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. Author manuscript; available in PMC: 2013 Mar 1.
Published in final edited form as: Nat Neurosci. 2012 Aug 5;15(9):1245–1254. doi: 10.1038/nn.3181

Figure 3. 5-HT2A-dependent increased expression of HDAC2 in frontal cortex by chronic atypical antipsychotics.

Figure 3

(a–b) Chronic clozapine modulates the expression of Hdac2 mRNA in mouse frontal cortex. 5HT2A+/+ (a) and 5HT2A−/− (b) mice were chronically (21 days) injected with vehicle (black) or 10 mg/kg clozapine (red), and sacrificed one day after the last injection (5HT2A+/+, n = 12; 5HT2A−/−, n = 6 mice per group). Expression of Hdac1, Hdac2, Hdac3, Hdac4, Hdac5, Hdac6, Hdac7, Hdac8, and Hdac9 mRNAs was assayed by qRT-PCR. Effect of chronic clozapine on Hdac2 mRNA expression in 5HT2A+/+ mice: t = 3.40. The α value was corrected for multiple independent null hypotheses by using the Holm’s sequentially rejective Bonferroni method. *P < 0.006; two-tailed Student’s t-test.

(c–e) Chronic clozapine up-regulates protein levels of HDAC2, and not HDAC1 or HDAC4, in mouse frontal cortex. 5HT2A+/+ (c) and 5HT2A−/− (d) mice were chronically (21 days) injected with vehicle (black) or 10 mg/kg clozapine (red) and sacrificed one day after the last injection (n = 6 mice per group). Effect of chronic clozapine on HDAC2 expression in 5HT2A+/+ mice: t = 3.17. The α value was corrected for multiple independent null hypotheses by using the Holm’s sequentially rejective Bonferroni method. *P < 0.017; two-tailed Student’s t-test. Representative immunoblots are shown (e).

(f–h) HDAC2, and not HDAC1 or HDAC4, is increased in postmortem human brain of atypical antipsychotic-treated, and not untreated, schizophrenic subjects. Western blots showed no change (f) of HDAC2 protein levels in frontal cortex of untreated schizophrenics compared to matched control subjects; and up-regulation (g) of HDAC2 protein levels in frontal cortex of atypical antipsychotic-treated schizophrenics compared to matched control subjects (t = 4.22). The α value was corrected for multiple independent null hypotheses by using the Holm’s sequentially rejective Bonferroni method. *P < 0.017; two-tailed Student’s t-test. Representative immunoblots are shown (h).

(i–j) Clozapine reverses the 5HT2A-dependent repression of HDAC2 promoter activity by serotonin. HEK293 cells were transfected with a HDAC2 promoter-luciferase construct and/or with a plasmid encoding the 5HT2A, treated with serotonin (5HT), clozapine, and/or vehicle, and then analyzed for luciferase activity (n = 3 independent experiments performed in triplicate). Concentration response of 5HT in mock-transfected cells (i), F[3,8] = 1.81, P > 0.05; concentration response of 5HT in 5HT2A-transfected cells (i), F[3,8] = 17.52, P < 0.001; one-way ANOVA. *P < 0.05; **P < 0.01; Bonferroni’s post hoc test of one-way ANOVA. Concentration response of clozapine in 5HT2A-transfected cells (j), F[5,12] = 15.01, P < 0.001; one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; Bonferroni’s post hoc test of one-way ANOVA.

(k) Dopamine D2-dependent signaling does not affect the promoter activity of HDAC2. HEK293 cells were transfected with a HDAC2 promoter-luciferase construct and/or with a plasmid encoding the D2 receptor, treated with dopamine (DA), clozapine, or vehicle, and then analyzed for luciferase activity (n = 6 independent experiments performed in triplicate). Effect of DA in mock-transfected cells (k), t = 0.30, P > 0.05; two-tailed Student’s t-test. Effect of DA in D2-transfected cells (k), F[3,8] = 1.77, P > 0.05; one-way ANOVA. Error bars represent s.e.m.