Bidirectional Regulation of Aggression in Mice by Hippocampal Alpha-7 Nicotinic Acetylcholine Receptors
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Neuropsychopharmacology (2018) 43, 1267–1275 © 2018 American College of Neuropsychopharmacology. All rights reserved 0893-133X/18 www.neuropsychopharmacology.org Bidirectional Regulation of Aggression in Mice by Hippocampal Alpha-7 Nicotinic Acetylcholine Receptors 1 1 1 1 2 ,1 Alan S Lewis , Steven T Pittenger , Yann S Mineur , Dawson Stout , Philip H Smith and Marina R Picciotto* 1 2 Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA; Department of Community Health and Social Medicine, CUNY School of Medicine, New York, NY, USA Humans with 15q13.3 microdeletion syndrome (15q13.3DS) are typically hemizygous for CHRNA7, the gene coding for the α7 nicotinic acetylcholine receptor (nAChR), and manifest a variable neuropsychiatric phenotype that frequently includes persistent aggression. In mice, nAChR activation by nicotine is anti-aggressive, or ‘serenic,’ an effect which requires α7 nAChRs and is recapitulated by GTS-21, an α7 α nAChR partial agonist. Pharmacotherapies potentiating 7 nAChR signaling have also been shown to reduce aggression in human α 15q13.3DS. These findings identify the 7 nAChR as an important regulator of aggressive behavior, but the underlying neurobiological α substrates remain to be determined. We therefore investigated the brain regions and potential neural circuits in which 7 nAChRs regulate aggressive behavior in male mice. As in 15q13.3DS, mice heterozygous for Chrna7 were significantly more aggressive compared to wild- type controls in the resident-intruder test. We subsequently examined the hippocampus, where α7 nAChRs are highly expressed, particularly in GABAergic interneurons. Resident–intruder interactions strongly activated granule cells in the dentate gyrus (DG). In contrast, GTS-21, which reduces aggression in mice, reduced DG granule cell activity during resident–intruder interactions. Short hairpin RNA knockdown of Chrna7 in the DG enhanced baseline aggression and eliminated the serenic effects of both nicotine and GTS-21 on α α attack latency. These data further implicate 7 nAChRs in regulation of aggression, and demonstrate that hippocampal 7 nAChR signaling is necessary and sufficient to limit aggression. These findings suggest that nAChR-mediated regulation of hippocampal excitatory–inhibitory balance could be a promising therapeutic intervention for aggression arising in certain forms of neuropsychiatric disease. Neuropsychopharmacology (2018) 43, 1267–1275; doi:10.1038/npp.2017.276; published online 6 December 2017 INTRODUCTION schizophrenia, and behavioral problems including aggression (Sharp et al, 2008; Shinawi et al, 2009). Similar phenotypes Improved understanding of neural circuits contributing to are reported from smaller deletions encompassing only aggression is necessary to develop more effective therapeutic CHRNA7 and in at least some cases a portion of a options for patients, including those with neuropsychiatric neighboring gene, OTUD7A (Hoppman-Chaney et al, 2013; disorders. Syndromes associated with well-defined abnorm- Masurel-Paulet et al, 2010; Mikhail et al, 2011; Shinawi et al, alities at the genetic level can provide an important starting 2009). Furthermore, children and adolescents with aggressive point to understand fundamental pathophysiological me- behaviors severe enough to warrant antipsychotic treatment chanisms. 15q13.3 microdeletion syndrome (15q13.3DS; were recently reported to be far more likely to harbor OMIM #612001) is a rare condition that results from the deletions of CHRNA7 than the general population, with loss of part of the proximal portion of chromosome 15, an CHRNA7 deletion conferring an odds ratio of 184 for unstable genetic region prone to deletions and duplications aggression (Gillentine et al, 2017). Mouse models of (Sharp et al, 2008; Shinawi et al, 2009). The most common 15q13.3DS recapitulate multiple phenotypic aspects of the form of 15q13.3DS results from an ~ 1.5 megabase deletion disorder, including abnormal electroencephalography and that encompasses roughly six genes, including CHRNA7, the neuronal excitability, abnormal spatial memory and atten- gene encoding the excitatory ligand-gated α7 nicotinic tion, and aggressive behavior following mild stressors (Fejgin acetylcholine receptor (nAChR). This deletion is highly et al, 2014; Nilsson et al, 2016). Taken together, evidence penetrant and results in a variable phenotype that frequently from human and animal studies suggests that abnormal includes intellectual disability, seizures, neuropsychiatric genetic dosage of CHRNA7 and related neuronal signaling disorders including autism spectrum disorder (ASD) or disturbance likely plays a causal role in some neurobeha- vioral aspects of the 15q13.3DS, including aggression *Correspondence: Dr MR Picciotto, Department of Psychiatry, Yale regulation. University School of Medicine, 34 Park Street, 3rd Floor Research, New α Haven, CT 06508, USA, Tel: +203-737-2041, Fax: +203-737-2043, In a mouse model of aggression, we recently found the 7 E-mail: [email protected] nAChR was necessary for the anti-aggressive, or ‘serenic’ Received 8 July 2017; revised 6 October 2017; accepted 30 October effects of systemic administration of the pan-nAChR agonist 2017; accepted article preview online 7 November 2017 nicotine, and that GTS-21, an α7 nAChR partial agonist, Aggression regulation by hippocampal α7 receptors AS Lewis et al 1268 recapitulated the serenic effects of nicotine (Lewis et al, PBS only for all other experiments. Nicotine hydrogen 2015). In humans, galantamine, a positive allosteric mod- bitartrate was used for nicotine solutions, which were ulator at the α7 nAChR and an acetylcholinesterase inhibitor, adjusted to pH 7.0 ± 0.5 with 1 M sodium hydroxide and was reported to successfully reduce persistent, severe rage doses expressed as free base. All solutions were injected outbursts in a man with 15q13.3DS (Cubells et al, 2011), intraperitoneally (i.p.) 10–15 min prior to the start of while transdermal nicotine successfully reduced persistent resident–intruder tests at a volume of 10 ml/kg body mass. aggressive behavior in an adolescent with idiopathic ASD Drug administration was counterbalanced across animals for (Van Schalkwyk et al, 2015). Despite genetic and pharma- repeated-measures experiments. cological evidence linking CHRNA7 to pathological aggres- sivity, the brain regions and neural circuitry important for α aggression regulated by 7 nAChRs are not well understood. Resident–Intruder Test The α7 nAChR is highly expressed in the hippocampus of both mice (Baddick and Marks, 2011) and humans (Rubboli Resident–intruder tests were performed exactly as described et al, 1994), where it is preferentially expressed on previously (Lewis et al, 2015) and detailed in the GABAergic interneurons and can inhibit activity of principal Supplementary Materials and Methods. cells in multiple hippocampal regions (Frazier et al, 2003; Freedman et al, 1993; Ji and Dani, 2000). This organization α – suggests 7 nAChR signaling might regulate excitatory Locomotor Activity inhibitory (E/I) balance in the hippocampus, which is commonly altered in neurodevelopmental conditions (Gao Mice were habituated to the test room for 1 h, then placed in and Penzes, 2015) and directly linked to aggressive behavior a clean, clear plexiglass rat cage for 30 min. Activity was (Kohl et al, 2013; van der Kooij et al, 2014). measured by horizontal and vertical infrared sensors (Fusion We therefore investigated the brain regions critical to the HC version 1.5, AccuScan Instruments, Inc., Columbus, regulation of aggressive behavior by α7 nAChR signaling in OH, USA). mice, focusing on the hippocampus. Our results have important implications for better understanding how neuromodulatory systems might influence brain regions Hippocampal Infusion of Adeno-Associated Viral within larger networks important for aggression. Further- Vectors Expressing Small Hairpin RNA (shRNA) more, our results lend additional support to targeting E/I Constructs imbalance for treatment of pathological aggression, including pharmacological agents active at α7 nAChRs, which are well Design, purification, and validation of viral vectors are tolerated in human studies (Freedman, 2014). described in Supplementary Materials and Methods. Viral vectors were infused bilaterally into the hippocampus using a stereotaxic frame mounted with 5 μl Hamilton syringes MATERIALS AND METHODS under 2% isoflurane anesthesia. An aliquot of 2 μl of purified Animals virus was infused into each side of the intermediate hippocampus (coordinates: anterior/posterior = − 3.08 mm, Wild-type (WT) male C57BL/6 (B6) and Balb/c (B/c) mice lateral =±3.0 mm, dorsal/ventral = − 3.2 mm) over the were purchased from Jackson Laboratory (Bar Harbor, ME, course of 10 min, and the syringe left in place for an USA) and habituated to the animal facility for at least 1 week. additional 5 min to reduce backflow. Mice were subsequently Male mice heterozygous for a null mutation of the α7 subunit single housed and allowed to recover for 3 weeks prior to (HET) (Orr-Urtreger et al, 1997) maintained on the B6 behavioral testing. After testing, viral infusion sites were background for over 30 generations were bred with female B/ identified by GFP fluorescence in 40 μm brain slices from c mice to generate F1 WT and HET littermates. As B/c mice paraformaldehyde (PFA)-perfused animals. Mice with evi- are significantly more aggressive than B6 mice, (Schneider dence of GFP-positive cells in dentate gyrus (DG;