Interaction of Nitric Oxide and Serotonin in Aggressive Behavior
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Available online at www.sciencedirect.com R Hormones and Behavior 44 (2003) 233–241 www.elsevier.com/locate/yhbeh Interaction of nitric oxide and serotonin in aggressive behavior Silvana Chiavegattoa and Randy J. Nelsonb,* a Department and Institute of Psychiatry and Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of Sao Paulo Medical School, Sao Paulo, Brazil b Departments of Psychology and Neuroscience, Ohio State University, Columbus, OH 43210, USA Received 30 July 2002; accepted 19 February 2003 Abstract Nitric oxide (NO) modulates many behavioral and neuroendocrine responses. Genetic or pharmacological inhibition of the synthetic enzyme that produces NO in neurons evokes elevated and sustained aggression in male mice. Recently, the excessive aggressive and impulsive traits of neuronal NO synthase knockout (nNOSϪ/Ϫ) mice were shown to be caused by reductions in serotonin (5-HT) turnover and deficient 5-HT1A and 5-HT1B receptor function in brain regions regulating emotion. The consistently high levels of aggression observed Ϫ/Ϫ in nNOS mice could be reversed by 5-HT precursors and by treatment with specific 5-HT1A and 5-HT1B receptor agonists. The expression of the aggressive phenotype of nNOSϪ/Ϫ knockout mice requires isolated housing prior to testing. The effects of social factors such as housing condition and maternal care can affect 5-HT and aggression, but the interaction among extrinsic factors, 5-HT, NO, and aggression remains unspecified. Taken together, NO appears to play an important role in normal brain 5-HT function and may have significant implications for the treatment of psychiatric disorders characterized by aggressive and impulsive behaviors. © 2003 Elsevier Inc. All rights reserved. Keywords: Aggression; Violence; Gene knockout; Mouse; Animal model; Isolation; Environmental factors Introduction pectoralis was discovered in the 19th century (reviewed in Parker, 1996), although the mechanisms by which nitrates Nitric oxide (NO), first identified as an endogenous reg- worked were not discovered until 1980 (Furchgott and ulator of blood vessel tone, also serves as a neurotransmitter Zawadzki, 1980). Relaxation of blood vessels in response to in both the central and the peripheral nervous systems acetylcholine requires that the endothelium secretes a sub- (Baranano et al., 2001; Baranano and Snyder, 2001). NO is stance initially termed endothelial-derived relaxing factor an endogenous gas that has several biochemical properties (Furchgott and Zawadzki, 1980), which was ultimately dis- of a free radical at body temperature. NO is very labile, with covered to be NO (Baranano and Snyder, 2001). NO is also a half-life of Ͻ5 s; consequently, many studies have ma- the active metabolite of nitroglycerin, as well as other ni- nipulated NO indirectly by affecting its synthetic enzyme, trates, and stimulates blood vessel dilation by activating nitric oxide synthase (NOS), that transforms arginine into guanylyl cyclase which induces cGMP formation (Toda, NO and citrulline. Studies that have used this approach have 1995). Thus, NO is an important endogenous mediator of determined that NO mediates many critical physiological blood vessel tone. and behavioral functions (reviewed by Nelson et al., 1997). A second biological function of NO emerged in the late As noted, the first biological function of NO was discov- 1970s from an independent line of research documenting ered in the circulatory system. The ability of nitroglycerin the carcinogenic risk of dietary nitrosamines. The discovery and other organic nitrates to alleviate the pain of angina that both humans and non-human animals produce urinary nitrates in greater amounts than consumed and that this * Corresponding author. Fax: ϩ1-614-451-3116. production increases during bacterial infections led to the E-mail addresses: [email protected] (S. Chiavegatto), [email protected] realization that an endogenous source of nitrates existed (R.J. Nelson). (Green et al., 1981). Macrophages convert L-arginine to 0018-506X/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2003.02.002 234 S. Chiavegatto, R.J. Nelson / Hormones and Behavior 44 (2003) 233–241 L-citrulline and a reactive species that kills tumor cells in ing throughout development when critical ontogenetic pro- vitro, namely NO (Hibbs et al., 1987). Thus, NO plays an cesses, including activation of compensatory mechanisms, important role in immune function. may be affected (Nelson, 1997). Furthermore, differences in It was soon discovered that NO was also released when genetic background might also contribute to the observed cerebellar cultures were stimulated with glutamate (Garth- changes in behavior of knockout mice (Wolfer et al., 2002). waite et al., 1988). Pharmacological inhibition of the syn- To address these criticisms, mice were treated with 7-nitroi- thetic enzyme NOS blocked the elevation of cGMP levels in ndazole (7-NI) (50 mg/kg ip), which specifically inhibits brain slices coincident with activation of the N-methyl-D- nNOS formation in vivo (Demas et al., 1997). Isolated mice aspartate subtype of glutamate receptor (Bredt and Snyder, treated with 7-NI displayed substantially increased aggres- 1989; Garthwaite et al., 1989). Since then, many studies of sion in two different tests of aggression compared to control NO affecting neural function have been reported (reviewed animals (Demas et al., 1997). Drug treatment did not affect in Baranano and Snyder, 2001; Wiesinger, 2001). By acting nonspecific locomotor activities. NOS activity in brain ho- on the neurons and endothelial cells in the circulatory sys- mogenates was reduced Ͼ90% and immunocytochemical tem, NO exerts profound effects on neuroendocrine func- staining for citrulline, an indirect marker for the NO syn- tion and behavior. In this review, we focus on the role of thesis, revealed a dramatic reduction in 7-NI-treated animals NO on aggression. (Demas et al., 1997). These pharmacological results confirm Consistent with its three main functions, three distinct and extend the behavioral results obtained in nNOSϪ/Ϫ isoforms of NOS have been discovered: (a) in neural tissue mice. Importantly, a combination of the traditional pharma- (nNOS; NOS-1), (b) an inducible form found in macro- cological approach and a targeted gene disruption approach phages (iNOS; NOS-2), and (c) in the endothelial tissue of to the study of aggression enhances the strengths and min- blood vessels (eNOS; NOS-3) (Baranano and Snyder, imizes the weaknesses of each single approach. 2001). Suppression of NO formation by either elimination Plasma androgen concentrations influence aggression. Ϫ/Ϫ of arginine or use of N-methyl-L-arginine, a potent NOS nNOS and WT mice do not differ in blood testosterone inhibitor, affects all three isoforms of NOS. concentrations either before or after agonistic encounters (Nelson et al., 1995). However, data on castrated nNOSϪ/Ϫ males suggest that testosterone is necessary, if not suffi- Behavioral studies of nNOS cient, to promote increased aggression in these mutants (Kriegsfeld et al., 1997). Castrated nNOSϪ/Ϫ mice dis- nNOS is localized in high densities within emotion- played low levels of aggression equivalent to the reduced regulating brain regions (Nelson et al., 1997). The specific aggression observed among castrated WT males. Androgen- behavioral role of neuronal NO was examined in nNOS replacement therapy restored the elevated levels of aggres- gene knockout (nNOSϪ/Ϫ) mice created by homologous sion in nNOSϪ/Ϫ mice. recombination (Huang et al., 1993). Informal observation of A complete battery of sensorimotor tests was conducted the newly arrived nNOSϪ/Ϫ mice in our laboratory revealed to make certain that the mutant mice did not suffer from high levels of aggression among male cage-mates. We now impairments (e.g., blindness, muscular weaknesses) that know that the combination of the missing nNOS gene and may have indirectly facilitated aggression (Nelson, 1997). the isolated housing conditions within the shipping contain- Although there were no obvious motor impairments in ers facilitated the extreme aggressiveness when the animals nNOSϪ/Ϫ mice when tested during the light portion of the were subsequently housed in groups of five. Behavior, as day, testing during the animals’ nocturnal activity period any phenotype, is the result of genotype–environment in- was critical to reveal motor deficits. Because the cerebellum teractions. Often, the environmental influences are subtle, has the highest numbers of nNOS neurons in the brain, it and careful behavioral analyses are necessary to untangle was surprising that presumed cerebellar functions such as lawful relationships regarding the contribution of environ- balance and coordination were grossly normal in nNOSϪ/Ϫ ment and genotype to the behavioral phenotype (Pfaff, mice. When tested during the night (active phase of the 2001). Male nNOSϪ/Ϫ residents engaged in three to four rodents), a striking impairment in balance and motor coor- times more aggressive encounters than wild-type (WT) dination performance was discovered in the nNOSϪ/Ϫ mu- mice when tested in the intruder–resident model of offen- tants, but not observed in the WT mice (Kriegsfeld et al., sive aggression (Nelson et al., 1995). Nearly 90% of the 1999). The temporal interaction between nNOS activity and aggressive encounters were initiated by the nNOSϪ/Ϫ ani- the serotonin system has not been described. mals. Similar results were obtained in diadic or group en- Importantly,