn-Kinase as a Determinant of Ethanol Sensitivity: Relation to NMDA-Receptor Function

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Science  24 Oct 1997:
Vol. 278, Issue 5338, pp. 698-701
DOI: 10.1126/science.278.5338.698

Abstract

Animals vary in their sensitivity to ethanol, a trait at least partly determined by genetic factors. In order to identify possible responsible genes, mice lacking Fyn, a non–receptor type tyrosine kinase, were investigated. These mice were hypersensitive to the hypnotic effect of ethanol. The administration of ethanol enhanced tyrosine phosphorylation of theN-methyl-d-aspartate receptor (NMDAR) in the hippocampus of control mice but not in Fyn-deficient mice. An acute tolerance to ethanol inhibition of NMDAR-mediated excitatory postsynaptic potentials in hippocampal slices developed in control mice but not in Fyn-deficient mice. These results indicate that Fyn affects behavioral, biochemical, and physiological responses to ethanol.

Ethanol (EtOH) is among the most widely abused drugs in the world, yet the neural mechanisms responsible for EtOH intoxication and dependence are largely unknown. Genetic factors affect the determination of the behavioral responses to EtOH in rodents and humans (1), but few specific genes that increase or decrease the drug actions have been reported (2, 3).

Tyrosine kinases phosphorylateN-methyl-d-aspartate (NMDA) and γ-aminobutyric acid A (GABAA) receptors and modulate the electrophysiological function of these receptors (4-6). The function of these receptors is also modulated by EtOH, and they are hypothesized to be targets through which EtOH exerts its behavioral effects (7). To investigate the possible involvement of Fyn tyrosine kinase in the behavioral sensitivity to EtOH, we assessed the hypnotic effect of EtOH on Fyn-deficient mice (8). At all doses tested, the duration of the loss of righting reflex (LORR) after EtOH administration was significantly longer for Fyn-deficient (homozygous Fyn-deficient,fyn z/fyn z) mice than for control (heterozygous Fyn-deficient, +/fyn z) mice (Fig. 1A). It is unlikely that the difference between the two groups was due to a difference in the general excitability of the central nervous system or in the general ability of a mouse to right itself, as no significant differences were found in the duration of the LORR induced by flurazepam (a benzodiazepine derivative) at any dose (Fig. 1B). Analysis of the blood EtOH concentration curve (8, 9) revealed no significant differences between the two groups (Fig. 1C). The enhanced sensitivity to EtOH is, therefore, likely due to changes in the sensitivity of the central nervous system rather than to changes in pharmacokinetic or metabolic factors. Thus, lack of Fyn tyrosine kinase exacerbates the hypnotic effects of EtOH.

Figure 1

Enhanced sensitivity to the hypnotic effect of EtOH in Fyn-deficient mice. Sensitivity was evaluated by measuring the duration of LORR after administration of (A) EtOH or (B) flurazepam (doses measured per kilogram of mouse body weight). Two-way analysis of variance (ANOVA) showed a significant difference [F(1,81) = 5.78, P < 0.02] in the sensitivity to EtOH. There was no significant effect in the sensitivity to flurazepam [F(1,100) = 0.39, P > 0.50]. The number of animals tested is shown in parentheses below each column. Asterisks indicate a significant difference between +/fyn z andfyn z/fyn z mice at each dose (simple main effect; *P < 0.05, **P < 0.01, ***P < 0.0001). (C) Blood EtOH concentration curve in +/fyn z andfyn z/fyn z mice (seven mice of each genotype). There were no statistically significant differences between the two genotypes (two-way repeated-measure ANOVA).

Because EtOH enhances tyrosine phosphorylation in A431 cells (a human epidermal carcinoma) (10) and in neural cell line PC12 cells (11), we examined whether such modulation of tyrosine phosphorylation by EtOH also occurred in the brains of +/fyn z andfyn z/fyn z mice (12). The level of tyrosine phosphorylation after saline treatment was not significantly different between the two groups. A significant enhancement in tyrosine phosphorylation of a 180-kD protein (p180) 5 min after EtOH administration was observed in the hippocampus of +/fyn z mice but not in the hippocampus offyn z/fyn z mice (Fig.2, A and B). The enhancement was also observed in C57BL/6, a standard inbred mouse strain. The lack of this up-regulation infyn z/fyn z mice indicates that it is mainly mediated by Fyn tyrosine kinase.

Figure 2

Up-regulation of protein tyrosine phosphorylation after EtOH administration. (A) Representative immunoblots of extracts prepared after the administration of saline or EtOH with a phosphotyrosine-specific antibody. Arrowheads indicate p180. (B) Quantification of the level of phosphotyrosine at 180 kD. Two-way ANOVA showed that the effect of gene and treatment×gene interaction were significant [F(1,32) = 5.88, P < 0.05;F(3,32) = 2.96, P < 0.05]. A significant enhancement in tyrosine phosphorylation was observed 5 min after EtOH administration in +/fyn z mice [Tukey's honestly significant difference (HSD) test, α = 0.05] but not infyn z/fyn z mice. (C) Tyrosine phosphorylation of NMDARɛ1 and ɛ2 and the amount of NMDARɛ1 and ɛ2 protein in the hippocampus 5 min after the administration of saline (lane 1, +/fyn z; lane 2,fyn z/fyn z) or EtOH (lane 3, +/fyn z; lane 4,fyn z/fyn z). Each sample with the same number was obtained from the same animal. Group effect was significant in tyrosine phosphorylation of NMDARɛ2 [F(3,20) = 4.18, P < 0.02]. It was significantly greater 5 min after the administration of EtOH than it was 5 min after saline administration [control, 1.00 ± 0.00; EtOH, 2.89 ± 0.58; Tukey's HSD test, α = 0.05] in +/fyn z mice but not infyn z/fyn z mice [control, 0.84 ± 0.35; EtOH, 1.09 ± 0.66]. There were no significant group effects in the tyrosine phosphorylation of NMDARɛ1 [1.00 ± 0.00, 0.61 ± 0.12, 1.33 ± 0.25, and 0.67 ± 0.33, in the same order as that of the representative blots] nor in the amounts of the NMDARɛ1 [1.09 ± 0.11, 1.12 ± 0.25, 1.25 ± 0.21, and 1.15 ± 0.12] and NMDARɛ2 [1.06 ± 0.05, 1.00 ± 0.07, 0.97 ± 0.06, and 1.12 ± 0.11] proteins (P > 0.05).

In the central nervous system of the rats, a tyrosine-phosphorylated protein band of molecular mass 180 kD in the postsynaptic density fraction contains NMDAR2B (corresponding to NMDARɛ2 in mice) (13) and NMDAR2A (NMDARɛ1) (5). The tyrosine phosphorylation of NMDAR2B is up-regulated in response to lesions with 6-OH-dopamine (14), taste learning (15), and the induction of long-term potentiation (16). In addition, NMDARɛ1 and ɛ2 are colocalized with Fyn in the postsynaptic density and phosphorylated by Fyn (5). Consequently, we investigated the level of tyrosine phosphorylation of NMDARɛ1 and ɛ2 after saline and EtOH treatment in the hippocampus, where the up-regulation occurred (17). Enhanced tyrosine phosphorylation of NMDARɛ2 5 min after EtOH treatment was observed in +/fyn z mice but not infyn z/fyn z mice (Fig. 2C, first row). On the other hand, +/fyn z mice andfyn z/fyn z mice had similar amounts of NMDARɛ2 after saline and EtOH administration (Fig.2C, second row). Concerning NMDARɛ1, neither tyrosine phosphorylation nor the amount of receptor protein was up-regulated (Fig. 2C, third and fourth rows). It is therefore likely that the enhanced tyrosine phosphorylation of NMDARɛ2 after EtOH treatment accounts for the enhancement of tyrosine phosphorylation of p180.

The inhibition of NMDAR-mediated excitatory postsynaptic potentials (EPSPs) by EtOH is gradually reduced during the period of EtOH exposure in hippocampal slices (acute tolerance) (18). This acute tolerance might be caused by the up-regulation of tyrosine phosphorylation of NMDAR subunits, because NMDAR currents are potentiated by tyrosine kinases in hippocampal neurons (4). To test this possibility, we compared the effects of EtOH on NMDA-mediated EPSPs in the CA1 hippocampal neurons of +/fyn z andfyn z/fyn z mice (19). Bath application of EtOH initially suppressed NMDA-mediated EPSPs, but the amplitude of the EPSPs gradually recovered in +/fyn z mice during the application of EtOH, showing the development of acute tolerance (Fig.3, A, B, and D). By contrast, infyn z/fyn z mice, EtOH suppressed NMDA-mediated EPSPs with little sign of development of tolerance (Fig. 3, A, C, and D), and EPSPs recovered their original amplitude after EtOH was removed. Thus, modulation of NMDAR function by Fyn seems to be involved in the development of acute tolerance to EtOH. Furthermore, the acute tolerance was eliminated when EtOH was applied together with ifenprodil, an agent considered to be a selective antagonist of NMDAR containing NMDARɛ2 (20) (Fig. 3E). These findings are consistent with the notion that enhancement of tyrosine phosphorylation of NMDARɛ2 is a basis of the acute tolerance (21).

Figure 3

Acute tolerance to EtOH inhibition of NMDAR-mediated EPSPs. (A) Time course of maximal slope for EPSPs in the hippocampus of +/fyn z andfyn z/fyn z mice. The EtOH was bath-applied during the time indicated by the arrow. Five slices from four +/fyn z mice and seven slices from seven fyn z/fyn zmice were used. Representative traces of EPSPs in +/fyn z mice (B) andfyn z/fyn z mice (C) during the control period and 5 and 15 min after the start of EtOH application. These EPSPs were almost completely abolished by APV (10 μM). (D) Comparison of peak inhibition of EPSPs during the 15 min of EtOH application (Inhibition) and maximum reduction of inhibition after the peak inhibition of each slice (Tolerance) in +/fyn z andfyn z/fyn z mice. Two-way repeated-measure ANOVA showed a significant gene effect [F(1, 10) = 9.78, P < 0.02], tolerance effect [F(1,10) = 18.33, P < 0.005], and tolerance×gene interaction [F(1,10) = 9.07, P< 0.02]. Means with * are significantly different from the mean with # (Tukey's HSD test, α = 0.05). (E) Elimination of acute tolerance to EtOH inhibition by ifenprodil in +/fyn z mice. NMDAR-mediated EPSPs were inhibited by application of EtOH and ifenprodil (10 μM) in +/fyn z mice [n = 6, repeated-measure ANOVA, F(2, 15) = 13.39, P < 0.0001], but no significant difference was noted between the EPSP amplitudes at 5 min and those at 15 min after the start of the application (Tukey's HSD test, α = 0.05).

Data have been accumulated to support the hypothesis that the inhibition of NMDA-mediated currents underlies the behavioral effects of EtOH (22). Although it is not certain whether the hypnotic effects of EtOH are directly mediated by the hippocampus (from which some of the data, including ours, were derived), the hypothesis is further supported by our results: Fyn-deficient mice showed abnormalities in their behavioral sensitivity to EtOH together with abnormal responses of NMDAR to EtOH.

Mice lacking the γ isoform of protein kinase C (PKCγ) are more resistant to the behavioral effects of EtOH, and these effects could be mediated by modulation of GABAA receptor function (3). These findings and our present results indicate that kinases may regulate behavioral EtOH sensitivity by modulating the function of receptors that are targets of EtOH, such as NMDA and GABAA receptors.

  • * These authors equally contributed to the biochemical findings.

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