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Figure S1. M1 expression following chronic valproate treatment. Expression of M1 protein in frontal cortical tissue was compared between control- and valproate-treated male Wistar Kyoto rats. No significant difference was detected.

Figure S2. Lithium levels in the brain and serum. Male wild-type (WT) and M1 knockout (KO) mice were chronically treated with control and lithium-treated chow, and lithium levels were determined as described in the Materials and Methods. Lithium concentrations were significantly elevated in the serum (A) and brain tissue (B) of both WT and KO mice, and no significant effects of genotype on lithium levels were detected [Serum: F(1,20)treatment = 141.7, p < 0.0001; F(1,20)genotype = 0.1453, p = 0.7071; Brain: F(1,20)treatment = 234.9, p < 0.0001; F(1,20)genotype = 0.03903, p = 0.6766].

Figure S3. Effects of M1 ablation and chronic lithium treatment on frontal cortical phospho-MARCKS level. Adult male M1 knockout (KO) and wild-type (WT) littermates were bred, housed, and treated with lithium-treated or control chow as described in the Materials and Methods. One week after the last behavioral experiment, frontal cortical tissues were obtained and assayed to detect levels of phospho-MARCKS (pMARCKS) and total MARCKS. Representative blots of pMARCKS and MARCKS are shown in (A) and pMARCKS expression is presented quantitatively in (B). No significant effects on pMARCKS were found [F(1,16)genotype = 0.1381, p = 0.7150; F(1,16)treatment = 0.1344, p = 0.7187].

Figure S4. Effects of M1 ablation and chronic lithium treatment on additional outcome measures of the elevated-plus-maze test. Adult male M1 knockout (KO) and wild-type (WT) littermates were bred, housed, and treated with lithium-treated or control chow as described in the Materials and Methods, and then tested in the elevated-plus-maze test. (A) A significant genotype effect was detected for time spent in the closed arms [two-way ANOVA, F(1,20)genotype = 4.461, p = 0.0475; F(1,20)treatment = 0.7377, p = 0.4006; F(1,20)interaction = 0.9536, p = 0.3405]. (B) No significant difference was detected for distance traveled in the closed arms (two-way ANOVA). Data are means ± SEMs. * indicates p < 0.05. Letter G in the graph indicates a comparison between genotype only.

Supplementary materials and methods: Determining plasma and brain lithium levels.

Table S1. M1 is a predicted target of the mood stabilizer-responsive miRNAs let-7b and let-7c.

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