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, 129 (6), 1024-34

Inhibition of Striatal-Enriched Tyrosine Phosphatase 61 in the Dorsomedial Striatum Is Sufficient to Increased Ethanol Consumption

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Inhibition of Striatal-Enriched Tyrosine Phosphatase 61 in the Dorsomedial Striatum Is Sufficient to Increased Ethanol Consumption

Emmanuel Darcq et al. J Neurochem.

Abstract

The STriatal-Enriched protein tyrosine Phosphatase 61 (STEP61 ) inhibits the activity of the tyrosine kinase Fyn and dephosphorylates the GluN2B subunit of the NMDA receptor, whereas the protein kinase A phosphorylation of STEP61 inhibits the activity of the phosphatase (Pharmacol. Rev., 64, , p. 65). Previously, we found that ethanol activates Fyn in the dorsomedial striatum (DMS) leading to GluN2B phosphorylation, which, in turn, underlies the development of ethanol intake (J. Neurosci., 30, , p. 10187). Here, we tested the hypothesis that inhibition of STEP61 by ethanol is upstream of Fyn/GluN2B. We show that exposure of mice to ethanol increased STEP61 phosphorylation in the DMS, which was maintained after withdrawal and was not observed in other striatal regions. Specific knockdown of STEP61 in the DMS of mice enhanced ethanol-mediated Fyn activation and GluN2B phosphorylation, and increased ethanol intake without altering the level of water, saccharine, quinine consumption or spontaneous locomotor activity. Together, our data suggest that blockade of STEP61 activity in response to ethanol is sufficient for the activation of the Fyn/GluN2B pathway in the DMS. Being upstream of Fyn and GluN2B, inactive STEP61 in the DMS primes the induction of ethanol intake. We show that ethanol-mediated inhibition of STEP61 in the DMS leads to Fyn activation and GluN2B phosphorylation. (a) Under basal conditions, active STEP61 inhibits Fyn activity and dephosphorylates GluN2B. (b) Ethanol leads to the phosphorylation of STEP61 on a specific inhibitory site. The inhibition of STEP61 activity contributes to the activation of Fyn in response to ethanol, which, in turn, phosphorylates GluN2B. These molecular adaptations in the DMS promote ethanol drinking.

Keywords: addiction; alcohol; ethanol; phosphatase; phosphorylation; striatum.

Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

Figure 1
Figure 1. Ethanol consumption increases STEP61 inhibition, Fyn activation and GluN2B phosphorylation in the DMS
Mice underwent 6 weeks of intermittent-access 20% ethanol in a two-bottle choice drinking procedure. Control animals underwent the same paradigm but had access to water only. The DMS was removed 4 hrs after the initiation of the last ethanol drinking session (binge). (A) The level of STEP61 phosphorylation was determined by western blot analysis using anti-[pSer221]STEP antibodies. The nitrocellulose membrane was stripped and re-probed with anti-STEP to measure the total level of the phosphatase, and anti-GAPDH antibodies were used as loading control. (B–C) Anti-[pY418/420]Src/Fyn, and anti-Fyn antibodies (B) and anti-[pY1472]GluN2B and anti-GluN2B antibodies (C) were used to detect the activated form and the total amount of Fyn (B), and the phosphorylated and total amount of GluN2B (C), respectively. Optical density of immunoreactivity of phosphorylated-protein bands were normalized to total protein and plotted as percentage of the water only control group. Two-tailed unpaired t-test; **p < 0.01 and ***p < 0.001. (A–C) n = 6.
Figure 2
Figure 2. STEP61 phosphorylation in the DMS is long-lasting and is not observed in the DLS or NAc
The drinking paradigm was conducted as described in Fig. 1. (A) The DMS was dissected 24 hrs after the end of the last ethanol drinking session (withdrawal). (B–C) The DLS (B), and NAc (C) were removed 4 hrs after the initiation of the last ethanol drinking session (binge). (A–C) The level of STEP61 phosphorylation was determined as described in Fig. 1A. Optical density of immunoreactivity of phosphorylated-protein bands were normalized to total protein and plotted as percentage of the water only control group. Two-tailed unpaired t-test; ***p < 0.001. n=6.
Figure 3
Figure 3. Infection of the DMS of mice with lentivirus expressing short hairpin RNA that targets STEP61 produces a knockdown of the protein
Ltv-shSTEP or Ltv-shSCR control were bilaterally infused at a titer of 1.8 × 107 pg/ml into the DMS of mice. Striatal tissues were collected 4 weeks after virus infusions and used for immunohistochemistry (A), and western blot analysis (B–D). A, Ltv-shSTEP infects DMS neurons. Left, depicts the specificity of the site of infection. Slices were co-stained with anti-GFP and anti-NeuN antibodies. Scale bar, 1 mm. Right image depicts Ltv-shSTEP infection of neurons but not glia. Slices were co-stained with anti-GFP and anti-NeuN (top) or anti-GFAP (bottom) antibodies. Scale bar, 50 μm. B, Ltv-shSTEP infection decreases STEP61 expression in the DMS. The protein level of STEP61 was determined by western blot analysis (top panel) and GAPDH immunoreactivity was used as an internal loading control (bottom panel). C, Ltv-shSTEP in the DMS does not change the protein level of the phosphatase in the DLS. STEP61 protein levels were determined as described in B. D, Ltv-shSTEP does not change the protein level of Fyn and GluN2B in the DMS. The level of Fyn (top panel) and GluN2B (middle panel) were determined by western blot analysis, and GAPDH immunoreactivity was used as an internal loading control (bottom panel). The histograms depict the mean ratio of STEP61, Fyn or GluN2B/GAPDH ± SEM and data are expressed as the percentage of control (Ltv-shSCR infected mice). Two-tailed unpaired t-test; ***p < 0.001. n = 7–8 (B and D), n = 3 (C).
Figure 4
Figure 4. STEP61 knockdown in the DMS increases ethanol-mediated Fyn activation and GluN2B phosphorylation
Mice were infused with Ltv-shSCR or Ltv-shSTEP at a titer of 1.8 × 107 pg/ml in the DMS of mice. Four weeks after the virus infusion, animals were treated with an acute administration of saline (S) or ethanol (E, 2.0 g/kg, i.p.) and the DMS was collected 15 min later. Anti-STEP antibodies (A), Anti-[pY420]Fyn, and anti-Fyn antibodies (B) and anti-[pY1472] GluN2B and anti-GluN2B antibodies (C) were used to detect the total amount of STEP (A), the activated form and the total amount of Fyn (B), and the phosphorylated and total amount of GluN2B (C), respectively. Optical density of immunoreactivity of phosphorylated-protein bands were normalized to total protein and plotted as percentage of Ltv-shSCR+Saline treatment. Two-way ANOVA with SNK post hoc test (A) ###p < 0.001 versus Ltv-shSCR (B) **p < 0.01, ***p < 0.001 versus Ltv-shSCR+S and #p<0.05 versus Ltv-shSCR+E. (C) * p<0.05, ***p < 0.001 versus Ltv-shSCR+S and ##p < 0.01 versus Ltv-shSCR+E. n = 4–5.
Figure 5
Figure 5. Knockdown of STEP61 in the DMS of mice increases ethanol intake and preference
Mice were bilaterally infused in the DMS with Ltv-shSTEP or Ltv-shSCR at a titer of 1.8 × 107 pg/ml, and ethanol intake was determined using a continuous-access 10% ethanol for 6 days (A–C), continuous-access 20% ethanol for 6 days (D–F), and intermittent-access 20% ethanol for 6 sessions (G–H). Intake was averaged for each mouse across all days of access and data are presented as mean ± SEM of ethanol intake per 24 hrs (A, D and G), water intake per 24 hrs (B, E and H) and ethanol preference (C, F and I). Ethanol preference was calculated as the percentage of ethanol solution consumed relative to total fluid intake (ethanol + water). n=10–12
Figure 6
Figure 6. Knockdown of STEP61 in the DMS of mice does not alter saccharin or quinine intake or alter spontaneous locomotion
DMS neurons were infected with Ltv-shSTEP or Ltv-shSCR as described in the legends of Fig. 5 (A) STEP61 knockdown in the DMS does not alter saccharin or quinine intake. Experiments were conducted one week after the last ethanol drinking session. Saccharin (0.066%) or quinine (0.06 mM) solution was each provided for 3 successive days. Data are presented as average of daily saccharine or quinine intake. (B) STEP61 knockdown in the DMS does not alter spontaneous locomotor activity. Experiments were conducted one week after the last quinine drinking session. Mice were placed in locomotor activity chambers and the distance traveled was recorded for 60 min. Data are presented as cumulative locomotor activity (cm) during the testing period. Data are expressed mean ± SEM. n=10–12.
Figure 7
Figure 7. Ethanol-mediated inhibition of STEP61 in the DMS leads to Fyn activation and GluN2B phosphorylation
(A) Under basal conditions, active STEP61 inhibits Fyn activity and dephosphorylates GluN2B. (B) Ethanol leads to the phosphorylation of STEP61 on a specific inhibitory site. The inhibition of STEP61 activity contributes to the activation of Fyn in response to ethanol, which, in turn, phosphorylates GluN2B. These molecular adaptations in the DMS promote ethanol drinking.

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