The physiology of aggression: towards understanding violence

The physiology of aggression: towards understanding violence

The research reported in this thesis was carried out at the Department of Behavioral Physiology of the University of Groningen (Biology Center, Haren, The Netherlands). The completion of this thesis was supported by the educational program of the BCN (Graduate School of Behavioral and Cognitive Neurosciences, University of Groningen). Printing this thesis was sponsored by Noldus Information Technology BV, Data Sciences International, BCN, Faculty of Mathematics and Natural Sciences, and the University of Groningen.

Cover image:

Cover design: Lay-out: Printer:

“Cain and Abel” (1542-44), Titian, Oil on canvas Santa Maria della Salute, Venice, Italy Doretta Caramaschi Dick Visser Drukkerij van Denderen BV, Groningen, NL

ISBN: 978-90-367-3985-6

© 2009 by Doretta Caramaschi No part of this book may be reproduced or transmitted in any form or by any means without prior written permission of the author.

RIJKSUNIVERSITEIT GRONINGEN

The physiology of aggression: towards understanding violence

PROEFSCHRIFT

ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op vrijdag 9 oktober 2009 om 14.45 uur

door

Doretta Caramaschi geboren op 23 november 1979 te Suzzara, Italië

Promotor: Copromotor:

Prof. dr. J. M. Koolhaas Dr. S. F. de Boer

Beoordelingscommissie:

Prof. dr. B. Olivier Prof. dr. A. J. W. Scheurink Prof. dr. A. Sgoifo

“Mankind must evolve for all human conflict a method which rejects revenge, aggression, and retaliation. The foundation of such a method is love." Martin Luther King Jr., December 11, 1964

Table of contents CHAPTER 1

General introduction

9

CHAPTER 2

Is there co-selection for aggressiveness, coping strategy and emotionality in mice? 45 CHAPTER 3

Development of violence in mice through repeated victory along with changes in prefrontal cortex neurochemistry

61

CHAPTER 4

Is hyper-aggressiveness associated with physiological hypo-arousal? A comparative study on mouse lines selected for high and low aggressiveness

81

CHAPTER 5

Differential role of the 5-HT1A receptor in aggressive and non-aggressive mice: an across-strain comparison.

99

CHAPTER 6

Changes in serotonin-1A receptor functionality with social experience in mouse lines selected for high and low aggression

119

CHAPTER 7

Tryptophan-free diet lowers fronto-cortical serotonin levels with no effect on mouse aggression

133

CHAPTER 8

Dynamic intracellular distribution of serotonin-1A receptors in mice predisposed to violence

147

CHAPTER 9

General discussion

161

Reference list

175

Nederlandse samenvatting

199

Sintesi della tesi in italiano

205

Acknowledgements

212

1

CHAPTER

General introduction

CHAPTER 1

Aggression is a behaviour expressed in a social context, when two individuals are in conflict with each other. From a biological point of view, aggression is necessary to achieve social dominance and thereby procure resources and the means to reproduce. Thus, a certain level of aggressiveness is generally considered to contribute to higher fitness. In non-human animals, aggression in competition over resources is considered a normal behaviour. In human societies, in contrast, aggression is often unwanted and can reveal itself as a symptom of psychopathologies. Human aggression can entail violent acts that cause suffering and eventually death and that are therefore punishable by law. Despite various political and financial efforts to reduce human violence, little is known about which prevention and intervention programmes are the most effective (Krug et al., 2002). Part of the problem lays in the fact that we still lack a clear mechanistic explanation of violence. Most of the animal models for aggression that have tried to address this issue cannot be translated easily into the violent human phenotype. The aim of this thesis is to identify central and peripheral physiological mechanisms associated with extreme aggressiveness using animal models, with the ultimate aim to contribute to more evidence-based intervention and prevention programs for human violence. Knowledge of these mechanisms may also have implications for the guidance of public and judicial policies.

AGGRESSION AND VIOLENCE Definitions A considerable problem in the study of aggression across species is the confusion between the definitions of aggression and violence. The two constructs overlap, but there are important differences. Both terms were first used to describe human behaviour. In the Oxford English Dictionary, aggression is defined as “Hostile or destructive tendency or behaviour, held to arise from repressed feelings of inferiority, frustration or guilt. In addition, feeling or energy displayed in asserting oneself, in showing drive or initiative, aggressiveness, assertiveness, forcefulness. (Usu. as a positive quality.)”. Violence, on the other hand, is “The exercise of physical force so as to inflict injury on, or cause damage to, persons or property; action or conduct characterized by this; treatment or usage tending to cause bodily injury or forcibly interfering with personal freedom.” (Simpson and Weiner, 2000). It is noticeable that these definitions cannot easily be translated in nonhuman animal behaviours, since we can neither measure objectively “feelings of inferiority”, “drive or initiative” and “assertiveness” nor know the motivation “to inflict injury”. Another point of confusion is the positive or negative connotation that aggression assumes depending on the circumstances. Often, in humans, it is 10

GENERAL

INTRODUCTION

almost overlapping with violence, indicating an unwanted behaviour. Alternatively, it is sometimes used to describe a positive behaviour when it overlaps with assertiveness and drive. Since none of these concepts is applicable to nonhuman animal behaviour, translational (across-species) research on aggression and violence needs operational definitions that consist of objectively measurable behaviours both in human and non-human studies. Non-human ethological research has recognized the function of agonistic interactions in survival and reproduction for decades. Aggression is primarily an adaptive behaviour and is part of a more general proactive coping strategy aimed at mastering challenging environmental situations. Due to its important biological function, a certain degree of aggression is evolutionarily conserved. This functional aggression (Natarajan et al., 2009), as described in non-human animals, shows moderate intensity, context-dependency and sensitivity to inhibitory cues. Violence, as a more extreme form of this behavioural expression, goes beyond these contextual inhibitory characteristics; it often results in maladaptive outcomes such as injuries to partner and progeny. The pathological characteristics of this exaggerated behaviour make it more comparable to human violence. A growing number of studies report examples of non-human animal violent behaviour, even among invertebrates. The distinction between functional aggression and violence is an important issue to consider in the study of violence, since the physiological determinants of aggression might not overlap completely with the pathological determinants of violence, and the interpretation of data obtained in such studies might differ drastically. Therefore, when studying violence in a non-human animal model it is important to think carefully about definitions. Can we define violence in a rodent model? What should the criteria be to allow us to compare it to human violence? I touch upon this issue particularly in Chapter 3, although it is a major theme throughout the thesis. Types of aggression Another confusing point in the study of aggression is the fact that the term “aggression” refers both to the life-stable behavioural trait and to the discrete actual aggressive act or state. Aggression, as a behavioural trait, is the propensity to engage in agonistic interactions. As a behavioural state, aggression, or the aggressive act, can be categorized in different types. Most of the research on aggression in non-human species is carried out in rodents. Depending on the stimulus eliciting the aggressive act, different types have been described in rodents, namely predatory, intermale, fear-induced, territorial, irritable, sex-related, maternal and instrumental (Moyer, 1968). Except for predatory aggression, which is typically interspecies, from a predator to its prey, the other types are 11

CHAPTER 1

expressed in intraspecific interactions. Mothers defending their progeny exhibit maternal aggression, while the other types are usually found in males. Fearinduced and instrumental aggression differ mainly in the accompanying arousal level, which is higher in the former than the latter. Further research in rodents identifies a major distinction between offensive and defensive aggression, where the difference lies in the presence or absence of a perceived threat immediately before the expression of aggression (Blanchard and Blanchard 1977; Blanchard and Blanchard 2003). Similarly, in humans, there is a consensus about two distinct types of aggression, namely reactive aggression (affective, emotional or hostile aggression) and the premeditated, instrumental or proactive aggression (Vitiello and Stoff 1997). It seems that most aggressive children display mainly reactive aggression, while a smaller proportion shows both reactive and instrumental aggression. Reactive aggression is conceptually close to impulsive aggression, yet individuals that engage in instrumental aggression are very often also highly impulsive in other contexts; yet they form a separate group from the impulsive aggressive individuals. Reactive and proactive types of aggression are often described in children and are thought to be the result of the activation of distinct neural pathways and to correlate with different levels of physiological reactivity. In this thesis I focus on male offensive aggression, although the characterization of female aggression and non-offensive subtypes of aggression in rodents needs to be explored further.

PERIPHERAL PHYSIOLOGY OF AGGRESSION Stress hormones There is a strong intuitive connection between aggression and stress. In certain individuals, aggression is displayed as part of the effort to cope with a social challenge or stressor (Benus et al., 1991b; Koolhaas et al., 1999). At the peripheral level, any perceived stressor activates two main physiological pathways: the sympatho-adrenomedullary (SAM) system and the hypothalamic-pituitaryadrenocortical (HPA) axis. Aggressive individuals tend to respond physiologically to stress with a high activation of the SAM system, while low-aggressive or reactive individuals show a higher HPA-axis activation (Benus et al., 1991b; Koolhaas et al., 1999). In mice, both offensive and defensive aggression give rise to high HPA and SAM activation during the social encounter (Bartolomucci et al., 2003). In humans, individuals with a Type A personality, e.g. those who are hostile and irritable, are more prone to develop cardiovascular problems associated with high sympathetic or poor parasympathetic cardiac control (Ward et al., 1986; Lee and Watanuki 2007). Often, display of aggression is associated with the feeling of 12

GENERAL

INTRODUCTION

anger and a high emotional response. In contrast to this reactive, hostile, emotional aggression, instrumental or cold-blooded aggression is executed in the absence of emotional arousal. Individuals that frequently engage in antisocial behaviour using instrumental aggression show a reduced autonomic baseline tone and low HPA axis tone and activation (Virkkunen, 1985; Raine et al., 1990; Scarpa and Raine 1997; Raine et al., 2000; Raine, 2002a; Raine, 2002b; Popma et al., 2006). A long-lasting physiological activation in response to stress can also be one of the causes of the development of aggressive personality. Glucocorticoids and noradrenaline released in the blood stream during a stressful situation may affect neural substrates by binding to certain receptors, specifically those on brain circuits involved in neuroplasticity, i.e. the hippocampus, and in the regulation of social behaviour, i.e. the serotonin and vasopressin systems. In vulnerable individuals, stress can lead to a subtype of depression characterized by anxiety, anger and aggressive outbursts (van Praag, 2004). Moreover, adverse early life experiences such as maltreatment or neglect can lead to the development of aggressive temperamental disturbances (Caspi et al., 2002; Van Goozen et al., 2007). Recent studies in rats have also demonstrated pro-aggressive effects in juveniles and adults that are maternally separated or early socially deprived (Veenema et al., 2006; Toth et al., 2008). In adolescent hamsters, social stress can lead to accelerated development of adult forms of agonistic behaviour via activation of the HPA axis (Wommack and Delville 2007a; Wommack and Delville 2007b). However, reduced HPA-axis functioning in adult rats causes an increase in pathological aggression, expressed as a shift to a predominance of injurious attacks on vulnerable body regions rather than ritualized, less injurious ones (Haller et al., 1998; Haller et al., 2000; Haller et al., 2001). It has been proposed that pronounced stress activation in the early phases of life tunes or primes the functioning of the neuronal fear circuit, leading to desensitization in individuals that develop antisocial behaviour (Van Goozen and Fairchild 2008). Stress-related physiological mechanisms might be a good indicator of different types of aggression and of the severity of the risk of developing violence. But are aggressive and violent mice physiologically similar to violent humans? I investigate this topic in Chapter 4. Gonadal hormones Testosterone, the male sex hormone, exerts its main function in determining the male sex during prenatal life and at puberty. Since in many mammalian societies, aggressive behaviour is typically expressed in male intrasexual competition for females and social dominance, it is logical to think of a role for testosterone and/or its metabolites in the development of an aggressive behavioural phenotype. With this in mind, previous studies have looked for an association and a 13

CHAPTER 1

causal relationship between testosterone and aggression. Basically, one of the conceptual sources for studying the neurochemical basis of social behaviour derives from the endocrine depletion-repletion research paradigm that was introduced in 1849 by Arnold Berthold. He removed the gland of interest (testis) that secreted the source of the endogenous substance (testosterone) that he suspected to be necessary for the display of a specific behaviour (aggressive displays) and consequently that behaviour disappeared. Thereafter he replaced the glandular material and recorded the return of the original behaviour (Berthold, 1849). The near-total decline in aggressive displays and fighting after castration and the effective restoration of this behaviour after administration of testosterone has been replicated in several invertebrate, fish and avian species thereby indicating the obligatory role of androgens in aggressive behaviour. However, in mammalian species like rodents and primates, instead of being obligatory in their function, androgens exert a modulatory effect on aggressive behaviour. Castrated mice and rats without prior to aggressive experience rarely fight when confronted by a male conspecific. However, when aggressive behaviour is fully established in the behavioural repertoire, castration gradually reduces but does not prevent aggression against a conspecific male (De Bold and Miczek, 1981). A positive correlation between aggression and testosterone has been shown in humans e.g. see for review (Archer, 2006) and non-human primates (Higley et al., 1996). However, the correlation is not always found, leading some to propose that testosterone is more related to dominance, which can also be expressed without aggression e.g. see for review (Archer, 2006). A cross-strain genetic analysis in mice also revealed a positive correlation between testosterone and aggression (Roubertoux et al., 2005). In line with a causal relationship is the finding that a reduction of testosterone levels in rodents leads to a decrease, and enhancement to an increase, of aggression e.g. see for review (Nelson and Trainor 2007). This effect could occur through different mechanisms. First, testosterone may act either directly or as one of its active metabolites, dihydrotestosterone (DHT), dihydro-epiandrosterone (DHEA) and estradiol or a combined androgenic/estrogenic action (see for review (Simon et al., 1996)). Second, testosterone might stimulate vasopressin synthesis and in this way enhance aggression (Ferris et al., 1989; Ferris and Delville 1994; de Vries, 2008). Vasopressin itself can also promote aggression indirectly by reducing serotonin neurotransmission in the hypothalamus (Ferris and Delville 1994). Similarly, androgens might modulate 5-HT and GABA and therefore affect aggression (Cologer-Clifford et al., 1997; Cologer-Clifford et al., 1999; Miczek et al., 2002; Mitchell et al., 2008). Another testosterone-dependent mechanism which might exert its effects through the serotonin pathway involves nitric oxide (Nelson and Trainor 2007). 14

GENERAL

INTRODUCTION

NEUROBIOLOGY OF AGGRESSION Neuroanatomy and neurophysiology A schematic overview of a putative neural network involved in aggression is presented in figure 1.1 (Koolhaas et al., in press). As an emotional behaviour, aggression involves the activation of brain structures involved in emotion control and appraisal (Dalgleish, 2004). The expression of an aggressive state, studied in detail in cats, is associated with modulation by the midbrain on sympathetic activation (Bard, 1928). Electric stimulation of the hypothalamus in cats, rats and monkeys is sufficient to induce aggression together with neuroendocrine and autonomic activation. During an aggressive state, brain areas related to the organization of general stress responses like the stria terminalis, mediocentral amygdala, hypothalamus, pituitary, septum, locus coeruleus, dorsal periacqueductal grey and orbitofrontal cortex become activated e.g. see (Gregg and Siegel 2001). Recently, using modern brain imaging techniques (fMRI) in awake rats that were triggered to aggressive motivation, this suspected putative neural circuitry of aggression was indeed activated together with the unexpected intense activation of anterior thalamic nuclei (Ferris et al., 2008).

Gonadal steroids

Olfactory input

Sensory input

VNO

Thalamus

Aob Bnst

Aha

Poa

Mea

LS

PFC

Motor cortex

Basal ganglia

PAG Neuroendocrine output

Autonomic output Nociception

Motor output

Figure 1.1 Schematic diagram of a putative neural network involved in offensive aggression (Koolhaas et al., in press).VNO= vomeronasal organ, Aob= anterior olfactory bulb, Mea= medial amygdala, LS= lateral septum, PAG= periacqueductal gray, Bnst= bed nucleus of stria terminalis, Poa= hypothalamic preoptic area, Aha= anterior hypothalamic area, PFC= prefrontal cortex'

15

CHAPTER 1

Instrumental aggression is not associated with emotional arousal and might be more related to developmental impairments in amygdala-based aversive conditioning, as well as dysfunctions in error-monitoring and emotional appraisal in the anterior cingulate cortex, e.g. see for review (Crowe and Blair 2008). Instrumental aggression, in particular its unemotional and callous characteristic, might also be related to low empathy and brain dysfunctions in areas involved in empathy (Patrick and Zempolich 1998). In general, prefrontal cortical brain regions are responsible for behavioural planning, execution, inhibition and emotional and sensorimotor integration. Therefore my thesis focuses on this anatomical area. Neurochemistry: serotonin The best-known neurochemical mechanism associated with violence is the brain serotonergic system. Initially the association was found in isolated mice that became aggressive while reducing their serotonin turnover, which is an indicator of serotonin neurotransmission and consequent degradation (Garattini, 1967; Giacalone et al., 1968; Modigh, 1973). Similar studies were then performed in human and non-human primates, confirming a negative correlation between serotonin metabolite levels in the cerebrospinal fluid and high, impulsive aggression levels (Brown et al., 1979; Brown et al., 1982; Linnoila et al., 1983; Virkkunen et al., 1989; Limson et al., 1991; Mehlman et al., 1994; Higley et al., 1996; Fairbanks et al., 2001). The negative correlation between serotonin turnover and aggression became known as the serotonin-deficiency hypothesis of aggression. A proper functioning of the serotonergic system involves the integration of several processes: serotonin synthesis, release, receptor activation, re-uptake and degradation. From the serotonin-deficiency hypothesis as a starting-point, followup research has pursued the idea that changes in one or more of these elements might explain the low serotonin metabolite levels in aggressive/violent individuals. The functioning of the enzyme tryptophan hydroxylase (TPH) tightly regulates serotonin synthesis. Manipulating serotonin synthesis by reducing the availability of tryptophan, the essential precursor of serotonin, increases aggressiveness in various species and contexts (Gibbons et al., 1979; Chamberlain et al., 1987; Kawai et al., 1994; Cleare and Bond 1995; Bjork et al., 2000; Bond et al., 2001; Bell et al., 2001). Early studies on the effects of the TPH inhibitor parachlorophenylalanine documented increases in aggression in rats (Sewell et al., 1982; Vergnes et al., 1986). Moreover, a single-nucleotide polymorphism in the coding region of the TPH gene has been associated with anger and aggression in healthy human subjects (Rujescu et al., 2002; Hennig et al., 2005). Serotonin release is reduced in the prefrontal cortex of rats after fighting against an intruder (van Erp and Miczek 2000), while stimulation of serotonin 16

GENERAL

INTRODUCTION

release with fenfluramine reduces aggression in human subjects, especially those with a history of aggression (Coccaro et al., 1996; Cherek and Lane 1999; Cherek and Lane 2001). Serotonin release is under the control of the inhibitory autoreceptors 5-HT1A and 5-HT1B, located distally and proximally, respectively, to the synapses (Pineyro and Blier 1999). Activation of 5-HT1A or 5-HT1B receptors on the serotonergic neurons reduces aggression in rats and mice (de Boer et al., 2000; Bannai et al., 2007). 5-HT1A receptors are described in more detail in the box “The 5-HT1A receptor: functions and regulation” and figure 1.2. Serotonin release is triggered by action potentials in the serotonergic cells and is modulated by heteroreceptors, for example NMDA, GABAA/B, α1/2 and D2, which are activated by release of their specific neurotransmitters by neuronal projections originating from different brain areas (Pineyro and Blier 1999). It is not yet clear how these heteroreceptors on serotonergic neurons are involved in aggressive behaviour. Once released, serotonin is transiently available in the synaptic cleft for neurotransmission. The extracellular availability of serotonin depends on the functionality of the serotonin transporter (5-HTT) and the monoamino-oxidase (MAO) enzyme, responsible for reuptake and degradation, respectively. Acutely, inhibition of 5-HTT with SSRIs leads to reduction of aggression in several animal models (Olivier et al., 1989; Ferris and Delville 1994; Dodman et al., 1996), and mice and rats lacking 5-HTT show reduced aggressiveness (Holmes et al., 2002;

Figure 1.2 Schematic representation of a serotonin neuron and the position of the inhibitory 5HT1A receptors on it (autoreceptors) and on a non-serotonergic neuron (heteroreceptors).

17

CHAPTER 1

Homberg et al., 2007). It therefore seems that the longer serotonin is available at the synaptic cleft, the lower the aggressiveness. However, during development, low activity of the MAO-A enzyme due to genetic polymorphisms is associated with a highly aggressive personality and an increased predisposition to develop antisocial behaviour and conduct disorder (Brunner et al., 1993; Manuck et al., 2000; Caspi et al., 2002; Foley et al., 2004). Accordingly, deletion of the MAO-A gene in mice results in increased aggressiveness (Cases et al., 1995). These studies suggest that serotonin availability at the synapses might have differential roles during different developmental stages. Serotonin binds to at least 14 subtypes of specific postsynaptic membranebound receptors, each with a different location and function. In vivo pharmacological studies have provided evidence for decreased functionality of poststynaptic 5-HT2A/C and 5-HT1A heteroreceptors, probably hypothalamic, in aggressive individuals (Coccaro et al., 1989; O'Keane et al., 1992; Coccaro et al., 1995; Moeller et al., 1998; Cherek et al., 1999; Cleare and Bond 2000). Similarly, decreased 5-HT2-induced activation of the prefrontal cortex and the anterior cingulate and a decreased amount of 5-HT1A receptors in the anterior cingulated, prefrontal cortex, amygdala and dorsal raphe have been found in aggressive human subjects (Parsey et al., 2002; New et al., 2002). Although there seems to be a consensus that low serotonergic neurotransmission is linked to high aggressiveness, other studies show a positive correlation or no correlation between serotonin neurotransmission and aggressiveness. In most of the cases, contradictory reports can be explained by examining in detail the different definitions of aggression used and the choice of subjects. Typically, research on aggression in non-human animals and in healthy humans has generated results that are in apparent conflict with data on pathologically violent human offenders. The latter suggests that the role of the serotonergic system might differ between the different subtypes of aggression and it is therefore important to take into account the specific type of aggressive behaviour studied. The paradigms used to assess components of the serotonergic system also seem to matter in adding complexity to the interpretation. Indeed, the serotonergic system is a highly complex and dynamic network that is not restricted to a modulatory function in the central nervous system, but has also a key role in the periphery, where serotonin serves physiologically essential functions. Is serotonin related specifically to violence and therefore the most pathological cases of aggression, or to aggressive tendency in general? How is serotonergic neurotransmission characterized in aggressive and violent individuals? What are the role and the dynamics of the inhibitory 5-HT1A receptors in violent individuals? Chapters 5 , 6, 7, and 8 provide some answers to these questions.

18

GENERAL

INTRODUCTION

Other neurochemical correlates Other neurotransmitter systems have been linked to aggression, such as dopamine, GABA, glutamate and noradrenaline (Nelson and Trainor 2007). Dopaminergic D2 antagonists are used clinically to treat violent outbursts. D2 receptor agonists (but not D1) induce defensive behaviour in cats. Elevated frontocortical dopamine precedes attacks and defensive behaviour in rats and amphetamine-induced dopamine release can increase aggressive behaviour in mice and rats. However, the specificity of dopamine in the control of aggression remains speculative since many of the dopaminergic compounds enhance or reduce general activity (agitation, sedation, fatigue)(see for review DE Almeida et al., 2005). GABA is generally found to be at a low level in aggressive individuals. However, compounds that modulate the activity of GABAA receptors, including alcohol, may have opposite effects on aggression, also depending on type of aggression. The anti-aggressive effects of benzodiazepines seem to be due to sedation, except for escalated/pathological forms of aggression in mice and rats in which there is a more selective antiaggressive effect (see for review De Almeida et al., 2005). Glutamate excitation of the PAG elicits defensive rage behaviour in cats and is involved in exaggerated emotional reactivity leading to seizures (Gregg and Siegel 2001). Again, the specificity of the antiaggressive effect of compounds acting on glutamate NMDA receptors is dependent on the aggression model used. Belozertseva and Bespalov (1999) reported that in isolation-induced mouse aggression it was mainly due to sedation, whereas it seemed to be more specific for aggression in the morphine-heightened aggression model (Belozertseva and Bespalov 1999). Since all these neurotransmitters exert modulatory actions on the serotonergic neurons and vice versa, their effects on aggression might due to the indirect interaction with components of the serotonergic system. Convergence on serotonin as unifying mechanism The aforementioned neural and endocrinological mechanisms proposed to explain aggression in mammals share a common feature: the involvement of serotonin along their upstream or downstream pathways. A summarized overview is depicted in Figure 1.3.

19

CHAPTER 1

Figure 1.3 Schematic diagram of hormones and neurotransmitters found to be related to aggression and serotonin. See text for abbreviations and details.

20

GENERAL

INTRODUCTION

Box 1.1: THE 5-HT1A RECEPTOR: FUNCTIONS AND REGULATION The serotonin-1A receptor (5-HT1A-R) is the most studied of the serotonin receptors, since it was the first to be visualized in tissue, cloned and manipulated pharmacologically. The 5-HT1A protein is coded by an intronless gene, Htr1a, located on chromosome 13 in the mouse genome and on chromosome 5 in the human genome. Htr1a codes for a protein of 421-422 aminoacids, with high homology between mouse and human (Sundaresan et al., 1989). Upstream non-coding regions have been identified as domains for transcriptional regulation for 5-HT1A (Le Francois et al., 2008). Although there is no high-resolution image of the protein, its predicted structure consists of seven putative hydrophobic transmembrane domains, with the amino terminus oriented facing the extracellular space, three hydrophilic intracellular loops and three extracellular loops. N-glycosylation sites at the extracellular terminus suggest that the protein is transferred from the ER, where it is produced, to the Golgi, where it is glycosylated (Raymond et al., 1999). Due to a specific motif in the C-terminus and to interaction with other proteins, 5-HT1A-R is then translocated to specialized regions of the cell membrane (Carrel et al., 2006; Carrel et al., 2008). 5-HT1A-R tends to be localized in specific membrane microdomains (Kalipatnapu and Chattopadhyay 2005), where it exerts its function of G-protein couple receptor, GPCR. 5-HT1A-R is expressed in several regions of the central nervous system, specifically the lateral septum, hippocampus, frontal and enthorinal cortices, anterior raphe nuclei, neocortex, several thalamic and hypothalamic nuclei, nucleus of the solitary tract, dorsal tegmentum, nucleus of the spinal tract and of the trigeminal nerve, and in the spinal cord (Palacios et al., 1990; Chalmers and Watson 1991; Kia et al., 1996b). It is mainly localized on the soma and dendrites of neurons, and possibly also on glial cells. Peripherally, 5-HT1A-R is also expressed in the skin, gut and blood mononuclear cells (Gershon et al., 1990; Yang et al., 2006; Nordlind et al., 2008). The receptor has a high affinity for serotonin. As GPCR, the 5-HT1A receptor exerts its main cellular function through activation of G-proteins upon agonist ligand stimulation. The main signalling mechanisms in which 5-HT1A-R is involved are: (i) inhibition of adenylyl

21

CHAPTER 1

cyclase, and consequently decrease of cyclic AMP, through coupling with Gi/o proteins; and (ii) activation of phospholipase C and other phospholipases, and consequently PKC, through G-protein βγ-subunits. Through these mechanisms, and depending on the cells in which it is expressed, 5-HT1A-R stimulates: (i) inward current and intracellular mobilization of Ca2+; (ii) Erk, Akt and nuclear factor κB, and consequently acts on proliferation and cell survival pathways; (iii) production of reactive oxygen species and nitric oxide; and (iv) active ion transport. 5-HT1A-R also activates G-protein-gated inwardly rectifying K+ channels (GIRK channels), with consequent neuronal hyperpolarization, see for review (Raymond et al., 1999). Differences in functions exerted through different brain regions might depend on a differential coupling to G proteins. Functional mutations in the second intracellular loop might influence the coupling itself (Mannoury la Cour et al., 2006; Kushwaha et al., 2006). In the brain, 5-HT1A-R exerts important functions, such as the regulation of circadian rhythms, mood, eating, fear conditioning and social behaviour (de Boer et al., 2000; Albert and Lemonde 2004; Horikawa and Shibata 2004; Ebenezer et al., 2007; Shields and King 2008). At the physiological level, the 5-HT1A receptor is involved mainly in temperature and cardiovascular regulation (Hjorth, 1985; Nalivaiko and Sgoifo 2008; Audero et al., 2008). As mentioned above (see Serotonin deficiency), 5-HT1A-R is involved in these functions by acting as a postsynaptic heteroreceptor in non-serotonergic cells and as a presynaptic autoreceptor in serotonergic cells. The intracellular functions in which the 5-HT1A autoreceptor is implicated have important consequences for the serotonin-producing neurons and are therefore a candidate pathway for the research on aggression and violence. The functionality of 5-HT1A-R is dependent, as mentioned earlier, on its expression levels, i.e. transcriptional regulation. Moreover, 5-HT1A-R can be phosphorylated and therefore desensitized by PKA, PKC and GRK (G-proteincoupled receptor kinase) (Raymond et al., 1999). Finally, 5-HT1A-R functionality is attenuated by regulators of G-protein signaling (RGS) proteins (Raymond et al., 1999). The functional desensitization of 5-HT1A-R is also obtained through agonist-mediated endocytosis/internalization (Riad et al., 2001; Riad et al., 2004). In relation to aggression research, malfunctioning at any of these regulation levels may have important consequences for the functioning of the serotonergic system.

22

GENERAL

INTRODUCTION

INDIVIDUAL VARIATION Individuals showing different personality types may differ largely in aggressive traits. Human personalities are generally described in terms of five independent factors (Digman, 1990). Several behavioural variables are correlated with each other and cluster within one of these factors. These five dimensions are Neuroticism, Extraversion, Conscientiousness, Agreeableness and Openness to Experience. Aggression seems to be associated with both high Neuroticism and low Agreeableness (or high Antagonism). The neurotic aggressive type is characterized by emotional instability, anger and irritability, while the antagonist aggressive type is hostile and antisocial. A recent meta-analysis (Bettencourt et al., 2006) found positive correlations between high laboratory aggression and high self-reported trait aggressiveness and irritability both under unprovoking and provoking conditions. Other personality traits such as trait anger, type A personality, narcissism and impulsivity were only associated with aggression under provocation. The authors concluded in the first instance that there is a strong association between certain personality dimensions and physical aggression and, secondly, that trait aggressiveness and trait irritability include a mixture of proactive and reactive aggression, while trait anger, type A personality, narcissism and impulsivity relate to reactive aggression. Longitudinal studies confirm the idea that individuals show life-stable aggressive traits from early life and that some individuals show a propensity to commit violent crimes from youth (Nagin and Tremblay 1999; Loeber and Pardini 2008). Although it is clear that certain personality types are more prone to exhibit aggression, the question is whether these individuals are at risk of showing violence, defined as an extreme and injurious form of aggressive behaviour usually not acceptable in our society. From prison settings and criminal records, it seems that individuals with antisocial personality disorder and/or psychopathic personality are the most common violent types. Antisocial personality disorder is defined by the Diagnostic and Statistical Manual of Mental Disorder (fourth edition) as a “pervasive pattern of disregard for, and violation of, the rights of others that begins in childhood or early adolescence and continues into adulthood”. It is characterized by child conduct problems, impulsivity, irresponsibility, absence of long-term goals and poor behavioural control. Psychopathy partially overlaps with these behavioural deviances, but adds an extra dimension of emotional detachment and has various physiological correlates. A low proportion of individuals with antisocial personality disorder expresses signs of psychopathy (antisocial psychopaths). Individuals with antisocial personality disorder are partly defined by their criminal behaviour and therefore they are typically violent. In prisons, high psychopathy is associated with a high incidence of violent crimes and recidivism, especially in 23

CHAPTER 1

low-IQ and socially withdrawn psychopaths (Patrick and Zempolich 1998). However, in the population there is also a proportion of individuals characterized by unemotional behaviour that is not responsible for violent crimes or is not imprisoned. Interestingly, the psychopathic personality, or at least some of its most salient features, can be assessed already in children. Therefore, these features could potentially be used to identify a risk of becoming a violent offender. Given the difficulty in defining and quantifying behavioural traits that would predict a risk for the development of a violent personality, it is necessary to identify stable individual differences in physiological and neurochemical traits that correlate with stable individual differences in aggression and violence. I try to elucidate this association throughout the thesis.

ANIMAL MODEL: MOUSE SELECTION LINES FOR HIGH AND LOW AGGRESSIVENESS In order to address the issues mentioned above, I used a unique tool: three pairs of mouse lines selected for high and low aggression. A few decades ago, three independent laboratories bred aggressive mouse lines through artificial selection for certain aggression parameters. This resulted in three different animal models for aggression, each of them represented by a highly aggressive and a low/nonaggressive line. The details of the selections are summarized in Table 1.1. In Turku, Finland, Lagerspetz and Sandnabba developed the Turku-Aggressive (TA) and the Turku-Nonaggressive (TNA) lines from an outbred colony of Swiss albino mice (Lagerspetz, 1961). The mice from each line were kept in different rooms, weaned at 21 days of age and individually housed. At 60 days of age, males were tested for aggression in a standard 7-minute dyadic test in a neutral arena (clean glass container) against standard opponents (pretested nonaggressive animals). The aggressive behaviour was rated on a 7-point scale as follows: 1) The animal shows no interest in its test partner except occasional nosing. Tries to escape, squeaks, is immobile, if attacked by the partner. 2) Frequent nosing. Escapes, but tries occasionally to protect itself from the attacks of the partner. 3) Frequent vigorous nosing. The animal assumes occasionally a position of readiness for fight. The animal does not attack the test partner but protects himself when attacked. 4) Tail rattling, vigorous nosing. The animal follows and occasionally attacks the test partner. 5) Slight wrestling and occasional powerful attacks. The animal attacks its partner and bites. Tail rattling. 24

Wild-type mice captured in Groningen, The Netherlands

Van Oortmerssen and Bakker, 1981

Lagerspetz, 1961 Lagerspetz and Lagerspetz, 1971

Cairns et al., 1983

SAL LAL

TA TNA

NC900 NC100

Out-bred ICR (Institute of Cancer Research)

Out-bred colony of Swiss albino mice

Background strain

Obtained Reference lines

Table 1.1 Overview of the three selection programs.

Housed in relative isolation. At 45 days of age. In a neutral partition cage, against a group-reared test partner. 5 minutes with slide closed and 10 minutes with slide open (video recording).

Housed individually. At the age of 60 days. In a neutral cage against a non-aggressive mouse pre-tested.

Housed with a female. At the age of 14 weeks. On the edge of the home cage against a docile albino intruder (MAS-Gro). Resident-intruder paradigm repeated on three consecutive days.

Aggression test

Attack-frequency/ Attack latency score up to 900

7-point scale ratings (see text)

Average of the three Attack Latency Tests= Attack Latency Score (ALS)

Parameters scored

Brothers-sisters mating avoided

Brothers-sisters mating avoided

Brother-sisters mating only in the first four generations

Breeding

GENERAL INTRODUCTION

25

CHAPTER 1

6) Fierce wrestling and biting during most of the period. 7) Fierce wrestling. The animal bites the partner hard enough to draw blood. Males with high aggression scores were used for breeding together with females, sisters of high-scoring males, avoiding brother-sister mating (Lagerspetz and Lagerspetz 1971). The selection produced an aggressive line in the 11th generation, while the other line was already non-aggressive in the first generation. However, mice that showed some aggression were always present in the TNA line. Breeding pairs of both lines (10 of each, 77th generation) were transferred to Groningen, The Netherlands in 2003 and are currently in their 83th generation of selection. In Groningen, the Netherlands, the selection was carried out in a colony randomly bred from wild house mice trapped in a mansion near Groningen (van Oortmerssen and Bakker 1981). To breed the Short Attack Latency (SAL) and Long Attack Latency (LAL) lines, mice were kept after weaning (3–4 weeks) in unisexual litters until sexual maturity (7–9 weeks), at which point they were male-female paired in small cages. At the age of 14 weeks, each male mouse was tested for aggression in a resident-intruder test carried out in a large cage divided into 4 compartments (A, B, C and D). The resident experimental animal was housed in A and B, while the compartment C was the test arena. After an hour of exploration of compartment C, the experimental animal was tested with a naive albino intruder (MAS-Gro), previously confined to compartment D (sensory precontact allowed by a perforated Plexiglas slide). From the point of slide removal onwards, the attack latency was recorded. Later studies confirmed that this parameter correlates negatively with other parameters of aggression (Benus et al., 1991b). The final score used to define a SAL or a LAL mouse was the Attack Latency Score (ALS), i.e. the average of the attack latency times measured over three consecutive days of testing. When an animal did not attack in 600 sec, it was considered a non-attacking mouse, and the test was stopped. While the SAL line appeared in the 4th generation already and remained quite stable, the selection of a docile LAL line failed at the first few attempts and was obtained only at the 23rd generation of the control line. In North Carolina, U.S., Cairns et al. selected a base population of out-bred NCR mice on a dual criterion, namely increased attack and heightened reactivity to stimulation (Cairns et al., 1983). In this way, a highly aggressive line and an immobile line were created (I-lines). A second attempt focused only on aggression as a selection criterion and generated the NC900 and NC100 lines. The testing procedure was as follows: after weaning at 21 days, the mice were housed in relative isolation. At 45 days of age, aggressiveness was measured in a standard 10min dyadic test carried out in a neutral Plexiglas box, after 5-min of adaptation in which physical contact was not allowed. The attack frequency (in 5-sec intervals) 26

GENERAL

INTRODUCTION

and the attack latency were measured and combined together with other 31 variables in a scoring system up to 900. The result was the selection of an aggressive line, NC900, and a non-aggressive line, NC100. Breeding pairs of both lines (10 of each, 48th generation) were transferred to Groningen, The Netherlands, in 2004 and are currently in the 52th generation of selection. Behavioural phenotypes Aggression as a behavioural trait is only part of a more general suite of correlated behaviours that an individual performs in order to cope actively with the environment, i.e. the proactive coping strategy. The presence of alternative coping strategies and of correlations between behavioural traits has been extensively investigated in the SAL/LAL animal model, while in the other two models studies have addressed certain behaviours in relation to depression/anxiety etc. A summary of the literature concerning this issue is presented in Table 1.2. The six mouse lines were developed based on high and low intermale aggression levels. However, in view of our current distinction between aggression and violence, one may wonder which of these selection lines show signs of violence. Although the attack latency of SAL and LAL is highly correlated with other measures of aggressiveness, it does not determine dominance. Body weight appears to be an important determinant in the acquisition of dominance (Van Zegeren, 1980; van Oortmerssen et al., 1985). Recently, the aggressiveness of SAL and LAL mice was compared in terms of pathological/maladaptive aspects. SAL mice were found to be more violent than LAL, since SAL attacked more intensely the opponent in vulnerable regions and caused more wounds to the opponents (Haller et al., 2006). In TA and TNA mice, aggression has been mainly studied by examining the role of odour/olfaction communication in the induction of aggressiveness. The territorial marking pattern of urination differs in these two lines. In reaction, male mice tend to avoid the TA-marked places, while females choose those of TNA males (Sandnabba, 1985). TA odour on castrates has been found to elicit aggression from NMRI and Swiss albino mice of the parental strain used for the selection, but to a higher extent when the TA mice have been trained to high aggressiveness compared to TA-defeated mice (Sandnabba, 1986a; Sandnabba, 1986b). Male and female odour preferences of the two lines are associated with aggressiveness and dominant/subordinate social position (Sandnabba, 1986c). TA and TNA mice differ in their territoriality when tested in colonies, where TA mice fight excessively and often became dominant, while TNA mice stay in their original territories with less fighting (Sandnabba, 1997). These experiments suggest some correlation between dominance, female preference and TA aggressiveness. Recently, using a more detailed statistical approach, the social behaviour of TA mice was described as high in consummate aggression (e.g. box, bite, threat, chase), and that of TNA 27

28

Reference

Veenema et al., 2003

Hogg et al., 2000

Sluyter et al., 1995 Sluyter et al., 1996

Benus et al., 1988

Benus et al., 1990

Benus et al., 1990

Benus, 1988 Benus et al., 1989

Van Oortmerssen et al., 1985 Compaan et al., 1993 Benus et al., 1987

SAL/LAL Van Zegeren, 1980

Lines

Experiment Isolated SAL and LAL, no effect of attack latency in dominance acquisition, but effect of body weight Socially housed SAL and LAL, effect of attack latency rather than body weight in the acquisition of dominance TP aggressive effect on SAL; E no effect

Results

SAL learned earlier than LAL; SAL latency to explore was not affected by an extra object in the maze, while LAL explored; SAL learned slower when a different maze was presented every day. Social stress Defeat SAL flee more and LAL freeze more Controllable Two way active shock avoidance SAL escaping more, but within LAL three groups of non-social stress different speed and learning, one group similar to SAL Uncontrollable Inescapable shock LAL activity is suppressed, while SAL are hardly non-social stress influenced Reversal learning Y-maze SAL more difficulties in changing their motor behavioural patterns, more routine-formation Circadian rhythmicity Light-dark inversion SAL adjusted their rhythm to the inversion much slower Dark-Dark than LAL; SAL had a free-running period similar to the solar period, while the one of LAL was shorter Coping strategy Nest-building SAL showed more nest building than LAL Coping strategy Shock-probe/Defensive burying LAL showed more immobility than SAL in both situations; in home cage and fresh sawdust LAL did not adopt defensive burying in fresh sawdust, while SAL actively did it. Anxiety Hexagonal-tunnel maze SAL less anxious than LAL in the hexagonal-tunnel maze, Light-dark box while no differences were found in the LD box Anxiety and social -Elevated plus maze LAL less active than SAL in all the tests and showed more stress -Sudden silence test freezing and immobility; -Open field Increase in grooming in LAL and decrease in digging -Forced swim test in two paradigms: in SAL in the sensory contact paradigm; -Sensory contact Decrease in immobility, decreases in activity after -Sensory contact + daily defeat defeat paradigm in both lines.

Aggressive behaviour Population studies in semi-natural conditions Aggressive behaviour Population studies in semi-natural conditions Female aggression Testosterone propionate or estradiol neonatal treatment Learning Standard maze

Behaviour studied

Table 1.2 Overview of the behavioural characteristics (endophenotypes) of the aggressive and non-aggressive lines.

CHAPTER 1

Sex related coping styles Anxiety

Kvist et al., 1997

Metabolism Maze learning Open field Elevated plus-maze Staircase Light-dark box 7-min dyadic interaction

Cricket intruder Colony

NC900/ Hood & Quigley, 2007 Exploration NC100

Novel arena Light/dark box Elevated plus maze Neohypophagia

Aggression RTS (reactivity tactile stimulation) Social and Non-social Dyadic interactions Reactivity Immobility NC900/ Bauer & Gariépy, 2000 Freezing behaviour in 10-min dyadic tests in juvenile mice NC100 dyadic interaction

NC900/ Gariépy et al., 1988 NC100

Vekovischeva et al., 2007

Aggression

Predatory aggression Territorial behaviour

Nyberg et al., 2003

Resident-intruder test

Aggression test Sexuality test Marking behaviour Investigation of TA/TNA marked behaviour by NMRI mice Urinary marking pattern analysis Odor discrimination Open field with odours of other mice Learning Maze learning Passive avoidance Open field ambulation Base-line open field After maze learning open field

Sexual behaviour

Aggressive behaviour/violence

Sandnabba, 1995 Sandnabba, 1997

Selander & Kvist, 1991

Kvist, 1989

Sandnabba, 1986

TA/TNA Lagerspetz & Hautojärvi, 1967 Sandnabba, 1985

Haller et al., 2006

NC900 more reactive and less freezing than NC100. Freezing as a conservation/withdrawal response that reduces emotional arousal NC900 slower approach and lower levels of exploratory behaviours. NC900 slowest to enter light box, remain longer in dark box. NC900 less drinking in novel environment

NC900 and NC100 same reactivity NC100 more immobility

TA more consummatory aggression TNA more non-aggressive social interaction

TA marked territory was avoided by NMRI mice TA mice marked the whole bottom of the cage, while TNA urinated in fewer places with bigger spots TNA avoid TA odor; TA search for TA odor, but with loosing experience they tend to avoid it TA better in maze learning (active learning) TNA better in passive avoidance (passive learning) TA more locomotor activity and shorter latency to move than TNA. TA less emotionality (defecation) than TNA All these measures were suppressed together with suppression of aggression in the TA and returned to initial levels after rest. TA show more predatory aggression than TNA TA established hierarchies quickly through fights TNA remained in their original territories Differences in coping styles are found inter- and intrastrains between males and females TA less anxious than TNA

No difference in sexual behaviour between the strains

Attacks at vulnerable regions in SAL more than in LAL

GENERAL INTRODUCTION

29

CHAPTER 1

as socially explorative (e.g. sniffing, investigation, non-aggressive contact) (Vekovischeva et al., 2007b). TNA were found to be slightly aggressive, although their aggression was low in intensity (Vekovischeva et al., 2007b). Intermale aggression is associated with other forms of aggression. TA aggressive mice show more predatory aggression than TNA mice when exposed to a cricket intruder (Sandnabba, 1995). TA and TNA maternal and testosteroneinduced aggression levels differ in accordance with TA and TNA male-male aggressiveness, suggesting a similar genetic background for female and male aggression (Lagerspetz and Lagerspetz 1975; Lagerspetz and Lagerspetz 1983; Sandnabba, 1993b; Sandnabba et al., 1994). Similar findings have been obtained in the NC lines. Maternal aggression and inter-female aggression were found to be higher in NC900 than in NC100 females, similarly to the difference observed in males, even though this difference was expressed later in life (Cairns et al., 1983; Hood and Cairns 1988). During the SAL/LAL selection, female aggression resulted in a similar pattern to male aggression in the first generations but did not show any differences later on, suggesting a major role of environmental factors. From this it was concluded that in SAL and LAL mice, female aggression had a different genetic basis to that of male aggression (van Oortmerssen and Bakker 1981). However, when treated with testosterone in adulthood, SAL females show greater aggression than LAL females, with a major role played by a difference in the conversion of testosterone to estradiol by the enzyme aromatase (Compaan et al., 1993b). In the T lines, low aggressiveness is also associated with high sexuality (Lagerspetz and Hautojarvi 1967; Hautojarvi and Lagerspetz 1968; Lagerspetz and Lagerspetz 1971; Lagerspetz and Lagerspetz 1975), but contradictory results have been found (Sandnabba, 1993a; Sandnabba and Korpela 1994), suggesting a more complex relationship between sex and aggression. In the SAL/LAL model, several studies have investigated whether selection for aggression reflects a more general dichotomy in coping strategies by investigating behavioural strategies in non-social challenging situations. SAL mice show overall a proactive style, i.e. aimed at mastering the stressors, while LAL mice show a reactive/passive strategy. When exposed to social or non-social escapable stress, SAL males tend to flee whereas LAL males freeze more, with more variability among the LAL group (Benus et al., 1989). When exposed to inescapable nonsocial stress, LAL mice suppress their activity, while SAL mice are hardly affected (Benus et al., 1990a). SAL mice, in general, cope actively with their home environment, as shown by defensive burying of a shock-probe in the home cage (Sluyter et al., 1996a) and nest building (Sluyter et al., 1995b). On the other hand, SAL mice cope worse than LAL in a variable environment, showing more rigid and routine-like behaviour and less flexibility (Benus et al., 1988; Benus et al., 1990b). TA and TNA mice have been tested for coping strategies in terms of 30

GENERAL

INTRODUCTION

learning performances. TA mice perform better in solving a maze (active learning), while TNA mice perform better in a shuttle box (passive learning) (Kvist, 1989; Ewalds-Kvist et al., 1997). NC900 and NC100 have been tested for reactivity to stimuli and immobility/freezing behaviour, as indicative of active and passive strategies, respectively. NC100 mice are highly immobile in social and non-social contests, but no difference in reactivity to stimulation from the experimenter or from an intruder mouse has been documented (Gariepy et al., 1988). Freezing during social contests is more pronounced in NC100 juveniles, and it helps in reducing emotionality and enhancing affiliative behaviour (Bauer and Gariepy 2001). Regarding exploratory behaviour, the results are contradictory, since high motor activity and open-field exploration are associated with high aggressiveness in the TA line and with low aggressiveness in the LAL line (Lagerspetz, 1961; Selander and Kvist 1991; Veenema et al., 2003a). When tested for emotionality, mice from the Groningen and the Turku aggressive lines show less anxious behaviour (or defecation in case of the Turku lines) than their non-aggressive counterparts (Lagerspetz, 1961; Selander and Kvist 1991; Hogg et al., 2000; Nyberg et al., 2003; Veenema et al., 2003a). An early study showed that NC900 were considered similar to NC100 in terms of behavioural reactivity and defecation as measures for emotionality, although their urination response was higher (Gariepy et al., 1988). More recently, NC900 were found to be less explorative in a novel arena and in the light compartment of the light-dark box, and drank less milk in a novel environment compared to NC100 (Hood and Quigley 2008). In general, it seems that NC900 are more emotional than NC100. We can conclude that there are some similarities between the aggressive lines in their proactive strategy and between the non-aggressive lines in their reactive/passive coping style. However, the paradigms used are very different from each other and the heterogeneity of studies does not allow a solid conclusion. Is it possible that the selections generated individuals with different types of aggression? Are mouse personality types related to aggression and violence? Which mouse line is exhibits a form of aggression more similar to human violence? In Chapters 2 and 3 I attempt to answer these questions by subjecting all the six mouse lines to the same behavioural paradigms, and look for similarities and differences. Genetics The three models prove that aggression has a hereditary component. Realized heritability has been calculated at 0.34 for the TA and 0.30 for the SAL line at the 11th generation. Since aggressive behaviour was reported in SAL males but not in 31

CHAPTER 1

females, it was hypothesized that genes on the Y chromosome are associated with aggression in the SAL/LAL lines. Using F1 hybrid lines and congenic lines, it has been shown that the non-pseudo-autosomal region of the Y chromosome is involved in determining aggressiveness in terms of attack latency, although the effect may depend on the combination with the pseudo-autosomic region and the autosomal background (van Oortmerssen and Sluyter 1994; Sluyter et al., 1994b). The Y chromosome does not affect behavioural flexibility, nest-building behaviour or the dopaminergic system in terms of sensitivity to apomorphine (Sluyter et al., 1995a; Sluyter et al., 1996b; Sluyter et al., 1997). An effect of the non-pseudoautosomal region of the Y chromosome has been found in the defensive burying behaviour, but the effect is somehow masked by the autosomal background (Sluyter et al., 1999). Although correlated with aggression, the nonpseudoautosomal region of the Y chromosome is not correlated with adult testosterone levels and anatomical differences in the hippocampus (Van Oortmerssen et al., 1992; Hensbroek et al., 1995). In conclusion, genes on the Y chromosome do play a role in the determination of the aggressive behavioural phenotype of male SAL mice, although the effect is not associated with other components of the SAL proactive coping style, nor with some of their characteristic neuroendocrine and neurochemical features. Beside these classic genetic studies, a molecular approach has been used to investigate the genetic characteristics that underlie the aggressive phenotype of SAL mice and differentiate it from LAL mice. Since the project was part of a major research line on depression, the brain region of choice was the hippocampus. Both genome-expression profiles generated by Serial Analysis of Gene Expression and GeneChip analysis have identified a general down-regulation in SAL mice, compared to LAL, of transcripts related to the cytoskeleton, members of a specific calcium/clamodulin signal transduction cascade (e.g. ERK2, raf-related oncogene), and several MAPK related genes involved in learning and memory (Feldker et al., 2003a; Feldker et al., 2003b). Interestingly, the only up-regulated transcript identified is gas5 (growth-arrest-specific-5), a gene coding for small nuclear RNAs (Feldker et al., 2003b). In conclusion, genes present on the Y chromosome play a role in determining individual differences in aggressiveness. However, the genetic basis of the more general suite of behavioural and physiological traits, in which the aggressive behaviour is embedded, seems to lie in the genes related to structural changes in brain structures, as shown in the hippocampus. Phenotypic plasticity Environmental manipulations carried out in mice from the SAL/LAL, TA/TNA and NC900/NC100 lines are summarized in Table 1.3. 32

Reference

Period

Exposure to aggression in a glass container Exposure to aggression through a wire mesh Encounter with more aggressive and less aggressive individuals Housing with male siblings or isolation Ethanol injection to TNA and to inhibited TA Repeated defeat Only visual, olfactory and auditory stimuli of the defeat After repeated defeat, animals in isolation or in sensory contact with the residents Isolation rearing or female-paired rearing Intruder-resident, neutral cage and resident-intruder female

Post-weaning

Pre-weaning Post-weaning

Post-weaning

Post-weaning

Post-weaning

Cairns et al., 1983

Post-weaning

Isolation or group-rearing

Cross-fostering Cross-fostering + endotoxin exposure

Testosterone injection Isolation-rearing Cross-fostering

Pre-weaning Post-weaning Pre-weaning

Post-weaning

Cross-fostering

Embryo transfer Cross-fostering

Experiment

Pre-weaning

NC900/ Cairns et al., 1983 NC100 Hood and Cairns, 1989 Pre-weaning Granger et al., 2001 Pre-weaning

Nyberg et al., 2004

Benus and Rondings, 1990 Compaan et al., 1992 Van Oortmerssen and TA/TNA Lagerspetz and Wuorinen, 1965 Sandnabba et al., 1993 Lagerspetz, 1961, 1964 Lagerspetz and Lagerspetz, 1971 Lagerspetz and Ekqvist, 1978 Lagerspetz and Sandnabba, 1982

SAL/LAL Van Oortmerssen et al., 1985 Sluyter et al., 1996 Prenatal Sluyter et al., 1995 Pre-weaning

Lines

No effect Endotoxin diminished aggression in NC900, no effect of cross-fostering Elicited aggression in isolated mice

Isolation elicits aggression also against a female Previous experience affects aggressive behaviour

Only after isolation aggression levels restored

Inhibited aggression No inhibition of aggression

Alcohol does not change aggression

Isolated mice showed higher aggressiveness

No effect Enhanced aggression Temporary change

No effect No effect on attack latency Effect on the evolution of attack latency No influence on pup growth of the different mother’s behaviour Reduction in aggression in LAL No effect Marginal effect, reduced aggression in both lines

Effect on aggression

Table 1.3 Overview of the environmental effects on the aggressive behaviour in the three selection programs.

GENERAL INTRODUCTION

33

CHAPTER 1

It is known that prenatal events can affect the behavioural phenotype in adulthood. Unfortunately, only in the SAL/LAL mice has this possibility been explored. Experiments in which embryos of SAL, LAL and their reciprocal F1’s were transferred to NMRI females have shown that the genotype, but not the prenatal environment, influences intermale aggression (Van Oortmerssen et al., 1992; Sluyter et al., 1994b; Sluyter et al., 1996c). Postnatal environmental stimuli before and after weaning can affect the adult behavioural phenotype. Preweaning experiments have been performed in all the selection lines using cross-fostering and other manipulations. In SAL and LAL parental lines, no effect of cross-fostering was observed on the attack latency (van Oortmerssen et al., 1985), whereas in their reciprocal F1’s only the evolution of attack latency over three days is affected (Sluyter et al., 1995c). In general, the genotype is the main determinant of the behavioural difference between SAL and LAL, even though SAL and LAL mothers show significantly different behaviour toward the pups (Benus and Rondigs 1996). Similarly, differences in the maternal behaviour of TA and TNA mothers only marginally affects the aggressive behaviour of TA and TNA males in adulthood, without masking the genetic factor (Lagerspetz and Wuorinen 1965). Cross-fostering does not have any effect on the aggressiveness of NC900 and NC100 and their replicate lines I900 and I100 (Cairns et al., 1983; Hood and Cairns 1989). Other preweaning manipulations have been carried out in the Dutch and North Carolina mice. Artificially enhanced testosterone on the day of birth reduces aggression in LAL males, but does not affect aggression levels in SAL males (Compaan et al., 1992), suggesting a higher neonatal sensitivity to testosterone in the more aggressive line. Exposure to E. coli endotoxin at a young age diminishes aggression in NC900 aggressive mice, and the effect is associated with enhanced HPA-axis activity in adulthood (Granger et al., 1996; Granger and Hood 1997; Granger et al., 2001). This has been explained as a higher immune reactivity and sensitivity to early stressors in the aggressive line, presumably through an effect on the mother’s maternal behaviour (Hood et al., 2003). Handling for a period of 3 weeks increases aggression in NC900 mice and the change is not directly associated with changes in plasma corticosterone and dopamine1 receptors (Gariepy et al., 2002). As with preweaning experience, postweaning manipulations have been performed in the aggressive and non-aggressive mouse lines. Isolation- versus group-rearing is a major focus for all the three mouse models for aggressiveness, since isolation-induced aggression is a widely used model for aggression research. Van Oortmerssen and Bakker’s (1981) selection of SAL aggressive mice was performed on animals reared with siblings until sexual maturation. After weaning, SAL and LAL mice were paired-housed with a female throughout the whole experiment. Social isolation at weaning or 1 month before testing did not 34

GENERAL

INTRODUCTION

have any effect on the aggressive behaviour of the SAL mice (van Oortmerssen and Bakker 1981). In contrast, TA and TNA mice were selected using an isolation period before the test for aggressiveness. When reared with brothers, neither TA nor TNA males showed any aggressiveness at all. The line difference appeared again after further isolation, and the aggressiveness of TA mice increased proportionally to the length of the isolation period (Lagerspetz and Lagerspetz 1971). Despite the high dependence on genotype and early life events, TA and TNA behavioural phenotypes were demonstrated to be highly influenced by isolationrearing conditions. When socially housed, experience of victory and defeat play a role, at least temporarily, in determining the aggression level of TA and TNA mice (Lagerspetz, 1961). TA mice were found to be particularly sensitive to the type of experience. Exposure to aggression in youth results in increased adult aggression, to a greater extent in TA than TNA mice (Sandnabba, 1993b). TA mice become non-aggressive after repeated defeats, whereas ethanol injection does not increase aggressive behaviour in TNA mice (Lagerspetz and Ekqvist 1978). Continuous physical contact with aggressive mice, rather than mere sensory contact, reduces the aggressiveness of TA mice, especially when the physical contact precedes the sensory contact period rather than vice versa (Lagerspetz and Sandnabba 1982). A more recent study showed that TA mice are always more aggressive than TNA in different social tests and rearing conditions, suggesting a strong genetic component. However, previous male-male interactions and housing with a female instead of group- or isolation-rearing interacts with genotype in a complex mechanism influencing the aggressive behaviour of the two lines (Nyberg et al., 2004). Group-rearing reduces the difference in aggression duration and frequency between NC900 and NC100 mice, compared to when the animals are reared in isolation (Cairns et al., 1983; Hood and Cairns 1989). When the rearing conditions are kept constant, repeated testing attenuates the difference in latency to attack between the two lines (Cairns et al., 1983). In general, despite the heterogeneity of paradigms and conditions, it can be concluded that animals reared in groups adjust their behavioural phenotype probably because of their previous experiences of victory and/or defeat. However, a strong genetic component plays a stable role in determining an aggressive phenotype in all three genetic selection lines. How does repeated social experience influence the aggression of these mouse lines? Can it contribute to the escalation to violence? I examine these issues in Chapter 3. Physiological and neurochemical correlates Bidirectional artificial selection for aggression results in the selection for alternative behavioural phenotypes, which reveal also distinct physiological and neurobiological mechanisms. A summary of the data on these aspects is in Table 1.4. 35

36

Sex hormones Serotonergic

Compaan et al., 1994 Korte et al., 1996

Serotonergic HPA axis

HPA axis

HPA axis and serotonergic

Serotonergic

Van Riel et al., 2002

Veenema et al., 2003

Veenema et al., 2003

Feldker et al., 2003

Van der Vegt et al., 2001 Serotonergic

Sexl hormones

Dopaminergic Serotonergic

SAL/LAL Benus et al., 1991 Olivier et al., 1990

Compaan et al., 1993

System studied

Reference

Lines

5-HT1A Northern blot analysis on hippocampus

Body weight and organ weight Corticosterone and ACTH levels, GR and MR mRNA in hippocampus and CRH mRNA in PVN (hypothalamus) after forced swimming test After acute and chronic social stress: Body weight and organ weight Corticosterone and ACTH plasma levels MR, GR and CRH in situ hybridization 5-HT1A receptor binding

5-HT1A in vivo functionality (alnespirone-induced hypothermia) Electrophysiological recordings after serotonin stimulation of hippocampal slices; 5-HT1A, MR, GR in situ hybridization; Plasma corticosterone levels

In vitro brain aromatase activity 5-HT1A in situ hybridization and ligand binding

Apomorphine stereotyped behaviour Catecholamine levels (HPLC) in the whole brain Testosterone in adult and young; Vasopressin immunoreactivity

Experiment

SAL more T in adulthood but less neonatally than LAL; SAL less VP density in LS and BNST than LAL POA aromatase activity lower in SAL than in LAL SAL more 5-HT1A mRNA in hippocampus and more ligand binding in hippocampus, lateral septum and frontal cortex SAL higher hypothermia than LAL (higher sensitivity of the receptor) In SAL higher hyperpolarization of CA1 cells after serotonin; SAL less corticosterone than LAL after novelty stress; SAL more 5HT1A mRNA in CA1; SAL less MR mRNA in DG SAL smaller thymus and spleen than LAL; SAL higher corticosterone (light phase) and ACTH levels (light and dark phase); LAL more MR mRNA production in CA2 after stress and CRH in PVN Prolonged body weight in LAL but not in SALdecrease after social stress; Higher corticosterone and ACTH stress response in LAL; Higher 5-HT1A binding in SAL hippocampus than in LAL SAL more mRNA than LAL

SAL are more sensitive to apomorphine than LAL SAL less serotonin than LAL

Results

Table 1.4 Overview of the neurochemical, endocrinological and immunological characteristics of the aggressive and non-aggressive lines.

CHAPTER 1

GABA-ergic

Immune Tumor susceptibility

Petitto et al., 1993

Dopaminergic

NC900/ Lewis et al., 1988 NC100 DeVaud et al., 1989

Weerts et al., 1992

Glutamatergic

Vekovischeva et al., 2007

Serotonergic Noradrenergic Testis and seminal vesicles

Neuronal activation

Haller et al., 2005

TA/TNA Lagerspetz et al., 1968

Serotonergic

Veenema et al., 2005

Reduction of aggression (biting), in particular the non-competitive one reduces boxing

Serotonin: TATNA in the brain stem Testis: TA>TNA Adrenalin in adrenals: TA>TNA

5-HT1A mRNA more in SAL than in LAL in CA1; 5-HT1A ligand binding more in SAL than in LAL in CA1 and DG; Less 5-HT in SAL than in LAL in brain stem; Lower 5-HIAA/5-HT in SAL than in LAL in striatum; Effects on behaviour of 5-HT1A agonists in SAL and in LAL In SAL pattern of activation different than LAL and different from territorial aggression models; activation of central amygdale and ventrolateral periaqueductal grey, similar to models of violence

HPLC nucleus accumbens and caudate ; NC100 lower dopamine and metabolites than nucleus NC900 in both regions; D1 and D2 receptors binding autoradiography Higher D1 and D2 density in NC100 in nucleus accumbens and caudate nucleus. Benzodiazepine effect on behaviour in a Benzodiazepine at high doses reduced aggressive dyadic interaction behaviour in NC900 and motor behaviour in Benzodiazepine binding in vivo NC100; GABA-dependent chloride uptake assay NC900 less benzodiazepine binding in cortex, hypothalamus and hippocampus; no difference in pons and medulla; NC900 less GABA-dependent chloride uptake Tumor induction NC100 more tumor than NC900 NK cell function NK cell function lower in NC100 than in NC900 Serum corticosterone assay Trend higher serum corticosterone level in NC100

Chemical detection of serotonin and noradrenaline in the forebrain and in the brain stem; adrenals, testis and seminal vesicles weight; adrenaline content in the adrenals. Effects of glutamatergic competitive and non-competitive antagonists on aggression

c-fos after aggressive encounter

HPLC for serotonin and 5-HIAA 5-HT1A in situ hybridization and autoradiography ligand binding Behavioural effects of 5-HT1A agonists in forced-swim test

GENERAL INTRODUCTION

37

38

Lines

Dopaminergic

Dopaminergic

Immune system and HPA axis Immune system and HPA axis

Dopaminergic

Immune system

Immune system and maternal behaviour Acute tolerance to ethanol

Lewis et al., 1994

Gariépy et al., 1995

Granger et al., 1996

Gariépy et al., 1998

Petitto et al., 1999

Granger et al., 2001

Reed et al., 2001

Granger et al., 1997

System studied

Immune

Reference

Petitto et al., 1993

Table 1.4 Continued. Experiment

Repeated intragastric ethanol injection and progressive recovery from motor impairment on isolated and group-reared mice

Dihydrexidine (full D1-agonist) effects in social interaction test and D1 binding in striatum in continuously isolated and isolated-grouped mice; NK cell activity in cells from mice with different post-weaning social experience Endotoxin to pups; Cross-fostering

Dihydrexidine (full D1-agonist) effects in social interaction test in group- and isolation-reared mice Perinatal endotoxin exposure effects on HPA axis and social behaviour Endotoxin effects on physiological parameters, social behaviour and HPA axis

In cells from isolated and group-reared mice: -Mitogen assay -Interleukin-2 assay -γ-interferon assay -NK cell activity Dihydrexidine (full D1- agonist) effects in social interaction test; D1-induced behaviour and effects of D1and D2-antagonist pretreatment

Results

NC100 less NK activity than NC900; No effect of the post-weaning experience Endotoxin decreased aggressive behaviour and social reactivity in NC900; no interaction effect with the fostering condition Genetic-environment interaction effect on females, but not on males.

Endotoxin affected social behaviour in both lines and diminished hypothalamic CRF in NC900 Lower threshold of temperature, body weight and corticosterone in NC900; decreased social reactivity and aggressiveness in NC900 and increased social reactivity in NC100 DHX effects on behaviour and D1 binding higher in continuously isolated mice

D1 agonist reduces aggression in NC900 and non-agonistic approach in NC100; no effect on freezing behaviour; D1-antagonist, not D2-antagonist, antagonized D1-agonist effects on social interaction. D1 agonist enhances social reactivity especially in the isolated; NC900 more reactive to social stimuli

NC100 less mitogen stimulation effect, IL-2 production, γ-interferon and NK activity than NC900; no housing by line interaction effect

CHAPTER 1

GENERAL

INTRODUCTION

In accordance with other models of proactive/reactive coping strategies, SAL mice show less corticosterone reactivity to corticotrophin-releasing hormone (CRH) challenge than LAL, as well as lower plasma corticosterone baseline levels, but more fluctuation in corticosterone levels across light-dark cycle (Korte et al., 1996; Veenema et al., 2003b). Furthermore, the ACTH response to stress is higher in SAL mice compared to LAL, while corticosterone response is much less pronounced and shorter, suggesting a reduced adrenocortical sensitivity in the former line (Veenema et al., 2003b; Veenema et al., 2004). The higher and longer-lasting stress response from the LAL mice is associated with higher mineralocorticoid receptor (MR) mRNA expression in the hippocampus and higher CRH in the hypothalamic paraventricular nucleus (Veenema et al., 2003b; Veenema et al., 2004). In LAL mice, more than SAL, the HPA hyper-responsivity after a chronic social stress paradigm results in a more pronounced body weight loss and an increase in hippocampal MR mRNA, with correlated increase in passive behavioural responses in anxiety tests (Veenema et al., 2003a). In NC mice, baseline and endotoxin-stimulated corticosterone activation during a dyadic interaction are higher in the aggressive than the non-aggressive line, while hypothalamic CRF does not differ (Granger et al., 1996; Granger and Hood 1997). These results indicate that proneness to aggression is associated with different HPA-axis functioning, although to what extent is difficult to extrapolate. It may be that this system is more generally related to the proactive coping style of SAL mice and is not well represented in the NC900 line, due to the lack of a distinct proactive phenotype. While stress research has described SAL mice as more resistant to stress than LAL, immunological findings have shown that NC900 mice are more resistant to the development of immune diseases and tumours (Petitto et al., 1993; Petitto et al., 1994; Petitto et al., 1999). The discrepancy in immune functioning could not be ascribed to a baseline difference in plasma corticosterone levels, but indications of a different corticosterone response to infections reveals an important role of the HPA axis in differentiating these lines (Granger et al., 1996; Granger and Hood 1997; Granger et al., 2001). A more consistent finding in the stress physiology of the three mouse models of aggression concerns the sympathetic-adrenomedullary system. It seems that aggression is related to high sympathetic reactivity to stress, as revealed by high adrenaline content in the adrenals of TA mice (Lagerspetz et al., 1968). In this thesis, I investigate the peripheral physiology of the six mouse lines for the first time in the same experiments (see Chapters 4 and 5). Since testosterone is generally known to correlate positively with aggressiveness, researchers have investigated the neuroendocrine circuit in which testosterone is involved. An early study reported that the selection for SAL mice corre39

CHAPTER 1

lated with high plasma testosterone levels due to high gonadal production (van Oortmerssen et al., 1987). The secretory capacity of Leydig cells is highest in SAL mice during pre-puberty and in adulthood, whereas in LAL it is highest neonatally (de Ruiter et al., 1993). Prenatal testosterone exposure is higher in SAL than in LAL, with consequent sensitization of the adult SAL male to testosterone, leading to an increased capacity to display aggressive behaviour (Compaan et al., 1992; de Ruiter et al., 1993). High circulating testosterone levels increase brain aromatase activity from the day of birth, while the two phenomena are not strictly associated prenatally (Compaan et al., 1994a; Compaan et al., 1994b). In the SAL/LAL mice, the vasopressin content of the lateral septum is testosteronedependent, with SAL having the lowest fibre number and vasopressin content (Compaan et al., 1993a). In contrast, testosterone does not seem to be associated with male aggression in the T lines (Sandnabba et al., 1994), although testes of TA mice weigh more than those of TNA mice (Lagerspetz et al., 1968) and not much more research has been done on this topic in these lines. Testosterone is also not associated with male aggression in the NC-lines (Gariepy JL et al., 1996). Typical neurochemical systems involved in the control of aggression are serotonin, dopamine, γ-aminobutiric acid (GABA) and noradrenaline. The tissue level of forebrain serotonin and its metabolite is lower in the aggressive lines than the non-aggressive ones (Lagerspetz et al., 1968; Olivier et al., 1990; Veenema et al., 2005a). The serotonin-deficiency in SAL mice is associated with enhanced sensitivity of the presynaptic and postsynaptic 5-HT1A receptor and its expression in cortico-limbic structures. In SAL mice, only the postsynaptic 5-HT1A receptor is more sensitive than that in LAL mice (Korte et al., 1996; van der Vegt et al., 2001; Feldker et al., 2003a; Veenema et al., 2005a; Veenema et al., 2005b). SAL and LAL show different behavioural sensitivity to 5-HT1A agonists regarding aggressive behaviour, but also different characteristics of their alternative coping strategies (Veenema et al., 2005a). Because of the differential HPA-axis and serotonergic stimulation converging on the hippocampus, and consequent hippocampal remodelling, SAL and LAL may develop different behavioural phenotypes in order to cope successfully with environmental stimuli (Van Riel et al., 2002; Veenema et al., 2004). Is this mechanism involved in trait aggression or more generally in the coping behavioural response? Chapters 3 and 5 compare the prefrontal serotonin levels and the 5-HT1A neurotransmission of the six mouse lines and give some evidence related to this issue. Following the results of the previous chapters, in Chapters 6, 7 and 8 I choose to focus on the serotonergic system of violence, rather than more generally aggression. Therefore I restricted my focus to the mouse lines that were more suitable for answering this question. Pharmacological and psychiatric studies show an involvement of dopamine in the initiation and execution of aggressive behaviour (Miczek et al., 2002). SAL 40

GENERAL

INTRODUCTION

mice show higher sensitivity than LAL to the dopaminergic agonist apomorphine, in terms of the increase in stereotyped behaviour caused by the drug (Benus et al., 1991a). In the NC lines, both agonistic and non-agonistic social behaviours are mediated by D1 receptors, with no difference between the two lines, but more so in isolated than group-reared animals (Lewis et al., 1994; Gariepy et al., 1998). It seems that a differential dopaminergic activity reflects a different coping style, and differential proneness to social behaviours. Preclinical data suggest a controversial function of GABA and the GABAA modulator benzodiazepine in the control of aggressive behaviour. Only one study has been performed on the GABAergic system in the selection lines. Benzodiazepine reduces motor behaviour in NC100 mice, while it produces a shift in the NC900 mice from aggressive behaviour to more social behaviour. The differential effect on behaviour is reflected by a lower benzodiazepine binding in corticolimbic regions and reduced cortical GABA uptake in the aggressive line, compared to the non-aggressive and medium-aggressive lines (Weerts et al., 1992). These results suggest a difference in the sensitivity of the GABAergic system in individuals with opposite personalities. Glutamatergic modulation of aggression has only recently been investigated in the selected mouse lines. Selective AMPA-type glutamate receptor antagonists acutely reduce offensive aggression of TA mice, with the non-competitive GYKI 52466 suppressing all aggressive behaviours. The competitive NBQX also increases social behaviour and threat in TNA mice, suggesting that its effect may depend on differential sensitivity to the drug (Vekovischeva et al., 2007a). Several neuroanatomical localizations have been proposed for the regulation of aggressive behaviour. In SAL mice, the hippocampal intra- and infrapyramidal mossy fibre (IIPMF) distribution differs from that in LAL mice (Sluyter et al., 1994a). However, these differences do not completely correlate with the aggressiveness displayed and the involvement of the Y chromosome (see Genetic studies). The neuronal activation pattern in SAL males during an aggressive encounter, measured with c-fos immunostaining, shows strong activation of amygdala and periaqueductal grey matter (Haller et al., 2006). Since this differs from the pattern of activation in LAL males and shows similarities with the pattern in violent humans, it is more related to the violent temperament of SAL mice than to mouse-typical territorial aggressiveness.

OUTLINE OF THIS THESIS The main aim of this thesis is to understand the physiological correlates of aggression and the development of violence. Under this theme, I chose to study the 41

CHAPTER 1

three pairs of mouse lines genetically selected for high and low aggressiveness. Due to the heterogeneity of the data generated in these mouse lines and the consequent difficulty to generalize them through comparative literature analysis, I investigated them together in the same experiments using the same approach. I studied their behavioural phenotype, their peripheral stress physiology and their central serotonergic system with emphasis on the serotonin-1A receptor, trying to answer the following research questions: – are there different mouse aggressive characteristics that could be related to different groups of aggressive individuals in humans? – are violent mouse types physiologically similar to violent humans? – in which specific type of mouse aggression is serotonin involved, if it is involved at all? – is the serotonin-1A receptor involved in aggression and violence? In Chapter 2 I investigate the behavioural phenotype of the three pairs of mouse selection lines with respect to non-social proactive behaviours and activity/exploration. In Chapter 3 I investigate the possibility of identifying a violent behavioural phenotype in the mouse lines selected for high and low aggressiveness. To assess mouse violence I consider aggression against females and lack of sensitivity to the cues of the mouse opponent. In Chapter 4 I explore baseline and stress-related autonomic correlates of aggression and violence. In particular, I ask whether the association between low autonomic arousal and violence can be replicated in the different mouse lines, in view of the idea that they represent different types of aggressive personality. In Chapter 5 I investigate whether differences in the serotonergic system, and particularly in the functionality of the serotonin-1A receptor, correlate with the high and low aggressiveness of the mouse lines. In Chapter 6 I test the hypothesis that the development of violence is associated with a change in the functionality of the serotonin-1A receptor. In Chapter 7 I explore the causal relationship between serotonin and violent aggression, through manipulation of dietary levels of the serotonin precursor and measurement of the behavioural effects. In Chapter 8 I investigate the idea that the difference in the functionality of the serotonin-1A receptor between violent and docile mice is related to differences in the receptor ultracellular distribution.

42

GENERAL

INTRODUCTION

43

2

CHAPTER

Is there co-selection for aggressiveness, coping strategy and emotionality in mice?

Doretta Caramaschi, Sietse F. de Boer and Jaap M. Koolhaas

ABSTRACT Personality, as a suite of correlated behavioural traits, has a genetic basis. Therefore, it is likely that selection for one trait leads to co-selection for other traits. We tested the association between aggressiveness and emotionality by measuring proactive/reactive coping with a nonsocial environmental challenge and exploration of a novel environment, in lines of mice (Mus musculus) selected for high and low aggression, namely SAL, LAL, TA, TNA, NC900, and NC100. We expected highly aggressive lines to show high levels of boldness, active coping and low levels of exploration, since these individuals would be better adapted to becoming dominants through fighting in their deme rather than to dispersal to found new colonies, and vice versa for the low-aggressive lines. The results show that, overall, high aggressiveness was related to greater mobility in the Novel Object and Shock Prod tests, while no association was found between aggressiveness and coping in the Forced-Swim test. Exploration levels in an Open Field were associated with low aggressiveness in the SAL-LAL lines, and with high aggressiveness in the TA, TNA, NC900 and NC100 lines. In conclusion, selection for high aggressiveness led to co-selection for boldness, while selection for low aggressiveness led to co-selection for fearfulness and, depending on the original strain, for high and low exploration levels.

CHAPTER 2

INTRODUCTION Mouse populations, like those of other animals, show individual differences in behavioural and physiological traits (Benus et al., 1991b; Koolhaas et al., 2007). These traits have a genetic basis and are apparently the product of natural selection. However, there are differing views on the adaptive nature of behavioural traits. Some behavioural traits may be co-selected and result in adaptive ‘packages’, consistent across contexts and stable throughout the life of an individual (Sih et al., 2004a; Wolf et al., 2007). Alternatively, selection of adaptive traits might result in the co-selection of less adaptive traits as by-products of genetic correlations that act as constraints (Arnqvist and Henriksson 1997). Furthermore, constraints might be found already at the physiological level (Koolhaas et al., 1999; Stamps, 2007). This paper will further analyze the general pattern of behavioural traits that are co-selected with aggression, using a comparison of three mouse strains genetically selected for high and low levels of aggressive behaviour. A growing body of evidence has pointed to the existence of behavioural ‘packages’, or personalities, that constitute the basis for individual differences. Although there is growing consensus on the existence of similar behavioural phenotypes across species and across populations, some behavioural traits might vary along distinct axes. For example, rodent personalities have been described in terms of a coping axis and an emotionality axis (Steimer and Driscoll 2003; Koolhaas et al., 2007). Moreover, suites of correlated traits have been described using different terms with very similar meanings. The term ‘behavioural syndromes’ has been proposed to represent collections of stable correlated traits (Sih et al., 2004b), while ‘coping styles’ gives the functional adaptive connotation of being able to cope with challenging situations (Koolhaas et al., 1999). ‘Temperament’ is a more general term that takes into account the repeatability of objectively measurable behaviours and their early onset in the life of an individual (Reale et al., 2007). Beside these more technical terms, ‘personality’ is the preferred one in the human literature, where it was first used. Laboratory studies in biomedical research have often neglected the existence of personalities in their mouse populations. However, recent studies show that there are individual differences in the vulnerability to diseases and in the response to pharmacological treatments, and that in several cases these differences are related to different personalities (Mehta and Gosling 2008). Moreover, in the study of human aggression, it is worth mentioning that aggressiveness may be typical of certain personalities (Ramirez and Andreu 2006) and differential neurobiological mechanisms may be behind these relationships (Siever, 2008). In nonhuman populations, aggressiveness is often correlated with proactive coping, boldness, risk-taking behaviours, fearlessness and low levels of activity/explo46

AGGRESSIVENESS,

COPING STRATEGY AND EMOTIONALITY

ration (Koolhaas et al., 1999; Groothuis and Carere 2005; Dingemanse et al., 2007). In human populations, aggressiveness is expressed either with an impulsive/affective connotation or within a social-cognitive/instrumental dimension. In the five-factor model of personalities (Digman, 1990), aggressiveness is particularly associated with high neuroticism/low agreeableness. Aggressive behaviour is expressed in a cold-blooded manner in people with high trait aggression/irritability, while it is expressed under provocation in people with high trait anger, Type A personality, rumination traits, emotional susceptibility, narcissism and impulsivity (Bettencourt et al., 2006). In conclusion, studying correlations between behavioural traits in animal models, and therefore aiming at a better behavioural characterization of individual differences, may have important translational implications in human research. Pairs of genetic mouse lines artificially selected for high and low aggressiveness from different original populations give a unique opportunity for investigating the functional association between aggression and other behavioural traits. The highly aggressive SAL (Short Attack Latency) and the low-aggressive LAL (Long Attack Latency) lines were selected on the basis of attack latency against a conspecific male intruder from a colony of wild-trapped house mice in the area of Groningen, the Netherlands (van Oortmerssen and Bakker 1981). The TA (Turku Aggressive) and TNA (Turku Non-Aggressive) lines were obtained on the basis of ratings of aggressiveness against a male conspecific from outbred Swiss Webster mice at the University of Turku, Finland (Lagerspetz, 1961). The NC900 and NC100 high- and low-aggression lines were selected based on aggressiveness ratings in male-male competition from ICR mice at the University of North Carolina, USA (Cairns et al., 1983). The SAL-LAL lines have been characterized extensively in their proactive/reactive alternative coping styles, with links to their neurobiological and neuroendocrine profiles (Koolhaas et al., 1999; Veenema et al., 2005a). In the other lines, a behavioural characterization based on alternative active/passive coping, without attempting to describe alternative personalities, seems to point in the same direction observed in the SAL-LAL lines (Gariepy et al., 1988; Kvist, 1989; Ewalds-Kvist et al., 1997; Bauer and Gariepy 2001; Vekovischeva et al., 2007b). Exploratory behaviour and motor activity showed a negative association with aggressiveness in the SAL-LAL lines (Veenema et al., 2003a) and a positive association in the TA-TNA lines and NC-lines (Selander and Kvist 1991; Hood and Quigley 2008). This study was designed to unravel the association between trait aggression, boldness, fearfulness, proactive/reactive coping and exploration in male mice. We defined trait aggression as the propensity of a male to attack an age-matched male intruder in his own territory. Boldness was the propensity to approach and explore a novel object. Proactive coping was defined as the capacity to avoid a 47

CHAPTER 2

shock from an electrified probe by covering it with bedding material, as reviewed in de Boer and Koolhaas (2003), as opposed to reactive/passive coping, which is characterized mainly by immobility. Across all these tests, immobility was taken as an index of fearfulness. Emotionality was defined as the exploration levels or inactivity in a large novel arena, which was intended to mimic the situation of a novel and potentially risky environment to explore in order to found a new colony. We aimed to replicate findings previously obtained in the SAL-LAL model of proactive/reactive coping and extend those to the TA-TNA and NC900-NC100 models of aggression. Based on previous research and on the idea that docile mice are better suited to leave their original colonies and disperse, we expected the highly aggressive lines SAL, TA, and NC900 to be bolder and proactive, and the low-aggressive lines LAL, TNA, and NC100 to be more cautious.

METHODS Animals Male mice (n=30) from the SAL, LAL, TA, TNA, NC900 and NC100 lines were used in this experiment. The mice were bred in our laboratory at the Biological Center, University of Groningen, Haren, the Netherlands, and weaned at 21 days of age. At around 40 days, each male mouse was housed with a sister, to avoid social isolation and intrasexual aggression, in Makrolon® Type II cages furnished with food shavings as bedding material, shredded paper EnviroDry® (BMI, Helmond, Netherlands) for nesting and a cardboard tube as cage enrichment. The mice had ad libitum access to food (AMII, ABdiets, Worden, The Netherlands) and water and were kept under standard laboratory conditions, at a temperature of 22 ± 2 °C and on a 12:12 light-dark cycle (lights on at 21.00). The tests started when the mice were 3 months old and consisted of the following behavioural tests: Attack Latency/Resident-Intruder, Novel Object, Shock Prod, Forced-Swim, Open Field (see below for detailed explanation of each test). The tests were conducted in the dark phase in the aforementioned order. At the end of the experimental session the mice were euthanized with a mixture of CO2/O2. All the procedures were carried out at the Biological Center, University of Groningen, Haren, the Netherlands, under approval of the Institutional Animal Care Committee of the University of Groningen (licence D4540D) and in compliance with the Dutch law on animal experimentation and the European Communities Council Directive of 24 November 1986 (86/609/EEC). Attack Latency/Resident-Intruder The resident-intruder test was performed according to standard procedures. 48

AGGRESSIVENESS,

COPING STRATEGY AND EMOTIONALITY

Briefly, two days prior to the test, the mice were housed together with their female partners in cages for testing aggression. On the third day, when the mice were habituated to the cage and had marked it as their own territory, the females were removed and an unknown, age-matched, socially naïve male intruder from the A/J strain was introduced in each cage. The time it took for the resident experimental mouse to attack was recorded as Attack Latency. The intruder was then removed and placed back in its home cage, while the female was returned to the testing cage. The test was repeated on the two following days, using the same procedure. Testing was conducted in such a way that each resident mouse always encountered a new opponent in its home cage. On the third day of testing, the intruder was left in the testing cage for 5 minutes to allow the social interaction to develop fully. The whole of this interaction was videotaped for later behavioural analyses. Novel Object and Shock Prod Three days after the last Resident-Intruder interaction, the Novel Object paradigm was performed, followed by the Shock Prod test one day later. In the Novel Object and the Shock Prod tests, each male mouse was confronted with the introduction in their home cage of a prod capable of giving electric shocks. In the Novel Object test the prod was switched off and the mice experienced the presence of an unknown object in their home cage, while in the Shock Prod test they received an electric shock of 0.7 mA intensity every time they touched the prod (de Boer and Koolhaas 2003). The Novel Object test lasted for 5 minutes after the first approach to the prod, while the Shock Prod lasted for 5 minutes after the first shock was received. Both paradigms were given in absence of the females and in fresh sawdust, and were video recorded for later behavioural analyses. Forced Swim Two days after the Shock Prod test, the Forced Swim test was performed. For this test each experimental mouse was introduced to a cylinder (diameter = 14 cm, height = 20 cm) containing tap water maintained at a temperature of 25 °C. The water level reached the height of 15 cm. Each mouse was left for 5 minutes in the water, during which time its behaviour was videotaped for later analyses. After removal from the water, each mouse was wrapped in a towel where it was kept for a few minutes to dry and then placed in its home cage. Open field A week after the Forced Swim test, each animal was tested in an Open Field. Briefly, the mouse was placed in the centre of a large, round arena (120 cm diameter) and was left undisturbed for 30 minutes, during which a video camera 49

CHAPTER 2

connected to a PC recorded its movement trajectory from above. The arena was washed thoroughly with water at the end of each test. Behavioural analyses The behaviours in the Resident-Intruder, Novel Object, Shock Prod and Forced Swim tests were analysed using The Observer 5.0 software (Noldus Information Technology bv, Wageningen, the Netherlands). From the Resident-Intruder videos, the following behaviours were scored as continuous behavioural states: Attack, Threat, Chase, Social exploration, Non-social exploration, Immobility and Body care. From the Novel Object and Shock Prod videos, Prod burying, Prod sniffing, Cage explore, Body care, Immobility and Tail rattle were scored as behavioural states. From the Forced Swim videos, Swimming, Climbing and Immobility were scored as behavioural states. The behaviour of the mice in the Open Field was analysed using the Ethovision 3 tracking system (Noldus Information Technology bv, Wageningen, the Netherlands). The distance moved in the whole arena during each 5-minute interval was extracted as a measure of emotionality levels. Statistical analyses Statistical tests were performed using SPSS 14.0 for Windows (SPSS Inc., Chicago, Illinois, USA). Attack latencies were analysed statistically with ANOVA for repeated measurements, including ‘day’ (3 levels: day1, day2, day3) as a within-subject factor and ‘type’ (2 levels: aggressive line and non-aggressive line) and ‘selection’ (3 levels: Groningen, Turku, North Carolina) as between-subjects factors. All the interaction effects were included in the analysis. The duration of the behaviours scored in each observation of the Resident-Intruder, Novel Object, Shock Prod and Forced Swim tests was considered for statistical analyses. Group means for the duration of each behaviour were analysed using a two-factor general linear model with ‘type’ (2 levels: aggressive line and non-aggressive line) and ‘selection’ (3 levels: Groningen, Turku, North Carolina) and a ‘type*selection’ interaction (6 levels: SAL, LAL, TA, TNA, NC900, NC100) as between-subjects effects. Post-hoc analyses were computed using Tukey’s multiple comparisons. The distance moved in the Open Field was analysed using a general linear model for repeated measurements with similar between-subjects effects as in the other analyses and ‘interval’ (6 levels: 0-5, 5-10, 10-15, 15-20, 20-25, 25-30) as a within-subject effect. Post-hoc analyses were performed using Tukey’s multiple comparisons in each interval.

50

AGGRESSIVENESS,

COPING STRATEGY AND EMOTIONALITY

RESULTS Attack latency/Resident-Intruder The propensity to engage in inter-male aggression was tested in the attack latency test. The attack latency (figure 2.1A) decreased significantly across the three days of testing (day effect: F(2,48)=6.65, p=0.003), as expected from previous studies. On average, the mice from the aggressive lines displayed significantly lower attack latencies than low-aggression lines (type effect: F(1,24)=128.61, p