Figure 1. RNA-Seq Identified mRNAs Whose Translation Is Increased by Alcohol and depends on mTORC1 Activity in the NAc.
(A) Mice experienced 7–8 weeks of IA20%-2BC. Control animals had access to water only. Three hours before the end of the last 24 hr of alcohol withdrawal, mice were systemically administered with 20 mg/kg of rapamycin or vehicle. The NAc from the four groups of mice (water/vehicle [blue], water/rapamycin [hatched blue], alcohol/vehicle [red], alcohol/rapamycin [hatched red]) were dissected 3 hr after the administration of rapamycin and were subjected to polysomal purification and RNA-seq analysis.
(B) RNA-seq data depicting fragments per kilobase of transcript per million mapped reads (FPKM) value of Prosapip1, TAFA-3, Gucy1a3, and Tsnax. Data are presented as the average FPKM value ± SEM and expressed as the percentage of water/vehicle. Two-way ANOVA showed a significant interaction between alcohol and rapamycin (respectively, Prosapip1 F(1,8) = 8.874, p = 0.018; TAFA-3 F(1,8) = 10.02, p = 0.013, Gucy1a3 F(1,8) = 7.98, p = 0.022; Tsnax F(1,8) = 11.83, p = 0.009) and post hoc Student–Newman–Keuls test detected a significant difference between water and alcohol within the vehicle group (respectively, Prosapip1 p = 0.003; TAFA-3 p = 0.002; Gucy1a3 p = 0.002; Tsnax p = 0.007) and a significant difference between vehicle and rapamycin within the alcohol group (respectively, Prosapip1 p = 0.018; TAFA-3 p = 0.003; Gucy1a3 p = 0.005; Tsnax p = 0.04). n = 3.
(C) A new cohort of animals that underwent the same paradigm described in (A) was used to measure polysomal mRNA level of Prosapip1, TAFA-3, Gucy1a3, and Tsnax by qRT-PCR. Data are presented as the average ratio of each transcript to GAPDH ± SEM, and expressed as the percentage of water/vehicle. Two-way ANOVA showed a significant interaction between alcohol and rapamycin (respectively, Prosapip1 F(1,16) = 5.943, p = 0.027; TAFA-3 F(1,16) = 4.654, p = 0.047, Gucy1a3 F(1,28) = 7.6, p = 0.01; Tsnax F(1,28) = 6.97, p = 0.013) and post hoc Student–Newman–Keuls test detected a significant difference between water and alcohol within the vehicle group (respectively, Prosapip1 p = 0.013; TAFA-3 p = 0.017; Gucy1a3 p = 0.006; Tsnax p = 0.005) and a significant difference between vehicle and rapamycin within the alcohol group (respectively, Prosapip1 p = 0.004; TAFA-3 p = 0.007; Gucy1a3 p = 0.007; Tsnax p = 0.007). n = 5 for Prosapip1 and TAFA-3; n = 8 for Gucy1a3 and Tsnax. *p < 0.05, **p < 0.01.