Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) regulates anxiety- and novelty-related behaviors

Abstract

The activity-regulated cytoskeleton-associated protein (Arc, also known as Arg3.1) regulates glutamatergic synapse plasticity and has been linked to neuropsychiatric illness; however, its role in behaviors associated with mood and anxiety disorders remains unclear. We find that stress upregulates Arc expression in the adult mouse nucleus accumbens (NAc)-a brain region implicated in mood and anxiety behaviors. Global Arc knockout mice have altered AMPAR-subunit surface levels in the adult NAc, and the Arc-deficient mice show reductions in anxiety-like behavior, deficits in social novelty preference, and antidepressive-like behavior. Viral-mediated expression of Arc in the adult NAc of male, global Arc KO mice restores normal levels of anxiety-like behavior in the elevated plus maze (EPM). Consistent with this finding, viral-mediated reduction of Arc in the adult NAc reduces anxiety-like behavior in male, but not female, mice in the EPM. NAc-specific reduction of Arc also produced significant deficits in both object and social novelty preference tasks. Together our findings indicate that Arc is essential for regulating normal mood- and anxiety-related behaviors and novelty discrimination, and that Arc's function within the adult NAc contributes to these behavioral effects.

Keywords: AMPAR; Arc/Arg3.1; anxiety; behavior; cellular neuroscience; knockout mouse; novelty; nucleus accumbens; stress; viral-mediated gene transfer.

Figure. 1.. Anxiety and locomotion in Arc KO mice.

a, Time spent (seconds) by male wild-type (WT) and Arc KO mice in the center of the open field (OF). b, Time spent (seconds) spent in the open arms of the elevated plus maze (EPM) in male WT and Arc KO mice. c, Time spent (seconds) in the light side of the light/dark (L/D) box by male WT and Arc KO mice. d, Activity (beam breaks) in 5-min bins for WT and Arc KO mice during 1 hr locomotor test. Cumulative activity (beam breaks) during the first 5 min (e) or 1 hr (f) of the locomotor test. Bars represent Mean and SEM; n = 20 – 24 mice/group; *p <0.05, ** p < 0.01.

Figure. 2.. Social and mood behavior in Arc KO mice.

a, Time spent in the different compartments of the 3-chamber social interaction chamber during a sociability test for male WT and Arc KO mice. b, Time spent in the different compartments of the 3-chamber social interaction chamber during the social novelty test for male WT and Arc KO mice. c, Time spent immobile (seconds) by male WT and Arc KO in the forced swim test. d, Sucrose preference (expressed as sucrose consumption as a percentage of total sucrose and water consumption) in male WT and Arc KO mice. Bars represent Mean and SEM; n = 14 – 21 mice/group (3-chamber), n = 19 – 24 mice/group (forced swim), n = 9 – 11mice/group (sucrose preference); * p < 0.05.

Fig. 3.. AMPAR localization in Arc KO mice.

a, Approximate location of tissue collection sites. b, Quantification of normalized (β-tubulin) internal (top) and total (bottom) GluA1 (left) and GluA2/3 (right) protein expression in the NAc of young adult male WT and Arc KO mice. b, Quantification of normalized intracellular (top) and total (bottom) GluA1 protein expression in the prefrontal cortex (PFC), dorsal Hippocampus (dHPC), and amygdala (Amyg) of young adult male WT and Arc KO mice. c, Quantification of normalized intracellular (top) and total (bottom) GluA2/3 protein expression in the PFC, dHPC, and Amyg of young adult male WT and Arc KOmice. Lower panels display representative blot images, β-tubulin loading control. d, Arc mRNA expression in the NAc of male WT (n = 5) and Arc KO (n = 6) mice. Arc mRNA levels were normalized to GAPDH mRNA and compared using RT-qPCR. Bars represent Mean and SEM; n = 3 – 6 mice/genotype (protein expression), n = 5 – 6 mice/genotype (mRNA expression); * p < 0.05, *** p < 0.001.

Fig. 4.. Stress regulation of Arc expression

NAc tissue (a) was collected 1, 2, and 4 hrs after an acute defeat experience. b, Arc mRNA expression (normalized to GAPDH mRNA) in male C57BL/6J mice exposed to gentle handling (control) or a single 10 min social defeat session (acute). c, Arc protein expression in the NAc 2 hrs (left) and 4 hrs (right) after a 10 min social defeat stress. Arc expression was normalized to β-tubulin and levels expressed relative to the control condition. Bars represent Mean and SEM; n = 5 mice/condition (mRNA expression), n = 4 – 5 mice/condition (protein expression); * p < 0.05, ** p < 0.01.

Fig. 5.. Behavioral effects of NAc-specific Arc expression manipulations

a, Representative image demonstrating viral-mediated Arc over-expression in the NAc of Arc KO B mice. 2.5X and 10X images of the same field showing anti-Arc antibody staining. 20X’ and 20X” images showing independent examples of Arc over-expression (Green) primarily in NeuN positive (Red)cells. Scale bar = 100 uM b, Time (seconds⁾ spent in the open arms for control (GFP-NAc) and Arc overexpressing (Arc-NAc) WT and Arc KO littermates. c, Time (seconds) spent in the center of theopen field for GFP-NAc and Arc-NAc WT and Arc KO littermates. d, Knockdown of Arc in virally-infected NAc tissue from C57Bl/6N WT mice. e, Time (seconds) spent in the open arm for female and male shControl-NAc and shArc-NAc mice. f, Time (seconds) spent in the center of the open field for female and male shControl-NAc and shArc-NAc mice. Bars represent Mean and SEM; n = 4 – 6 mice/condition (Arc rescue), n = 2 – 3 mice/condition (shRNA knockdown in vivo), n = 9 – 12 e/condition (Arc shRNA behavior); * p < 0.05, ** p < 0.01.

Figure 6.. Social and novelty behavior in Arc shRNA mice

a, Time spent in the compartments of the 3-chamber social interaction chamber for male and female shControl-NAc and shArc-NAc mice during a sociability test. b, Time spent in the compartments of the 3-chamber social interaction chamber for male and female shControl-NAc and shArc-NAc mice during the social novelty test. c, Time spent interacting with the novel or familiar object for male and fem ale shControl-NAc and shArc-NAc mice in the novel object recognition trial. d, Total time spent interacting with objects for male and female shControl-NAc and shArc-NAc mice during the novel object recognition trial. Novel object discrimination index by treatment (e) and by sex and treatment (f). Bars represent Mean and SEM; n = 9 – 12 mice/condition; * p < 0.05, ** p < 0.01, *** p < 0.001.