Article Figures & Data
Figures
- Figure 1.
SA/SA mutant mice show increased ethanol consumption. Volitional ethanol intake was measured in a two-bottle choice test between tap water and 5% or 10% ethanol for three weeks in Src Ser75 mutant mice. A, Consumption of a 5% or 10% ethanol solution by SA/SA (left) and SD/SD (right) mice and their WT/WT counterparts. Measurements for mutants were normalized to the average amount per kilogram body weight per measurement period (3 or 4 d) for the WT littermate controls. B, Ethanol preference ratios (volume of ethanol solution consumed per total volume of fluid consumed) of a 5% or 10% ethanol solution in SA/SA (left) and SD/SD (right) mice and their WT/WT counterparts. C, Total fluid intake of a 5% or 10% ethanol solution and water for SA/SA (left) and SD/SD (right) mice and their WT/WT counterparts. Measurements for mutants were normalized to the average amount per kilogram body weight per measurement period for WT littermate controls. Black bars, WT/WT; white bars, SA/SA or SD/SD. Data are mean ± SD. Sample numbers are shown in parentheses; *p < 0.05, **p < 0.01 versus WT/WT by unpaired two-tailed Welch’s t tests, except for consumption of 10% ethanol in SD/SD mice, which was analyzed by Mann–Whitney U test.
- Figure 2.
Taste preference ratios for sucrose and quinine in WT and SA/SA (A) and SD/SD (B) mutant mice. Taste preference was assessed via a two-bottle choice paradigm with tap water and a tastant, each for 14 d, in the following order: 0.033% (w/v) sucrose, 0.1% (w/v) sucrose, 0.02 mM quinine, and 0.04 mM quinine. C, Total intake of a 0.033% (w/v) or 0.1% (w/v) sucrose solution in SA/SA (left) and SD/SD (right) mice and their WT/WT counterparts. Measurements for mutants were normalized to the average amount of 0.033% (w/v) sucrose per kilogram body weight per measurement period for WT littermate controls. Data are mean ± SD analyzed by paired factorial ANOVA and Bonferroni’s post hoc tests.
- Figure 3.
Plasma ethanol levels assayed 1 and 3 h after WT/WT and SA/SA (left) or SD/SD (right) mice were injected with ethanol (3 g/kg). Blood samples (10 μl) were collected from each mouse at the postinjection time points and used for the alcohol dehydrogenase enzymatic spectrophotometric assay. Data are mean ± SD analyzed by paired factorial ANOVA and Tukey–Kramer post hoc tests.
- Figure 4.
LORR for SA/SA (left) and SD/SD (right) mice and their WT counterparts after they were injected with ethanol (3 g/kg). Data are mean ± SD analyzed by unpaired and two-tailed Welch’s t tests.
- Figure 5.
Downregulation of ROCK signaling in striatal tissues from SA/SA mutant mice. A, ROCK activity levels in the striatal tissues of SA/SA (left) and SD/SD (right) mutant mice measured by immunoassays. B, ROCK protein levels in striatal tissues from SA/SA (left) and SD/SD (right) mutant mice measured by Western blotting (upper). ROCK protein level (A.U.) in each tissue was normalized to that of β-tubulin (Tub, lower). C, ROCK kinase activity (mU) per milligram of striatal tissues and normalized to ROCK protein amount (bottom) in SA/SA (left) and SD/SD (right) mutant mice. Scatter diagrams of the two variables are shown in upper panels. D, Phosphorylation of 60-kDa Akt at Ser473 (P-Akt) in the striatal tissues of SA/SA mutant mice as measured by Western blotting. Akt phosphorylation levels were normalized to the level of total Akt. Data are mean ± SD; *p < 0.05, **p < 0.01 by unpaired two-tailed Welch’s t tests.
Tables
Figure/table Data structure Type of test Sample size Statistical data Fig. 1A Ethanol consumption WT and SA/SA mice (5%) Normal distribution Welch t test unpaired WT: n = 6, SA/SA: n = 7 CI(d): –2.681 and –0.273, t = –2.867, p = 0.0226 WT and SA/SA mice (10%) Normal distribution Welch t test unpaired WT: n = 5, SA/SA: n = 5 CI(d): –1.730 and –0.258, t = –3.562, p = 0.0184 WT and SD/SD mice (5%) Normal distribution Welch t test unpaired WT: n = 7, SD/SD: n = 6 CI(d): –1.705 and 0.765, t = –0.906, p = 0.3960 WT and SD/SD mice (10%) Non-normal distribution Mann–Whitney U test WT: n = 8, SD/SD: n = 8 U = 26, p = 0.5286 Fig. 1B Ethanol preference ratio WT and SA/SA mice (5%) Normal distribution Welch t test unpaired WT: n = 6, SA/SA: n = 7 CI(d): –0.444 and –0.056, t = –2.987, p = 0.0179 WT and SA/SA mice (10%) Normal distribution Welch t test unpaired WT: n = 5, SA/SA: n = 5 CI(d): –0.177 and –0.039, t = –3.724, p = 0.0080 WT and SD/SD mice (5%) Normal distribution Welch t test unpaired WT: n = 7, SD/SD: n = 6 CI(d): –0.311 and 0.187, t = –0.588, p = 0.5747 WT and SD/SD mice (10%) Normal distribution Welch t test unpaired WT: n = 8, SD/SD: n = 8 CI(d): –0.109 and 0.094, t = –0.155, p = 0.8791 Fig. 1C Total liquid intake WT and SA/SA mice (5%) Normal distribution Welch t test unpaired WT: n = 6, SA/SA: n = 7 CI(d): –0.130 and 0.296, t = 0.916, p = 0.3900 WT and SA/SA mice (10%) Normal distribution Welch t test unpaired WT: n = 5, SA/SA: n = 5 CI(d): –0.156 and 0.133, t = –0.190, p = 0.8546 WT and SD/SD mice (5%) Normal distribution Welch t test unpaired WT: n = 7, SD/SD: n = 6 CI(d): –0.272 and 0.117, t = –0.877, p = 0.3999 WT and SD/SD mice (10%) Normal distribution Welch t test unpaired WT: n = 8, SD/SD: n = 8 CI(d): –0.223 and 0.191, t = –0.166, p = 0.8712 Fig. 2A Taste preference ratio (WT and SA/SA) Sucrose Normal distribution Paired factorial ANOVA WT: n = 5, SA/SA: n = 5 Genotype: F = 0.5107, p = 0.4951 0.033% (w/v) Bonferroni’s post hoc CI(m): WT, 0.477 and 0.569; SA/SA, 0.489 and 0.580, p = 0.5901 0.1% (w/v) Bonferroni’s post hoc CI(m): WT, 0.430 and 0.521; SA/SA, 0.447 and 0.538, p = 0.4224 Quinine Normal distribution Paired factorial ANOVA WT: n = 5, SA/SA: n = 5 Genotype: F = 0.0331, p = 0.8602 0.02 mM Bonferroni’s post hoc CI(m): WT, 0.134 and 0.522; SA/SA, 0.139 and 0.527, p = 0.9484 0.04 mM Bonferroni’s post hoc CI(m): WT, 0.033 and 0.421; SA/SA, 0.060 and 0.448, p = 0.6783 Fig. 2B Taste preference ratio (WT and SD/SD) Sucrose Normal distribution Paired factorial ANOVA WT: n = 5, SD/SD: n = 5 Genotype: F = 0.8475, p = 0.3842 0.033% (w/v) Bonferroni’s post hoc CI(m): WT, 0.495 and 0.604; SD/SD, 0.447 and 0.557, p = 0.0533 0.1% (w/v) Bonferroni’s post hoc CI(m): WT, 0.518 and 0.627; SD/SD, 0.522 and 0.631, p = 0.8722 Quinine Normal distribution Paired factorial ANOVA WT: n = 5, SD/SD: n = 5 Genotype: F = 0.4920, p = 0.5029 0.02 mM Bonferroni’s post hoc CI(m): WT, 0.231 and 0.540; SD/SD, 0.190 and 0.499, p = 0.4454 0.04 mM Bonferroni’s post hoc CI(m): WT, 0.056 and 0.365; SD/SD, 0.003 and 0.312, p = 0.3294 Fig. 2C Total sucrose intake WT and SA/SA mice Normal distribution Paired factorial ANOVA WT: n = 5, SA/SA: n = 5 Genotype: F = 0.3578, p = 0.5663 0.033% (w/v) Bonferroni’s post hoc CI(m): WT, 0.442 and 1.558; SA/SA, 0.578 and 1.694, p = 0.4868 0.1% (w/v) Bonferroni’s post hoc CI(m): WT, 2.105 and 3.221; SA/SA, 2.258 and 3.375, p = 0.4345 WT and SD/SD mice Normal distribution Paired factorial ANOVA WT: n = 5, SD/SD: n = 5 Genotype: F = 0.2553, p = 0.6270 0.033% (w/v) Bonferroni’s post hoc CI(m): WT, 0.442 and 1.558; SD/SD, 0.214 and 1.329, p = 0.2855 0.1% (w/v) Bonferroni’s post hoc CI(m): WT, 2.359 and 3.475; SD/SD, 2.343 and 3.458, p = 0.9385 Fig. 3 Blood ethanol concentration WT and SA/SA mice Normal distribution Paired factorial ANOVA WT: n = 6, SA/SA: n = 5 Genotype: F = 0.7817, p = 0.3996 1 h Tukey–Kramer post hoc CI(m): WT, 2085.307 and 2908.035; SA/SA, 2108.846 and 3010.099, p = 0.6879 3 h Tukey–Kramer post hoc CI(m): WT, 1063.610 and 1886.337; SA/SA, 1298.783 and 2200.036, p = 0.0947 WT and SD/SD mice Normal distribution Paired factorial ANOVA WT: n = 5, SD/SD: n = 4 Genotype: F = 0.1381, p = 0.7212 1 h Tukey–Kramer post hoc CI(m): WT, 2115.538 and 3687.719; SD/SD, 2109.947 and 3867.699, p = 0.7418 3 h Tukey–Kramer post hoc CI(m): WT, 1211.065 and 2783.246; SD/SD, 769.016 and 2526.767, p = 0.2089 Fig. 4 LORR WT and SA/SA mice Normal distribution Welch t test unpaired WT: n = 13, SA/SA: n = 13 CI(d): –12.835 and 11.450, t = –0.118, p = 0.9072 WT and SD/SD mice Normal distribution Welch t test unpaired WT: n = 16, SD/SD: n = 13 CI(d): –6.790 and 28.367, t = 1.261, p = 0.2184 Fig. 5A ROCK activity WT and SA/SA mice Assuming normality Welch t test unpaired WT: n = 4, SA/SA: n = 4 CI(d): 18.691 and 158.488, t = 3.305, p = 0.0228 WT and SD/SD mice Assuming normality Welch t test unpaired WT: n = 4, SD/SD: n = 4 CI(d): –157.109 and 115.935, t = –0.421, p = 0.6956 Fig. 5B ROCK protein level WT and SA/SA mice Assuming normality Welch t test unpaired WT: n = 4, SA/SA: n = 4 CI(d): –0.015 and 0.014, t = –0.141, p = 0.8929 WT and SD/SD mice Assuming normality Welch t test unpaired WT: n = 4, SD/SD: n = 4 CI(d): –0.032 and 0.085, t = 1.248, p = 0.2782 Fig. 5C ROCK activity/protein. WT and SA/SA mice Assuming normality Welch t test unpaired WT: n = 4, SA/SA: n = 4 CI(d): 194.255 and 925.975, t = 3.747, p = 0.0096 WT and SD/SD mice Assuming normality Welch t test unpaired WT: n = 4, SD/SD: n = 4 CI(d): –1286.927 and 604.178, t = –0.937, p = 0.3929 Fig. 5D Phosphorylated Akt WT and SA/SA mice Normal distribution Welch t test unpaired WT: n = 5, SA/SA: n = 5 CI(d): –0.077 and –0.007, t = –2.751, p = 0.0256 Table 2 Body weight Fig. 1 (ethanol two-bottle choice test) WT and SA/SA mice (5%) Normal distribution Welch t test unpaired WT: n = 6, SA/SA: n = 7 CI(d): –0.003 and 0.002, t = –0.512, p = 0.6229 WT and SA/SA mice (10%) Normal distribution Welch t test unpaired WT: n = 5, SA/SA: n = 5 CI(d): –0.002 and 0.009, t = 1.561, p = 0.1651 WT and SD/SD mice (5%) Normal distribution Welch t test unpaired WT: n = 7, SD/SD: n = 6 CI(d): –0.001 and 0.007, t = 1.907, p = 0.0874 WT and SD/SD mice (10%) Normal distribution Welch t test unpaired WT: n = 8, SD/SD: n = 8 CI(d): –0.003 and 0.003, t = 0.029, p = 0.9770 Fig. 2 (taste preference test) WT/WT and SA/SA mice Normal distribution Welch t test unpaired WT: n = 5, SA/SA: n = 5 CI(d): –0.005 and 0.007, t = 0.435, p = 0.6769 WT/WT and SD/SD mice Normal distribution Welch t test unpaired WT: n = 5, SA/SA: n = 5 CI(d): –0.004 and 0.009, t = 0.847, p = 0.4226 Table 3 Putative average ethanol consumption WT and SA/SA mice 5% ethanol Normal distribution Welch t test unpaired WT: n = 6, SA/SA: n = 7 CI(d): –2.223 and –0.209, t = –2.809, p = 0.0240 10% ethanol Normal distribution Welch t test unpaired WT: n = 5, SA/SA: n = 5 CI(d): –1.707 and –0.305, t = –3.309, p = 0.0107 WT and SD/SD mice 5% ethanol Normal distribution Welch t test unpaired WT: n = 7, SD/SD: n = 6 CI(d): –1.692 and 0.833, t = –0.820, p = 0.4414 10% ethanol Non-normal distribution Mann–Whitney U test WT: n = 8, SD/SD: n = 8 U = 26, p = 0.5286 Table 4 Putative average total liquid intake WT and SA/SA mice 5% ethanol Normal distribution Welch t test unpaired WT: n = 6, SA/SA: n = 7 CI(d): –19.2958 and 42.1470, t = 0.8822, p = 0.4074 10% ethanol Normal distribution Welch t test unpaired WT: n = 5, SA/SA: n = 5 CI(d): –12.7726 and 19.1717, t = 0.4800, p = 0.6468 WT and SD/SD mice 5% ethanol Normal distribution Welch t test unpaired WT: n = 7, SD/SD: n = 6 CI(d): –29.0687 and 14.5159, t = –0.7377, p = 0.4766 10% ethanol Normal distribution Welch t test unpaired WT: n = 8, SD/SD: n = 8 CI(d): –25.3723 and 22.8610, t = –0.1142, p = 0.9111 CI(d), 95% confidence interval for the difference in population means, lower, and upper limits; CI(m), 95% confidence interval for the population means, lower, and upper limits.
- Table 2.
Summary of mean body weights during two-bottle choice tests for ethanol and for sucrose or quinine
Experiment Mean body weight (kg) p value Fig. 1 SA 5% ethanol WT/WT 0.028 ± 0.002 (n = 6) SA/SA 0.028 ± 0.001 (n = 7) 0.6229 10% ethanol WT/WT 0.031 ± 0.004 (n = 5) SA/SA 0.028 ± 0.003 (n = 5) 0.1651 SD 5% ethanol WT/WT 0.030 ± 0.004 (n = 7) SD/SD 0.027 ± 0.002 (n = 6) 0.0874 10% ethanol WT/WT 0.028 ± 0.001 (n = 8) SD/SD 0.028 ± 0.003 (n = 8) 0.977 Fig. 2 Sucrose/quinine SA WT/WT 0.036 ± 0.005 (n = 5) SA/SA 0.035 ± 0.003 (n = 5) 0.6769 SD WT/WT 0.033 ± 0.004 (n = 5) SD/SD 0.031 ± 0.005 (n = 5) 0.4226 Data are mean ± SD; p values are comparisons with WT/WT by unpaired two-tailed Welch’s t tests.
Two-bottle ethanol choice test Average ethanol consumption (g/kg/d) p value SA 5% ethanol WT/WT 0.952 ± 0.371 (n = 6) SA/SA 2.168 ± 1.073 (n = 7) 0.0240 10% ethanol WT/WT 1.193 ± 0.483 (n = 5) SA/SA 2.199 ± 0.478 (n = 5) 0.0107 SD 5% ethanol WT/WT 0.945 ± 0.487 (n = 7) SD/SD 1.375 ± 1.201 (n = 6) 0.4414 10% ethanol WT/WT 1.294 ± 0.565 (n = 8) SD/SD 1.352 ± 1.197 (n = 8) 0.5286 Volitional ethanol intake was measured in a two-bottle choice test between tap water and 5% or 10% ethanol for three weeks in SA and SD mice. The measured values were divided by the number of days for each or the six measurements to obtain putative daily averages. Data are mean ± SD; p values are comparisons with WT/WT obtained by unpaired two-tailed Welch’s t tests, except for consumption of 10% ethanol in SD/SD mice, which was analyzed by Mann–Whitney U test.
Two-bottle ethanol choice test Average total intake (g/kg/d) p value SA 5% ethanol WT/WT 139.3 ± 29.1 (n = 6) SA/SA 127.9 ± 13.8 (n = 7) 0.4074 10% ethanol WT/WT 132.1 ± 12.8 (n = 5) SA/SA 128.9 ± 7.7 (n = 5) 0.6468 SD 5% ethanol WT/WT 109.2 ± 17.7 (n = 7) SD/SD 116.4 ± 17.8 (n = 6) 0.4766 10% ethanol WT/WT 118.8 ± 15.8 (n = 8) SD/SD 120.0 ± 26.8 (n = 8) 0.9111 Total liquid intake of a 5% or a 10% ethanol solution and water over a period of three weeks by SA and SD mice were measured in the two-bottle choice test. The measured values were divided by the number of days to obtain putative daily averages. Data are mean ± SD; p values are comparisons with WT/WT obtained using unpaired two-tailed Welch’s t tests.
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