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he Effect of Neurokinin1 Receptor Blockaden Territorial Aggression and in a Modelf Violent Aggression

ozsef Halasz, Mate Toth, Eva Mikics, Erik Hrabovszky, Boglarka Barsy, Beata Barsvari, and Jozsef Haller

ackground: Neurokinin1 (NK1) receptor blockers were recently proposed for the treatment of anxiety and depression. Disparate datauggest that NK1 receptors are also involved in the control of aggressiveness, but their role is poorly known.

ethods: We evaluated the aggression-induced activation of NK1 neurons by double-labeling brain sections for NK1 receptors and c-Fosn two laboratory models of aggression. We also studied the effects of the NK1 antagonist L-703,606 in these models.

esults: Aggressive encounters activated a large number of NK1 receptor-expressing neurons in areas relevant for aggression control. Thectivation was aggression-specific, because the effects of psychosocial encounters (that allowed sensory but not physical contacts) werearkedly weaker. In the medial amygdala, the activation of neurons expressing NK1 receptors showed a marked positive correlation with

he occurrence of violent attacks. In resident/intruder conflicts, NK1 blockade lowered the number of hard bites, without affecting milderorms of attack. In the model of violent aggression, attacks on vulnerable body parts of opponents (the main indicators of violence in this

odel) were decreased to the levels seen in control subjects. Autonomic deficits seen in the model of violent aggression were alsomeliorated. The effects of the compound were not secondary to changes in locomotion or in the behavior of intruders.

onclusions: Our data show that neurons expressing NK1 receptors are involved in the control of aggressiveness, especially in thexpression of violent attacks. This suggests that NK1 antagonists— beyond anxiety and depression—might also be useful in the treatment

f aggressiveness and violence.

ey Words: Aggression, amygdala, glucocorticoid, NK1 receptor,ubstance P, violence

he Neurokinin1 (NK1) receptor is abundant in brain areasknown to control emotional behavior (1–3). Not surpris-ingly, compounds that inhibit substance P neurotransmis-

ion by NK1 receptor blockade are promising targets for thereatment of anxiety and depression (4 –7). However, substance and NK1 receptors can impact on many different measures offfective behavior, leading us to question whether it couldnfluence aggressive responding. Indeed, there are earlier studiesn cats implicating substance P in defensive aggression (8,9),hereas NK1 knockout mice show reduced aggression as com-ared with control subjects (6,10). However, as yet, no one hasone a thorough, ethologically relevant, neuropharmacologicaltudy on NK1 receptors and aggression. Therefore, we studiedere the effects of the specific NK1 antagonist L-703,606 inesident rats facing intruders in their home-cage. The aggression-nduced activation of NK1 positive neurons was also studied inrain areas that are crucial for the execution of aggressiveehavior.

We also studied the effects of L-703,606 in a recently devel-ped model of violent aggression. The model was based onuman studies showing that antisocial aggressiveness is associ-ted with low plasma cortisol levels (11–13), reduced adrenaline,utonomic and skin-conductance responsiveness (14 –17), and

rom the Department of Behavioral Neurobiology (JHala, MT, EM, BoB, BeB,JHall); and the Department of Endocrine Neurobiology (EH), Institute ofExperimental Medicine, Hungarian Academy of Sciences, Budapest1083, Hungary.

ddress reprint requests to J. Halasz, M.D., Ph.D., Department of BehavioralNeurobiology, Institute of Experimental Medicine, 1083 Budapest, 43Szigony str., Hungary; E-mail: halasz@koki.hu.

eceived November 10, 2006; revised April 6, 2007; accepted April 13, 2007.

006-3223/08/$34.00oi:10.1016/j.biopsych.2007.04.022

social deficits (18). Previously we have shown in rats thatmimicking the endocrine condition associated with antisocialaggression (i.e., low glucocorticoid levels) resulted in the devel-opment of three important symptoms of the disorder: 1) antiso-cial type of aggressiveness (attacks aimed at vulnerable bodyparts of opponents); 2) low autonomic arousal during fights, and3) social deficits (19,20). We suggested earlier that this approachcan be used as a model of antisocial violence (21,22).

The pharmacological treatment of antisocial aggressiveness isan unresolved issue (23,24). For example, selective serotoninreuptake inhibitors frequently used to reduce aggressiveness in avariety of disorders (25–27) provided equivocal effects in antiso-cial personality disorder (28 –31). Therefore, novel treatmentapproaches might be especially valuable in this condition.

Methods and Materials

AnimalsSubjects were 2–3-month-old Wistar rats (Charles River Lab-

oratories; Hungary) weighing 400 – 450 g. Because aggressionwas studied in the resident/intruder paradigm, initially group-housed rats were isolated in individual cages for a week beforeaggressive encounters in cages measuring 60 � 40 � 50 cm. Thewalls of the cages were opaque except for the front wall, whichwas transparent. Food and water were available ad libitum, whiletemperature and relative humidity were kept at 22 � 2°C and60 � 10%, respectively. Rats were maintained in a revertedlight/dark cycle of 12 hours with lights off at 10:00 AM. Acclima-tization to the day/night schedule lasted 2 weeks. All rats hadfree access to .9% saline solution after adrenalectomy withglucocorticoid replacement (ADXr) or sham operation.

Male Wistar rats weighing approximately 300 g were used asopponents in aggressive and psychosocial encounters. These ratswere group-housed but otherwise maintained under similar

conditions. Each intruder was used only once.

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Experiments were carried out in accordance with the Euro-ean Communities Council Directive of November 24, 198686/609/EEC) and were reviewed and approved by the Animalelfare Committee of the Institute of Experimental Medicine.

xperimental DesignWe investigated the aggression-induced activation of NK1

ositive neurons in areas relevant to aggression control (Exper-ments 1–2), the effects of NK1 receptor blockade on aggressive-ess (Experiments 3–4), and finally, the impact of NK1 receptorlockade on aggression-induced autonomic activation (Experi-ents 5–6). Note that ADXr rats show deficits in autonomic

ctivation (see introductory text). The locomotor effects of NK1eceptor blockade were studied in Experiment 7. The behavior ofats was video recorded throughout and was analyzed later byeans of a computer-based event recorder (see following text

or details). Group assignment was random throughout.Experiment 1. Rats either remained undisturbed in their

ome cage or were exposed to psychosocial or aggressivencounters. Psychosocially stimulated rats faced intruders thatere confined behind a transparent, perforated partition. Inggressive encounters, residents interacted with intruders freely.ncounters lasted 20 min. One hour after the termination ofncounters, brains were sampled for immunocytochemical anal-sis (see following for details). Control brains were sampled inlternation with those of rats exposed to encounters. Sample sizeas 6/group.

Experiment 2. Subjects were either adrenalectomized (andmplanted with a low-release corticosterone pellet; ADXr) orham-operated (Sham). After 1 week recovery, Sham and ADXrats remained undisturbed in their home cage or were exposed toggressive encounters for 20 min. One hour after the terminationf encounters, brains were sampled for immunocytochemicalnalysis. Sample size was six for control subjects and nine for ratsxposed to aggressive encounters.

Experiment 3. Intact rats were treated with 0 (vehicle), .1, ormg/kg L-703,606, a highly specific NK1 antagonist. The com-ound was obtained from SIGMA (Budapest, Hungary) and wasissolved in saline. Thirty minutes after injection, the subjectsere exposed to aggressive encounters for 20 min. The dose

hoice was based on earlier findings (32,33). Sample size wasight/group.

Experiment 4. The subjects of the study were ADXr rats. Toheck for the effects of adrenalectomy, sham-operated controlubjects were also included. The ADXr rats were treated with 0vehicle), .1, or 1 mg/kg L-703,606. Shams were treated withaline. Thirty minutes after injection, the subjects were faced withntruders for 20 min. In this experiment, the behavior of intrudersas also scored. Sample size was nine/group.

Experiment 5. The study was performed in subjects thatere not submitted to adrenalectomy. Rats were implanted with

elemetric E-mitters (Minimitter, Bend, Oregon; see following foretails). After 1 week recovery, they were injected IP with vehicler 1 mg/kg L-703,606. Thirty minutes after injection, rats werexposed to aggressive encounters for 20 min. In addition to heartates, locomotor activity was also monitored by the same E-itters. Sample size was six/group.

Experiment 6. The subjects of this study were ADXr rats.ham-operated rats were also included for comparison. Oneeek after adrenalectomy or sham operation, all the subjectsere implanted with E-mitters. After another week, the ani-als were injected IP with vehicle or 1 mg/kg L-703,606.

hams received vehicle. Thirty minutes after injection, rats

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were exposed to aggressive encounters for 20 min. In additionto heart rate, locomotor activity was also monitored. Samplesize was eight/group.

Experiment 7. After 1 week of individual housing, intact ratswere treated with 0 (vehicle), .1, and 1 mg/kg L-703,606. Thirtyminutes after injection, they were studied in the open-field testfor 15 min. Sample size was seven/group.

Adrenalectomy and Corticosterone ReplacementAdrenalectomy and sham operation were performed under

ketamine–xylazine–promethazine anesthesia (50–10–5 mg/kgIP) by the dorsal approach. Surgery was performed in ratsacclimatized to the inverted day/night schedule for 2 weeks.Sham operation consisted in revealing the adrenals withoutdamaging them. Because ADX without glucocorticoid replace-ment induces neuronal degeneration (34), we ensured low andstable plasma levels of glucocorticoids by implanting subcutane-ous corticosterone pellets immediately after surgery. The pelletweighed 100 mg and contained 25% corticosterone and 75%cholesterol (both from SIGMA). Such pellets maintain approxi-mately 90–100 nmol/L plasma corticosterone concentrations forat least 3 weeks (21). No subcutaneous pellet was implanted intoShams.

Implantation of Telemetric E-Mitters and Data AnalysisThe E-mitters (Minimitter) were placed into the abdominal

cavity of rats through a midline abdominal incision underketamine–xilazine–promethazine anesthesia (50–10–5 mg/kgIP). The negative and positive heart rate leads were attached tothe anterior right side of the chest (near the clavicle) and to thechest wall (left to the sternum and anterior to the last rib),respectively. Telemetric recordings were made by means of theVitalView system (Minimitter). Data were sampled once everyminute around the clock. The average heart rate measuredduring the 5 min that preceded the first experimental manipula-tion (e.g., IP injection) was considered the baseline for subse-quent measurements.

Encounters and Aggression-Related VariablesEncounters were performed under dim red illumination (pro-

vided by two 25-W red lamps) in the home cage of rats. Behaviorwas video recorded and scored later by an experimenter blind tothe treatments, by means of a computer-based event-recorder(H77, Budapest, Hungary).

“Psychosocial encounters” started by separating the home-cage of subjects into two unequal compartments by a perforatedPlexiglas wall. The larger compartment (40 � 40 � 50 cm)contained the subject. The intruder was introduced into thesmaller compartment 1 hour later. The Plexiglas wall allowedolfactory and visual contacts but prevented direct physicalcontacts. We recorded the duration and the frequency of “rest-ing” (no locomotion), “exploration/walking” (sniffing move-ments directed towards the floor, walls, and air as well aslocomotion), “proximity” (time spent in the immediate proximityof the separating Plexiglas wall), and “direct contact” (reciprocalsniffing through the holes of the wall).

“Aggressive encounters” also took place in the home cage ofsubjects. The following behavioral variables were assessed:exploration (sniffing directed toward the environment); socialinvestigation (sniffing directed towards the opponent’s flank,nasal, or anogenital region); grooming (self-grooming with fore-paws and scratching with hindlegs); offense (aggressive groom-

ing, lateral threat, offensive upright posture, mounting and

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hasing taken together); defense (defensive upright, defensiveick, fleeing and freezing taken together); dominant posturekeeping down the opponent while he is laying on his back);ubordinate posture (laying on back while kept down by thepponent). We mention that submission was very low in subjectsnd even the few cases occurred when the resident lost balancewing to a push from the intruder.

Attack episodes were analyzed at low speed (frame-by-framehen necessary) for identifying the type of attacks and attack

argets. An attack was identified as “hard bite” when it involvedicking (clinch fights) or induced a strong startle response in thentruder (large jumps or immediate submission). “Soft bites” wereot associated with kicking and induced no response or milduivering only. Similar approaches were employed earlier35,36). The following targets were attacked: head (areas anterioro the ears), throat (the ventral area below the ears), dorsal areasposterior to the ears), and belly (ventral areas between legs).ead, throat, and belly were considered vulnerable targets.

he Open-Field TestThe test apparatus was a circular wooden area (diameter 90

m) surrounded by a 40-cm-high metal wall. Both the floor andalls were dark grey. Rats were placed next to the wall and werellowed to explore the arena for 15 min. The apparatus wasimly lit by two 25-W red lamps and was cleaned thoroughlyetween subjects. The arena was divided into equal sub-areas byoncentric and radial lines. Locomotor activity was evaluated onhe basis of the number of line crossings.

mmunocytochemistryBrain Processing. Animals were terminally anesthetized with

odium pentobarbital (Nembutal, Sanofi, 50 mg/kg, IP) anderfused through the ascending aorta with 150 mL ice-cold .1ol/L phosphate-buffered saline followed by 300 mL 4% para-

ormaldehyde (in .1 mol/L phosphate-buffered saline). Therains were removed, post-fixed in the same solution for 3 hoursnd cryoprotected overnight by 20% sucrose in phosphate-uffered saline at 4°C. Six series of 30-�m frozen sections wereut in the frontal plane on a sliding microtome. Section planesere standardized according to the atlas of Paxinos & Watson

37).Double Labeling for c-Fos and NK1 Positive Neurons. The

-Fos protein was labeled with a rabbit polyclonal antibodyaised against the amino terminus of c-Fos p62 (Santa Cruziotechnology, Santa Cruz, California; sc-52) as described earlier38,39). This antibody is highly selective and shows no cross-eactions with other members of the Fos protein family. Therimary antibodies (1:10,000) were detected by biotinylatednti-rabbit IgG (1:1000) and streptavidin conjugated HRP1:1000) (Jackson Laboratories, Bar Harbor, Maine). The perox-dase reaction was developed in the presence of diaminobenzi-ine tetrahydrochloride (.2 mg/mL), nickel-ammonium sulphate.1%), and hydrogen peroxide (.003%) dissolved in Tris buffer.he sections stained for c-Fos were double labeled with specificabbit polyclonal antibodies raised against a 23 amino acidynthetic peptide corresponding to the C-terminus (amino acids85-407) of the rat NK1/Substance P Receptor (1:5000, AB 5060,hemicon, Temecula, California). The primary antibodies wereetected by biotinylated donkey anti-rabbit IgG (1:1000) andtreptavidin conjugated HRP (1:1000) (Jackson Laboratories).he peroxidase reaction was developed in the presence ofiaminobenzidine tetrahydrochloride (.5 mg/mL) and hydrogen

eroxide (.005%) dissolved in Tris buffer (pH � 7.6).

Quantification of Staining. Staining specificity was tested inpreliminary experiments. Signal distribution and intensity afterdouble labeling was identical with that seen after single labeling.In double-labeling studies, all three—single c-Fos, single NK1,and double labeling—were seen in the same areas. The c-Fosand the NK1 receptor signals were clearly separable, owing totheir color (black and brown, respectively) and localization(nucleus and cytoplasm, respectively). Signals were counteddirectly in the microscope at 500-fold magnifications by aninvestigator blind to treatments.

A preliminary analysis was performed to identify the areasthat were activated in the brain areas of interest. In the prefrontalcortex, the activation of NK1 positive neurons was weak, and asomewhat stronger activation was observed in the infralimbiccortex. Within the amygdala, the majority of double-labeledneurons were located in the medial nucleus. Considerably fewerdouble-labeled cells were found in the cortical and basolateralamygdala. The NK1 signal was patchy in the central nucleus;therefore, this area could not be analyzed. In addition, the NK1signal was weaker here then in the medial nucleus. Withinthe hypothalamus, most double-labeled neurons were foundin the hypothalamic attack area. This area was delimited earlieron the basis of its role in the control of attacks (40). A detailedthree-dimensional reconstruction of this area can be found inHrabovszky et al. (41).

On the basis of the preliminary screening, the infralimbiccortex, medial amygdala, and hypothalamic attack area wereselected for detailed analysis. The methodology has been de-scribed earlier (38,39). The size of the scanned area was .603mm2 for the infralimbic cortex (Bregma �3.2 mm), .335 mm2 formedial amygdala; and .318 mm2 for the hypothalamic attack area(both at Bregma �2.3 mm). Cells were counted bilaterally in twosections 180 �m apart.

StatisticsData were shown as means � SEM. Behavioral data were

analyzed by Kruskal-Wallis analysis of variance (ANOVA) fol-lowed by Mann-Whitney post hoc comparisons. Immunocyto-chemical data were analyzed by one- or two-factor ANOVA(Factor1: encounter [control vs. encounter-exposed]; Factor2:glucocorticoid background [sham vs. ADXr]). Heart rate changeswere analyzed by repeated measure ANOVA. The ANOVAs werefollowed by Newman-Keuls post hoc comparisons.

Results

Experiment 1In psychosocial encounters, most of the time was spent in

exploration. For about 6% of total time, rats were in directcontact, and an additional 70% was spent in the close vicinity ofthe Plexiglas wall. In aggressive encounters, rats attacked theintruder (attack counts: 1.7 � .9) and showed offensive (6.5 �.8% time) and dominant (5.9 � 2.5% time) behaviors.

The number of activated of NK1 neurons showed region-specific changes (Figure 1). In the infralimbic cortex, activationwas low and showed no group differences [F (2,15) � 1.55, p �.25]. In contrast, the encounters increased the activation of NK1positive cells in both the medial amygdala and the hypothalamicattack area [F (2,15) � 7.27, p � .006, and F (2,15) � 24.92, p �.0001, respectively]. In both areas, the effect of aggressiveencounters was larger than the effect of psychosocial encounters.A similar pattern of changes was noticed when the percentage of

activated NK1 positive neurons was considered (see Supplement 1).

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xperiment 2The behavior of Sham and ADXr rats was similar except for

ulnerable attacks that were significantly more frequent in ADXr ratss compared with Shams [ 1,18) � 10.26, p � .001] (Figure 2).

The NK1 cell activation was low in the prefrontal cortexFigure 2). Aggressive encounters induced a marginal increase Fencounter(1,26) � 3.51, p � .07], but this did not depend on

igure 1. Representative photomicrographs from animals submitted togonistic encounters and the number of activated Neurokinin1 (NK1) posi-ive neurons (sections were double labeled for c-Fos and the NK1 receptor).

hite arrow: single labeled NK1 receptor positive neuron (brown); greyrrow: single labeled c-Fos positive neuron (dark grey/black nucleus); blackrrow: double labeled activated neurons expressing NK1 receptors. PFC/IL,

nfralimbic part of the prefrontal cortex; MeA, medial amygdala; HAA, hypo-halamic attack area. *Significantly different from control; #Significantly dif-erent from psychosocial encounter (p � .05).

igure 2. The effect of agonistic encounters on NK1 receptor activation inham-operated (Sham) and adrenalectomy with glucocorticoid replace-

ent (ADXr) rats. Behaviors were also scored. (A) The duration of variousehaviors. (B) The number of bite attacks. (C) The share of vulnerablettacks (directed towards the head, throat, and belly). (D) The absoluteumber of activated NK1 neurons (see Supplement 1 for relative activa-

ions). (E) The correlation between NK1 neuron activation in the medialmygdala and the share of vulnerable attacks. Abbreviations as in Figure 1.Significant effect of encounter (p � .05); #Significant effect of glucocorti-

oid background (p � .05).

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glucocorticoid background [Fcort(1,26) � .09, p � .7;Finteraction(1,26) � .09, p � .7]. In the medial amygdala, thenumber of activated NK1 neurons was increased by both theencounter [Fencounter(1,26) � 12.45, p � .002] and ADXr[ Fcort(1,26) � 4.58, p � .04]. There was no interaction betweenfactors [Finteraction(1,26) � .01, p � .9]. In the hypothalamicattack area, aggressive encounters massively increased thenumber of activated neurons [Fencounter(1,26) � 88.01, p �.0001], but this did not depend on glucocorticoid background[Fcort(1,26) � .66, p � .4; F interaction(1,26) � .91, p � .4]. Asimilar pattern of changes was seen when the percentage ofactivated NK1 positive neurons was considered (see Supple-ment 1).

The share of vulnerable attacks showed a positive correlationwith both the number and percentage of activated NK1 neurons(Spearman R � .494, p � .04 and R � .534, p � .03, respectively).The correlation was especially strong in ADXr animals (Spear-man R � .729, p � .03).

Experiment 3In intact residents, L-703,606 selectively decreased the num-

ber and increased the latency of hard bites [2,24) � 5.50, p � .06and 2,24) � 6.31, p � .04, respectively] (Figure 3). Otherbehaviors were not affected. The total number of bites was alsodecreased [2,24) � 5.60, p � .06]; however, this was secondaryto the decrease in hard bites.

Experiment 4Vehicle-treated Sham and ADXr rats showed no significant

behavioral differences except for a large increase of vulnerableattacks in ADXr rats as compared with Shams (Figure 4). In theformer, NK1 blockade selectively decreased the share of bothhard bites and vulnerable bites [H (3,35) � 8.05, p � .05 andH (3,35) � 16.84, p � .001, respectively]. L-703,606 also increasedthe latency of hard bites [H (3,35) � 12.61, p � .006]. Otherbehaviors were not affected.

In this experiment, the behavior of intruders was also scored,

Figure 3. The effect of Neurokinin1 (NK1) receptor blockade on aggressive-ness in intact residents. (A) The duration of various behaviors. (B) Thelatency of hard bites. (C) The frequency of different attack types. (D) Theshare of vulnerable attacks. *Significantly different from saline control(p � .05).

to investigate the interaction between the behaviors of contes-

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ants. However, the behavior of intruders did not depend on theehavior of residents (Table 1).

xperiment 5Again, NK1 receptor blockade decreased the number and

ncreased the latency of hard bites [H (1,11) � 4.146, p � .042 and (1,11) � 5.280, p � .022, respectively] (Figure 5).Before treatments, heart rates were similar in the two groups

control subjects: 402.5 � 20.1 beats/min; L-703,606-treated:25.5 � 10.7 beats/min; F (1,9) � 1.12, p � .3]. Injections inducedtransient increase in heart rates [Ftime(28,252) � 5.84, p �

0001], which was significant between min 2–6 after injections. Athis time point, heart rates increased to 482.2 � 20.8 beats/min.owever, values returned to baselines before the aggressive en-ounter (409.6 � 13.7 beats/min). The increase was not affected byhe treatment [Ftreatment(1,9) � .04, p � .8]. The aggressive encoun-er increased heart rates significantly [Ftime(19,171) � 4.80, p �0001], but this did not depend on treatment [Ftreatment(1,9) � .38,� .5; Finteraction(19,171) � .99, p � .4]. Locomotor activity was notffected by L-703,606 [F (1,9) � .09, p � .7].

xperiment 6The number of hard bites was not affected by ADXr but was

ecreased by L-703,606 [H (2,23) � 7.72, p � .02; Figure 6].ompared with Shams, the share of vulnerable attacks increased

igure 4. The effect of NK1 receptor blockade on aggressiveness in ADXrats. (A) The duration of various behaviors. (B) The latency of hard bites.C) The share of hard bites in the total number of attacks. (D) The totalumber of attacks. (E) The share of vulnerable attacks. Abbreviations as inigure 2. *Significantly different from saline injected control (p � .05).

able 1. The Behavior of Intruders Facing Residents that Received Various

esident EXP SOC

ham, saline 65.7 � 2.4 13.5 � 1.5 .DXr, saline 70.6 � 2.7 15.1 � 1.6 .DXr, .1 mg/kg L-703,606 68.8 � 3.5 14.5 � 1.6 .DXr, 1 mg/kg L-703,606 68.5 � 3.0 17.0 � 1.3 .(3,35) 1.48 1.38� .6 .7

Data were expressed as % time. Intruders received no treatment. Thexploration; SOC, social interactions; GRO, grooming; OFF, offense; DOM, d

ith glucocorticoid replacement.

in ADXr rats [H (2,23) � 5.97, p � .05]. The NK1 receptorblockade abolished this effect.

Before treatments, heart rates showed no group differences[Sham, saline: 398.1 � 13.6 beats/min, ADXr, saline: 423.4 � 15.7beats/min, ADXr, L-703,606: 397.5 � 12.3 beats/min; F (1,20) �1.18, p � .3]. Injections induced a transient increase in heart rates[Ftime(28,560) � 5.09, p � .0001], which was significant betweenmin 3–6 after injections. At this time point, heart rates increasedto 458.9 � 14.3 beats/min. However, values returned to baselinesbefore the aggressive encounter (399.1 � 6.3 beats/min). Theincrease was not affected by the treatment [Ftreatment(1,9) � .04,p � .8].

Aggressive encounters increased heart rates significantly[Ftime(19,380) � 5.92, p � .0001]. The increase was smaller inADXr rats, but the difference was abolished by NK1 receptor

ments

OFF DOM DEF SUB

1.3 � .4 3.2 � 1.5 2.9 � .4 9.7 � 2.11.1 � .4 1.6 � .7 3.1 � .7 6.1 � 1.92.9 � 1.0 3.1 � 1.5 3.2 � .8 5.1 � 1.31.3 � .3 1.2 � .5 4.1 � 1.0 5.6 � 2.2

2.55 1.22 .58 3.69.5 .7 .9 .3

tments received by residents were shown in the left-hand column. EXP,ant postures; DEF, defense; SUB, submissive posture; ADXr, adrenalectomy

Figure 5. The impact of Neurokinin1 (NK1) receptor blockade on aggres-sion-induced autonomic activation in intact rats. Behaviors were alsoscored. (A) The frequency of various attack types. (B) The latency of hardbites. (C) Heart rate changes during the aggressive encounter. For theperiod that preceded the encounter see text. (D) Area under the curve (AUC)for the data presented in panel C. (E) Locomotor activity during the resi-dent/intruder test. Data were expressed in conventional units provided bythe Minimitter system. For brevity, the AUC was shown. �HR, heart ratereactivity. *Significantly different from saline control (p � .05).

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lockade [Ftreatment(2,20) � 3.87, p � .038; Finteraction(38,380) �.26, p � .2]. Locomotion was not affected [F (2,20) � 1.9, p � .2].

xperiment 7The NK1 receptor blockade did not affect locomotion in the

pen-field (Table 2).

iscussion

ain FindingsAgonistic encounters activated a large number of NK1 recep-

or-positive cells in areas crucial for the control of aggressiveehavior (e.g., the medial amygdala and the hypothalamic attackrea) (42). The activation was aggression-specific, because theffects of psychosocial stimulation were significantly smaller.he activation of NK1 positive cells was larger in the medialmygdala of rats submitted to the model of violent aggressionhan in rats submitted to resident/intruder conflicts. The NK1eceptor blockade dose dependently decreased hard bites in theesident/intruder paradigm, without affecting milder forms ofttacks and other behaviors. In the violent aggression model,oth hard bites and vulnerable attacks (the main symptoms ofiolence in this model) were decreased. Moreover, the NK1ntagonist ameliorated autonomic deficits seen in the model ofiolent aggression but did not affect heart rates in resident/ntruder conflicts. Thus, NK1 receptor blockade decreased vio-ent forms of attack in both territorial aggression and a model ofiolent aggressive behavior. The effects of the compound wereot secondary to changes in locomotion or in the behavior of

igure 6. Behavioral and radiotelemetric data (heart rate and locomotion) inham-operated and violent ADXr rats during territorial encounter treatedith saline or the NK1 antagonist L-703,606. (A) The number of bite attacks.

B) Percentage of attacks targeted toward vulnerable body parts (head,hroat, and belly) of the opponents. (C) Heart rate reactivity during theesident/intruder test. (D) Heart rate reactivity during the resident/intruderest, area under the curve (AUC). (E) Locomotion during the resident/in-ruder test, area under the curve, units used by the Minimitter system.bbreviations as in Figures 2 and 5. *Significantly different from salineontrol (p � .05).

ntruders.

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NK1 Receptors in Territorial and Violent AggressionThe NK1 receptors are widely expressed in brain areas that

are crucial for the regulation of emotional responses (1–3).Beyond decreasing anxiety and depression (4 –7), the presentstudy suggests that such antagonists also affect aggressiveness.This finding is supported by disparate earlier data on theinteraction between substance P neurotransmission and aggres-siveness (6,8 –10). To our knowledge, we are the first to showthat NK1 positive neurons are activated by and are tightly boundto the execution of attacks, because these were inhibited by NK1receptor blockade. Surprisingly, the effect was restricted to hardbites, whereas milder attacks or other forms of aggressivenesswere not disrupted.

We also studied the effects of L-703,606 in a recently devel-oped model of violent aggression that—as suggested earlier(21,22)— can be used as a model of antisocial violence (seeintroductory text). In rats submitted to this model, the activationof NK1 positive cells was increased in the medial amygdala;moreover, there was a positive correlation between the activa-tion of these cells and violent attacks. Therefore, one cannotexclude that the occurrence of violent attacks was at least in partdue to alterations in substance P neurotransmission. In thismodel, L-703,606 treatment reduced hard bites but, more impor-tantly, decreased the number of attacks directed towards vulner-able body parts of the opponents. The number of such attackswas decreased to the levels seen in sham-operated controlsubjects. This further suggests a tight link between substance Pneurotransmission and violent attacks. Importantly, the NK1receptor antagonist also reduced the autonomic deficit associ-ated with violent aggression. This deficit was shown earlier andwas replicated here (20).

NK1 Antagonists: A Novel Approach to the Treatment ofAntisocial Personality Disorder?

In a meta-analysis of 62 surveys involving roughly 23,000prisoners, Fazel and Danesh (43) found that the incidence ofantisocial personality disorder was 47%. The odds ratio ofviolence and aggressiveness is around 7 in this subgroup,whereas the odds ratio of homicide is above 10, demonstratingthe high danger that this disorder represents for society (44 – 46).Unfortunately, treatments lead to modest effects in this disorder,because neither psychotherapy nor pharmacotherapy providesreliable results in the long run (47–52).

Because no reliably efficient treatments are known for anti-social personality disorder, the pharmacological validation of ourmodel seems difficult at present. Nevertheless, earlier findingssuggested that there is a certain degree of pharmacologicalsimilarity between antisocial personality disorder and our model.The similarity concerns agents that affect serotonergic neuro-transmission. Such agents (e.g., buspirone and selective seroto-nin reuptake inhibitors) efficiently reduce aggressiveness in avariety of psychological disorders (25–27) but not in antisocialpersonality disorder where equivocal results were obtained(28 –31). The impact of serotonergic neurotransmission on ag-

Table 2. The Effect of NK1 Receptor Blockade on Locomotor Activity inthe Open Field Test

Saline.1 mg/kgL-703,606

1 mg/kgL-703,606 H(2,21) p

LineCrossings 301.9 � 25.2 338.7 � 20.3 290.1 � 17.2 2.42 .3

NK1, Neurokinin1; L-703,606, a specific NK1 antagonist.

g(tcse

obdsd

C

aNacmctpa

RH

t

1

1

J. Halasz et al. BIOL PSYCHIATRY 2008;63:271–278 277

ression is also compromised in our violent aggression model53). Moreover, buspirone—which decreased aggressiveness inhe resident/intruder paradigm–dramatically increased attackounts in glucocorticoid-deficient rats (54). This pharmacologicalimilarity suggests that data obtained with our model can bextrapolated to antisocially disordered humans.

The NK1 receptor blockers are currently developed as anxi-lytic and antidepressant drugs (4 –7). As soon as these agentsecome clinically available, their effects on antisocial personalityisorder-related aggressiveness can be evaluated—even moreo, because this disorder is frequently comorbid with majorepression and certain types of anxiety disorders (18,55,56).

oncluding RemarksThe aggression-induced activation of NK1 positive neurons

nd the behavioral effects of NK1 receptor blockade suggest thatK1 receptors are strongly involved in the control of violentggression. On the basis of multiple similarities between antiso-ial personality disorder-associated aggressiveness and ourodel of violent aggression, we suggest that NK1 blockers might

onstitute a novel approach to the treatment of aggressiveness inhis disorder. The effects of L-703,606 in the resident/intruderaradigm suggest that these blockers might be useful in otherggression-related problems as well.

This work was supported by Hungarian National Science andesearch Fund (OTKA) grants (F048467, T 046785) and aarry Frank Guggenheim Foundation grant to J. Haller.

The authors declare no relevant conflicts of interest in rela-ion to this work.

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