5-HT2CR antagonist/5-HT2CR inverse agonist recovered the increased isolation-induced aggressive behavior of BALB/c mice mediated by ADAR1 (p110) expression and Htr2c RNA editing

Abstract

Introduction: Social isolation enhances the aggressive behavior of animals, but the detailed mechanism remains unclear. Epigenetic studies have suggested that Htr2c RNA editing is closely related to aggressive behavior. This study aims to obtain a fundamental understanding of how social isolation impacts adenosine deaminase acting on RNA 1 (ADAR1, RNA editing enzyme) and Htr2c RNA editing, leading to aggressive behavior, and explore the effective solutions for the recovery of this behavior.

Methods: We evaluated 21-day-old BALB/c mice with and without isolation for aggressive behavior using a resident-intruder test. Immune-reactivity and protein expression of ADAR1 (p110) were measured using immunohistochemistry and Western blotting. Htr2c RNA editing was evaluated using pyrosequencing. In addition, the 5-HT _2C R antagonist SB243213/5-HT _2C R inverse agonist SB206553 was used to treat the isolated mice, and the performance of both treatments on the behavior, ADAR1 (p110) expression, and Htr2c RNA editing in isolated mice was examined.

Results: Both the protein expression and immune-reactivity of ADAR1 (p110) in the amygdala decreased, but the percentage of Htr2c RNA editing at A and B sites of amygdala only showed a moderate increase in isolated BALB/c mice with enhanced aggressive behavior compared to the age-matched group-housed BALB/c mice. Additionally, treatment with the 5-HT _2C R antagonist SB243213/5-HT _2C R inverse agonist SB206553 recovered the enhanced aggressive behavior of isolated mice and returned the protein expression and immune-reactivity of ADAR1 (p110) back to the normal level. Moreover, compared to the age-matched isolated mice treated with physiological saline, isolated mice treated with 5-HT _2C R inverse agonist SB206553 showed a lower percentage of Htr2c RNA editing at both A and B sites, and the same result occurred in isolated mice treated with 5-HT _2C R antagonist SB243213 at B site of Htr2c RNA editing.

Conclusions: The 5-HT _2C R antagonist SB243213/5-HT _2C R inverse agonist SB206553 recovered increased aggressive behavior of isolated BALB/c mice mediated by ADAR1 (p110) expression and Htr2c RNA editing.

Keywords: 5‐HT2CR; ADAR1; aggressive behavior.

Figure 1

Groups of mice with and without 2 weeks of social isolation, followed by treatment with 5‐HT _2CR antagonist SB243213/5‐HT _2CR inverse agonist SB206553. Sixty healthy male BALB/c mice at the age of 21 days old were randomly divided into six groups, with 10 mice in each group. Each mouse was placed in a plastic cage (five mice per cage). The mice treated with physiological saline (20 ml/kg, i.p.) were individually housed for 2 weeks. The animals were labeled SI group (social isolation group, n = 10). In addition, the recovery of the behavioral deficit by 5‐HT _2C R antagonist SB243213/5‐HT _2C R inverse agonist SB206553 treatment (0.5 mg/kg, i.p.) was also examined. The treated groups were labeled SI + SB243213 group and SI + SB206553 group (recovery groups with drug treatments, n = 10/group). The age‐matched gregarious mice treated with physiological saline (20 ml/kg, i.p.) was labeled C group (normal control group, n = 10); meanwhile, the age‐matched gregarious mice treated with the 5‐HT _2C R antagonist SB243213/5‐HT _2C R inverse agonist SB206553 (0.5 mg/kg, i.p.) were labeled C+SB243213 and C+SB206553 (drug treatment alone groups, n = 10/group) groups, respectively

Figure 2

Outline of the procedure for measuring Htr2c RNA editing. Total RNA was extracted from the homogenized amygdala of the mice brain using the TRIzol method. Both RNA extraction and cDNA synthesis were performed using PrimeScript Hi‐Fide RT‐RCR Kit. Then, the prepared cDNA samples (n = 5 cDNA samples/group) were methylated by bisulfite treatment using Bisul‐Methylation Universal Kit. Then, PCR amplification of Htr2c was performed, with the used primers designed by PyroMark Assay Design 2.0 and synthesized by Hua Da Gene Company, as shown in Table 1. Subsequently, serial pyrosequencing measures were performed with the substrate mixture, enzyme mixture, and four types of dNTP added in the reaction system. Finally, pyrosequencing detector and Pyro Q‐CpG software were used to measure the frequency for A, B, D, and C/E editing sites

Figure 3

The 5‐HT _2C R antagonist SB243213/5‐HT _2C R inverse agonist SB206553 recovered the isolation‐induced increased aggressive ability in BALB/c mice. (a) Latency to first bite measured before and after the treatments with physiological saline/drugs for isolated BALB/c mice; (b) duration of biting genitals measured before and after the treatments with physiological saline/drugs for isolated BALB/c mice; (c) latency to first bite in all groups; and (d) duration of biting genitals in all groups. All data were statistically calculated as the means ± SD; **p < .01; (n = 10/group)

Figure 4

Decreased ADAR1 (p110) immune‐reactivity in amygdala of social isolated BALB/c mice and its recovery by the 5‐HT _2CR antagonist SB243213/5‐HT _2CR inverse agonist SB206553. (a) The brain regions were analyzed on the basis of the mouse brain atlas of Paxinos and Franklin (1997). a/Green represents BLA:basolateral amygdaloid nucleus; b/Red represents LaDL: lateral amygdaloid nucleus dorsolateral part; c/Blue represents LaVL: lateral amygdaloid nucleus ventrolateral part; d/Yellow represents LaVM: lateral amygdaloid nucleus ventromedial part. (b) Decreased ADAR1 (p110) immune‐reactivity‐positive signals in the BLA of the isolated mice and its recovery after treatment with the 5‐HT _2CR antagonist SB243213/5‐HT _2CR inverse agonist SB206553. Scale bar = 50 μm. (c) Decreased ADAR1 (p110) immune‐reactivity‐positive signals in LaDL, LaVL, and LaVM of the isolated mice and its recovery after treatment with the 5‐HT _2CR antagonist SB243213/5‐HT _2CR inverse agonist SB206553. Scale bar = 50 μm. (d) Statistical analysis of the integrated optical density of ADAR1 (p110) immune‐reactivity‐positive signals in the amygdala of the isolated mice and its recovery after treatment with the 5‐HT _{2 C}R antagonist SB243213/5‐HT _{2 C}R inverse agonist SB206553. All data were statistically calculated as the means ± SD; **p < .01; (n = 5/group)

Figure 5

Decreased ADAR1 (p110) protein expression in the amygdala of socially isolated BALB/c mice and its recovery after treatment with the 5‐HT _{2 C}R antagonist SB243213. (a) Decreased ADAR1 (p110) expression in the amygdala of isolated mice and its recovery after treatment with the 5‐HT _{2 C}R antagonist SB243213. (b) Statistical analysis of the decreased ADAR1 (p110) in the amygdala of isolated mice and its recovery after treatment with the 5‐HT _2CR antagonist SB243213. ADAR1 (p110) protein expression was normalized to the internal control GADPH. The data were expressed as the means ± SD; **p < .01 (C vs. SI); (n = 5/group)

Figure 6

Hypothesis of how social isolation induces increased aggressive behavior mediated with ADAR1 (p110) and moderate changes in Htr2c RNA editing. (a) Htr2c RNA editing isoforms include no editing (INI), partial editing (VNV, VSV, and the other 28 amino acid isomers; Werry et al., 2008) and complete editing (VGV). (b) The isolated BALB/c mice with increased aggressive behavior showed the decreased protein expression of ADAR1 (p110) and moderate increased tendency of Htr2c RNA editing percentage at A and B sites of amygdala. (c) The treatment with 5‐HT _2CR antagonist SB243213/5‐HT _2CR inverse agonist SB206553 recovered the aggressive behavior of isolated mice and brought ADAR1 (p110) back to the normal level. Moreover, 5‐HT _2C R antagonist SB243213/5‐HT _2C R inverse agonist SB206553 decreased the percentage of Htr2c RNA editing at both A and B sites in isolated mice