potentiating role of interleukin 2 (il-2) receptors in the midbrain periaqueductal gray (pag) upon...

Behavioural Brain Research 167 (2006) 251–260

Research report

Potentiating role of interleukin 2 (IL-2) receptors in the midbrainperiaqueductal gray (PAG) upon defensive rage behavior

in the cat: Role of neurokinin NK1 receptors

Suresh Bhatta, Allan Siegela,b,∗a Department of Neurology & Neurosciences, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA

b Department of Psychiatry, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA

Received 2 August 2005; received in revised form 13 September 2005; accepted 14 September 2005Available online 20 October 2005

Abstract

Feline defensive rage is a form of aggression occurring in nature in response to a threatening condition and is elicited under laboratory conditionsby electrical stimulation of the medial hypothalamus or midbrain periaqueductal gray (PAG). Since it has recently been shown that cytokinesc -2) into theP nificantlyf followingfi treatmento anotherf cts of IL-2w tinge Rd ough an NKr©

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an modulate neurotransmitter release, the present study was designed to determine the effects of administration of interleukin 2 (ILAG upon defensive rage elicited from the medial hypothalamus. Microinjections of relatively low doses of IL-2 into the dorsal PAG sig

acilitated defensive rage behavior elicited from the medial hypothalamus. The specificity of this phenomenon was supported by thendings: (1) IL-2 induced effects were dose- and time-dependent, (2) the facilitative effects of IL-2 could be completely blocked by pre-f the injection site with either anti-IL-2 or anti-IL-2 receptor antibody and (3) IL-2 administration into the PAG showed no effect upon

orm of aggression, namely predatory attack, elicited from the lateral hypothalamus. The findings further demonstrated that the effeere mediated by an NK1 receptor mechanism since pre-treatment of the PAG with an NK1 receptor antagonist completely blocked the facilitaffects of IL-2. Immunocytochemical observations supported these findings by demonstrating an extensive pattern of labeling of IL-2� in theorsal PAG. The present study thus demonstrates that IL-2 in the dorsal PAG potentiates defensive rage behavior and is mediated thr1eceptor mechanism.

2005 Elsevier B.V. All rights reserved.

eywords: Aggression; Defensive rage; Cat; PAG; Immunocytochemistry; NK1 receptor; IL-2 receptor

. Introduction

Defensive rage behavior in the cat is characterized by archingf the back, retraction of the ears, piloerection, unsheathing of

he claws, pronounced hissing, marked pupillary dilatation andaw striking[37]. This behavior occurs in nature in response toperceived threat, and is induced in the laboratory by electricalr chemical stimulation of the medial hypothalamus or midbraineriaqueductal gray[68]. In this context, the periaqueductal grayPAG) constitutes the most caudal region of the central nervousystem from which integration of defensive rage behavior takeslace[7,68]. This function of the PAG is made possible for threeeasons. The first is that it receives a primary input from regions

∗ Corresponding author. Tel.: +1 973 972 4471; fax: +1 973 972 3291.E-mail address: [email protected] (A. Siegel).

of the medial hypothalamus that mediate defensive rage beh[19,20,24,59]. The second is that the PAG projects its axonregions of the lower brainstem associated with the somatomand autonomic components of the defensive rage respons[61].The third, and perhaps, the most persuasive argument, iblockade of hypothalamic inputs to the PAG, either by lesof specific regions of the medial hypothalamus[20] or PAG[33], or by blockade of NMDA receptors in the PAG whreceive medial hypothalamic afferent fibers[40,59], effectivelyeliminates defensive rage behavior.

Because of the importance of the PAG for the expressiodefensive rage behavior, considerable attention has beenover the past two decades in identifying the neurochemical perties of this region that mediate and modulate defensivebehavior. Both excitatory and inhibitory receptor mechanhave been identified. Activation of NMDA, CCKB, NK1 and 5-HT2 receptors in the PAG has been shown to facilitate defe

166-4328/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2005.09.011

252 S. Bhatt, A. Siegel / Behavioural Brain Research 167 (2006) 251–260

rage [23,40,41,59,64], while activation of�-opioid, GABAAand 5-HT1A receptors have been shown to potently suppressdefensive rage[61–63,65,66].

A number of investigators have provided evidence thatcytokines can influence functions associated with the centralnervous system[1,4,13,15,71,73], including specific alterationsof neuroendocrine activity[32,69,74]. Interleukin 2 (IL-2) isone such pleiotropic cytokine that is widely distributed in var-ious brain regions including the hypothalamus, hippocampus,septal area, cortex and striatum[6,9] and is synthesized cen-trally [16,35,50]. In addition to its multiple immunoregulatoryfunctions and biological properties related to T-cells, IL-2 hasbeen shown to affect neuroendocrine functions[27,39], enhancehypothalamic norepinephrine[81], inhibit in vivo release ofdopamine within the nucleus accumbens[3,70], and stimulatethe release of the arginine vasopressin and corticotropin releas-ing hormone from the hypothalamus and amygdala[54,55].Moreover, in mesencephalic cultures, increases in dopaminerelease evoked by NMDA and kainate were observed followingIL-2 treatment[2]. It has been further shown that IL-2 modu-lated K(+)-evoked[3H]DA is released in a biphasic manner, withlow concentrations of IL-2 potentiating and higher concentra-tions inhibiting K(+)-induced[3H]DA release[52]. In a similarmanner, low doses of IL-2 potentiated acetylcholine release inhippocampal slices, but showed an inhibitory effect at higherdoses[6,26,60].

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sive rage behavior in the PAG was mediated through an interac-tion with NK1 receptors.

2. Materials and methods

2.1. Animals

Ten adult female cats weighing between 2 and 4 kg (Liberty laboratories,Waverly, NY) were utilized in this study. Cats were housed individually intheir home cages and had free access to food and water. All experiments wereapproved by the Institutional Animal Care and Use Committee of the New JerseyMedical School. The strengths associated with the use of this model for studyingthe defensive rage have been discussed previously[9,23].

2.2. Surgery

All procedures during surgery were performed aseptically. Cats were deeplyanesthetized with isoflurane (1–2%). Twenty-four stainless steel guide tubes(17 gauge, 10 mm in length) were stereotaxically mounted bilaterally (accord-ing to the atlas of Jasper and Ajmone-Marsan)[34] over holes drilled through theskull overlying the medial hypothalamus, lateral hypothalamus and dorsal mid-brain PAG and were cemented using dental acrylic cement (Lang Dental Mfg.Co., Wheeling, IL). Then, guide tubes were filled with bone wax (Ethicon Inc.,Somerset, NJ). Three stainless steel stylets were attached to the skull and servedas indifferent electrodes. One steel bolt was placed in a hole drilled into the nasalsinus of the cat and two nylon bolts were anchored to the skull with dental acryliccement. A plastic cap secured by these bolts protected the entire assembly.

2.3. Elicitation of defensive rage behavior and predatory attack

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Despite the neuromodulatory actions of IL-2, little is knobout the behavioral effects of this cytokine. The evidenceuman studies regarding the behavioral effects of IL-2 is de

rom patients diagnosed with depression or schizophreniaave been shown to have increased levels of plasma as welrospinal IL-2[5,12,42]. Furthermore, IL-2 immunotherapy hlso been found to be associated with cognitive impairm

ncluding confusion and deficiencies in spatial abilities[36,75].everal animal studies have revealed that the central admin

ion of IL-2 induced behavioral changes, including variationleep and arousal patterns[46,47]and increased novelty-inducxploratory activity and locomotion[82–84].

Recently, our laboratory has begun to examine the roytokines in the regulation of defensive rage and predatory aehavior in the cat. This approach was initiated on the basis

ines of research described above. Several experiments rehat IL-1� microinjected into the medial hypothalamus (frhich defensive rage could be elicited) potentiated defen

age elicited from the PAG[30,31]. In contrast, microinjectionf IL-2 into the same region of the medial hypothalamus poteuppressed PAG-elicited defensive rage, an effect that waso be mediated through GABAA receptors[9]. Since the effectf IL-2 have been shown to be site specific[6,9] and that theAG is critical for the expression of defensive rage, the pretudy was undertaken to determine the role of IL-2 in modulaefensive rage behavior from the PAG and the neurocheechanisms underlying this phenomenon.Because it has recently been shown that NK1 receptors pla

powerful role in the mediation of aggression[8,23] and otheelated processes in the PAG[14], the present study was extendo determine whether the process of IL-2 modulation of de

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Defensive rage behavior was induced by utilizing the procedures identhose used previously in our laboratory[8,9,29–31]. Experiments were carrieut in awake cats. Cats were freely moving except for the brief period ofelivery at which point the cat was placed in a restraining cat bag with itsecured to a head holder. Cats experienced no stress or discomfort foradual habituation to the experimental cage, veterinary restraining baead holder over the course of several days. Cats appeared comfortababituation as evidenced by the fact that they were quiet and purred and diso attempt to escape from the restraining bag or experimental chamberxperimental procedures.

At least 2 weeks following post-operative recovery from surgery, aniere placed in a wooden experimental chamber (61 cm× 61 cm× 61 cm) inhich they could be viewed through a clear Plexiglas wall. An cannula elec

23 gauge, insulated throughout its length except at 0.5 mm from the tip (Pne, Roanoke, VA) was lowered into the PAG and a monopolar stimulating

rode (51.5 mm long insulated throughout the length and exposed 0.5 mmhe tip, Plastics One) was lowered into the medial hypothalamus. The calectrode was used both for injection of drug and for stimulation. Electrodes

owered in 0.5 mm increments through guide tubes implanted on the skulying either the medial hypothalamus or PAG. While the cat was freely molectrical stimulation was applied at each of these increments with a bipectangular electrical pulses (0.2–0.8 mA, 63 Hz, 1 ms per half cycle duralectrical stimulation was generated by grass S-88 stimulators, whichonnected through differential amplifiers (Tektronix ADA400A) to the caektronix TDS 3012 digital oscilloscope was used to monitor peak-to-peaent. Once a defensive rage site was located and response was repeatedlyn a stable manner within 15 s following onset of stimulation, the monopolaannula) electrode was cemented in place using dental acrylic cement.

Predatory attack behavior, elicited by stimulation of the lateral hypamus, is characterized by initial stalking of the anesthetized rat followeigorous biting of its neck[76]. Aside from the presence of pupillary dilaion, other signs of autonomic activation are not apparent in this responsethods for inducing predatory attack in the laboratory have been pubreviously[9,25] and the methods used in the present study for the anf this form of aggression are identical to those described for defensiveehavior.

S. Bhatt, A. Siegel / Behavioural Brain Research 167 (2006) 251–260 253

2.4. Measurement of defensive rage and predatory attack

Response latencies for elicitation of hissing, which was used as a standardmeasure of defensive rage, or biting of the back of the neck of an anesthetizedrat as the measure for predatory attack behavior was defined as the time requiredfor the cat to express the specific form of aggressive response following onset ofelectrical stimulation. The hissing response was used as the principle measureof defensive rage since hissing constitutes an integral part of defensive rageresponse and occurs whenever defensive rage is elicited[8,9], while biting attackwas the response measure used as the principle measure of predatory attack[76].

The duration of stimulation was limited to 15 s on all trials. If a responsecould not be elicited within 15 s, a response latency score of 15 s was recordedfor that trial even though stimulation was ineffective in generating defensive rage(or predatory attack). If the response was elicited within 15 s, stimulation wasterminated and the response latency was recorded. The stimulating current wasadjusted to the levels that would induce hissing responses (or biting for predatoryattack) between 5 and 10 s, which enabled a determination of the suppressing aswell as facilitating effects of drug administration.

2.5. Dual stimulation

A dual stimulation procedure, in which 10 paired trials of single stimula-tion of the medial hypothalamus and dual stimulation of the PAG and medialhypothalamus were administered (at 63 Hz with a 4 ms delay separating biphasicpulses delivered to each region) in an A–B–B–A fashion in which ‘A’ representedstimulation of the medial hypothalamus alone and ‘B’ dual stimulation of themedial hypothalamus plus PAG, was utilized. The dual stimulation procedureidentified the sites in the PAG at which stimulation could be shown to facilitatethe occurrence of defensive rage behavior elicited from the medial hypothala-mus. This approach was used to ensure that there was a functional relationshipb ppliet sholdf d fore leveo thes tedm meno

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2 min. Experimental sessions were separated by at least 48 h in order to minimizethe possible interfering effects of prior drug exposure.

In the design of the experiment, each animal was utilized as its own controlwith respect to drug dose. In this manner, each animal received all doses of drugsand were administered the same treatments (described below). The order of drugdose and treatment delivery was determined by a randomization procedure foreach animal in order to control for the effects of drug exposure. In this manner,a total of five cats received microinjections of each dose of drug and were alsosubjected to the drug interactions described below. Thus, each cat was exposed tothe following experimental stages: (a) a dose–response analysis which includedadministration of three doses of IL-2 (500 pg, 1 and 5 ng) and saline; analysis ofthe effects of: (b) an anti-IL-2 monoclonal antibody (94.7 nmol); (c) an antibodydirected against the IL-2 receptor� subunit (IL-2R�) (48.7 nmol) and (d) theNK1 receptor antagonist, GR82334 (8 nmol) (Sigma). The order of treatmentswas randomly determined. The final experiment conducted on each animal wasdesigned to test the efficacy of the defensive rage sites. It included a comparisonof the effects of pre-treatment with antibody against administration of IL-2plus vehicle. In each of the instances in which IL-2 was administered togetherwith vehicle, the hissing response was clearly present, indicating the continuedefficacy of these sites.

2.7. Behavioral specificity of IL-2

To determine the behavioral specificity of IL-2, we investigated its effectson predatory attack behavior (n = 3). The same paradigm used for the study ofdefensive rage was employed for the study of predatory attack behavior.

2.8. Histology

Cats were perfused transcardially with 9.25% sucrose solution in PBS (w/v)(r lutionat 00) at2 drieda

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etween the PAG and hypothalamic attack sites in each cat. The current ao the modulating site in the PAG was maintained at a level below threor elicitation of hissing. Response latencies for hissing were determineach trial. At-test for paired observations was employed to determine thef significance between paired trials of single and dual stimulation withignificance level set atp < 0.05. Sites in the PAG that significantly modulaedial hypothalamically elicited defensive rage were utilized for the placef microinjections of IL-2 compounds and an NK1 antagonist (see below).

.6. Drugs and drug administration

Selective doses of IL-2, anti-human IL-2 monoclonal antibody (Pepronc., Rocky Hill, NJ), anti-human IL-2 receptor alpha (IL-2R�) neutralizing antiody (R&D Systems, Minneapolis, MN) and NK1 receptor antagonist GR823Sigma, St. Louis, MO) were utilized in this study. Drug dose levels were seln the basis of findings obtained from pilot experiments. Prior to drug adm

ration, baseline response latencies following medial hypothalamic stimuere determined. Since the same site was used for different drug dosetimulation was employed after every four to five microinjections to tesfficacy of the site.

On an experimental day, the cat was brought from its home cage to themental chamber where it remained 1 h prior to the initiation of the experin order to reduce general levels of stress. For drug delivery, the cat waestrained in a veterinary restraining bag. Then, its plastic head cap was rend the head was gently attached to a brass head holder on a stereotaxic asing the three bolts mounted on its head during surgery. A 0.5�l microsyringeSGE, Austin, TX) was lowered through the cannula electrode into thefrom which hissing could be elicited) and placed 0.5 mm below the elecip. A drug or saline in a total volume of 0.25�l was injected over a periodmin. Following microinjection, the syringe was left in place for 1 min to a

or diffusion, and was then slowly removed. For experiments in which bothnd: (a) anti-IL-2 monoclonal antibody, (b) anti-IL-2 receptor� antibody orc) GR82334 was used, the total injection volume was 0.5�l, delivered with aelay of 5 min between microinjections. The cat was then removed from theolder and returned to the experimental chamber. The cat was given fivef stimulation over each of the following four blocks of time: 30–40, 60–20–130 and 180–190 min post-injection, with an average inter-trial inter

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pH 7.2) followed by 4% paraformaldehyde (pH 7.4) at 4◦C. Brains wereemoved from the skull, blocked, and stored in a 4% paraformaldehyde sot 4◦C overnight. Then, brains were placed in 30% sucrose solution at 4◦C until

hey sunk to the bottom. Brain sections were cut in a cryostat (Leica CM190–25�m at−20◦C. Sections were mounted on gelatin coated slides, airnd stored at−20◦C.

.9. Immunohistochemistry

For IL-2 receptors staining, sections stored at−20◦C were brought to roomemperature. To recover the antigenicity of the tissue section, citrate buffeen retrieval method was employed[9]. Sections were washed 3× 5 min and

reated with 0.3% H2O2 for anti peroxidase activity. After washing 3× 5 min,he sections were blocked with blocking buffer (2% BSA, 2% goat serum,riton X-100 in PBS, pH 7.4) for 1 h. They were then incubated overnight

he primary antibody directed against the IL-2 receptor� subunit (15�g/mlf murine anti-human IL-2R� antibody used for behavioral data) at 4◦C in aoisturizing chamber. Primary antibody dilutions were carried out in bloc

uffer. Sections were washed 3× 5 min and then incubated with 1:333 dilutif the secondary antibody (biotinylated goat-anti mouse, Santa Cruz Biology, Santa Cruz, CA), for 2 h at RT in a moisturizing chamber. Sections

hen washed and treated with avidin–biotin complex (ABC kit, Vector Latories, Burlingame, CA) for 45 min and then treated with diaminobenzDAB, Vector Laboratories) for color development. Slides were further waith distilled water and dehydrated with alcohol and xylene. Photomicrogf sections were taken with an Olympus AX-70 microscope using an Optricrofire digital camera.

Omission controls were processed simultaneously, where pre-selectedere omitted for incubation with the primary antibody, while all other steps

dentical.

.10. Statistical analysis

A t-test for paired observation was used to determine the effects of dualation of the medial hypothalamus and PAG against single stimulationedial hypothalamus in order to identify sites in the PAG that modulate d

ive rage elicited from the medial hypothalamus. Since pre-injection ba


Fig. 1. Stimulation and injection sites. Upper panel: location of tips of cannulaelectrodes used to elicit defensive rage and for microinjections into the PAG(filled stars). Three cannula eleectrodes were located on the left side and four onthe right side of the dorsolateral PAG at approximately middle levels along itsrostro-caudal axis. Lower panel: tips of electrodes used to elicit defensive ragefrom the medial hypothalamus (filled circles) and predatory attack from the lat-eral hypothalamus (filled squares). Three electrodes were located on the left sideof the dorsomedial hypothalamus (for defensive rage) and four on the right sidefor this response. For predatory attack, one electrode was located in the ventrolat-eral aspect of the lateral hyupothalamus of the left and two in the same region ofthe right side.Abbreviations: AH, anterior hypothalamus; FX, fornix; LH, lateralhypothalamus; OT, optic tract; PAG, periaqueductal gray; VMH, ventromedialhypothalamus (adopted from atlas of Jasper and Ajmone-Marsan[34]).

latencies differed among animals, the data were transformed from responselatency scores to a percentage change in response latencies relative to base-line response latencies for the remaining statistical analyses. Percentage changewas calculated as follows: percentage change = [(pre-drug latency− post-druglatency)/pre-drug latency]× 100.

A two-way randomized blocks ANOVA was used to analyze the effectsof different doses of drugs (variable A) upon response latencies over the pre-injection and four post-injection time periods (variable B). One-way ANOVAswere also used to determine the level of significance of individual doses of drugover four periods of time. A Newman–Keuls multiple comparisons test wasemployed to determine the difference in responses at two different points oftime with the significance level set atp < 0.05 for all experiments.

3. Results

3.1. Anatomical localization of defensive rage sites

As shown inFig. 1, all electrode tips used to elicit defensiverage by medial hypothalamic stimulation for behavioral phar-macological experiments were located in or near the anteriormedial and dorsomedial hypothalamus immediately dorsal tothe ventromedial nucleus. Electrode tips used to elicit predatorattack were located in the middle third of the lateral hypothala-mus. Three of the seven electrode tips used for microinjectioninto the PAG were located on the left side and four on the rightside of the dorsal aspect of the PAG.

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Fig. 2. Single and dual stimulation: defensive rage behavior elicited from thePAG was facilitated by dual stimulation (medial hypothalamus + PAG) in each ofthe cats tested (p < 0.0001,N = 7). For this and subsequent figures, bars indicateS.E.M.’s.

d.f. = 100.93,p < 0.0001). Following dual stimulation, the aver-age reduction in response latencies was 75% (range 59–87%).The facilitating effects of dual stimulation reflect the presence ofa functional excitatory relationship between the medial hypotha-lamus and PAG sites used in this study that provided the rationalefor the microinjection of IL-2 and NK1 compounds.

3.3. Facilitation of defensive rage followingmicroinjections of IL-2 into PAG

Three doses of IL-2 (500 pg, 1 and 5 ng) and saline were testedover five blocks of time (pre-injection, 30–40, 60–70, 120–130and 180–190 min) to determine the effects of IL-2 microin-jections into the PAG upon medial hypothalamically eliciteddefensive rage behavior. The results of a two-way ANOVA indi-cated that microinjections of IL-2 into the PAG resulted in asignificant facilitation of hissing as seen inFig. 3[F(3,9) = 51.52,p < 0.0001]. Post hoc analysis showed that the 5 ng of IL-2induced a significant facilitation of hissing when comparedto the effects of administration of 1 ng, 500 pg or saline. Themaximal facilitation, induced by 5 ng of IL-2, was observed at

F IL-2f e- andt er

.2. Modulation of defensive rage following dualtimulation

Dual stimulation of the PAG facilitated MH-elicited defeive rage in each of the cats tested as seen inFig. 2 (t = 19.04

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ig. 3. Effects of microinjections of IL-2 into the PAG upon defensive rage.acilitated defensive rage elicited from the medial hypothalamus in a dosime-dependent manner (p < 0.0001,N = 5). Maximal facilitation of defensivage was induced by 5 ng of IL-2.


60 min (25%), declined over time, but remained significantlyabove baseline levels even at 180 min, post-injection [one-wayANOVA: F(3,116) = 4.08,p < 0.01]. A dose of 1 ng of IL-2 alsoinduced a significant facilitation of hissing (one-way ANOVA:[F(3,96) = 4.90,p < 0.01]. Doses of either 500 pg or saline hadlittle or no effect on response latencies for hissing (one-wayANOVAs for 500 ng and saline, respectively [F(3,116) = 1.79,p = 0.15, NS andF(3,96) = 0.54,p = 0.66, NS].

Since many experimental trials included pre-injections ofantibody/antagonist followed by IL-2 treatment, one experimentwas conducted to determine the effect of volume on responselatencies. For this experiment, the effects of 0.25�l of IL-2(5 ng) on response latencies were compared to response laten-cies following microinjections of 0.25�l saline + 0.25�l IL-2(5 ng). The results revealed no significant differences betweenthese two volumes at all blocks of time tested [F(1,3) = 1.35,p = 0.24].

3.4. IL-2 induced facilitative effects on defensive rage areblocked by pre-treatment with anti-IL-2 antibody

To identify the specificity of the effects of administrationof IL-2, response latencies following delivery of IL-2 alonewere compared to those following pre-treatment with an anti-IL-2 antibody. The results, shown inFig. 4, indicated that pre-treatment of the PAG site with an anti-IL-2 antibody completelyb ,p nti-b onsel

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Fig. 5. Effects of an anti-IL-2 receptor antibody (anti-IL-2R�) upon defensiverage. Pre-treatment with anti-IL-2 receptor antibody into the PAG completelyblocked the facilitating effects of IL-2 upon defensive rage elicited from themedial hypothalamus (p < 0.0001,N = 5). Administration of the anti-IL-2 recep-tor antibody alone had no effect upon defensive rage behavior elicited from themedial hypothalamus (p = 0.91, NS).

into the same PAG site 5 min prior to microinjections of IL-2completely blocked the facilitative effects of IL-2 upon medialhypothalamically elicited hissing [F(2,6) = 62.38,p < 0.0001].Microinjections of the anti-IL-2R� antibody alone did not sig-nificantly alter response latencies [F(3,96) = 0.17,p = 0.91].

3.6. IL-2 induced facilitation of defensive rage is blockedby pre-treatment with an NK1 receptor antagonist

To determine whether IL-2 induced facilitation of defensiverage is mediated through NK1 receptors, the same site in thePAG which received microinjections of IL-2 was pre-treatedwith the NK1 receptor antagonist, GR82334. The results, shownin Fig. 6, indicated that the NK1 receptor antagonist blocked thefacilitative effects induced by IL-2 [F(2,6) = 71.75,p < 0.0001].Microinjections of GR82334 alone had no effect upon responselatencies for hissing over time [F(3,96) = 0.095,p = 0.96].

F tmentw atinge otha-l neh thala-m

locked the facilitative effects of IL-2 on hissing [F(2,6) = 62.67< 0.0001]. The results further indicated that the anti-IL-2 aody administered alone had no significant effect on the resp

atency [F(3,96) = 0.76,p = 0.51].

.5. IL-2 induced facilitating effects on defensive rage arelocked by pre-treatment with anti-IL-2 receptor antibody

To further identify the specificity of the effects of deliveryL-2, response latencies following administration of IL-2 alere compared to those following pre-treatment with an

L-2 receptor antibody. As indicated inFig. 5, microinjectionsf an anti-IL-2 receptor antibody directed against the� chain

ig. 4. Effects of an anti-IL-2 monoclonal antibody upon defensive ragereatment of the PAG with an anti-IL-2 monoclonal antibody prior to micrections of IL-2 completely blocked the facilitating effects of IL-2 upon meypothalamically elicited defensive rage (p < 0.0001,N = 5).

ig. 6. Effects of NK1 receptor antagonist upon defensive rage. Pre-treaith the NK1 receptor antagonist, GR82334, completely blocked the facilitffects of IL-2 upon defensive rage behavior elicited from the medial hyp

amus (p < 0.0001,N = 5). Administration of the NK1 receptor antagonist aload no effect upon defensive rage behavior elicited from the medial hypous (p = 0.96, NS).


Fig. 7. Immunocytochemistry. Distribution of IL-2 receptors in the PAG. (A) Low power view of the PAG indicating the regions from which photomicrographs weretaken and shown in panel (B) of the dorsal PAG, depicting relatively intense labeling of IL-2 receptors, and panel (C) of the ventral PAG, depicting relatively sparselabeling.Abbreviations: AQ, cerebral aqueduct; CNIII, occulomotor nucleus; SCC, superior colliculus. Scale bar = 0.5�m, shown for panels B and C.

3.7. Microinjections of IL-2 into the PAG has no effect onpredatory attack behavior elicited from the lateralhypothalamus

This experiment was conducted to compare the effects ofIL-2 upon defensive rage against another form of aggression,namely predatory attack behavior. Specifically, we determinedthe effects of microinjections of IL-2 into the PAG on predatoryattack elicited from the lateral hypothalamus. The results of aone-way ANOVA, where 5 ng of IL-2 were microinjected intothe PAG (i.e., the dose that was maximally effective againstdefensive rage) indicated no significant changes in responselatencies for predatory attack elicited from the lateral hypotha-lamus over all time periods examined [F(3,36) = 0.58,p = 0.62].The percent changes in response latencies following IL-2 admin-istration were as follows: 30 min, 0.4%; 60 min, 2.23%; 120 min,2.66% and 180 min, 3.54%. The results of this experimentthus provide evidence that the facilitative effects of IL-2 upondefensive rage behavior elicited from the medial hypothala-mus are specific to this form of aggression and not to otherforms.

3.8. Immunocytochemical labeling of IL-2

Immunocytochemical analysis, shown inFig. 7, indicatedt uni-fA thev bel

ing was displayed relative to that observed in the dorsal PAG(Fig. 7C).

4. Discussion

The present study provides novel evidence for regional brainspecificity by the actions of IL-2 upon a form of aggressivebehavior. The principle finding of the study is that IL-2, microin-jected into the dorsal PAG, potentiated defensive rage behaviorin the cat in a dose-dependent manner. The findings that thefacilitative effects of IL-2 were significantly blocked by pre-treatment with either anti-IL-2 antibody or anti-IL-2 receptorantibody provide documentation of the specificity of the effectsof IL-2 upon defensive rage. Here, it is reasonable to assumethat the mechanism involves an interaction in which IL-2 actsthrough IL-2 receptors. Because administration of the anti-IL-2receptor antibody alone into the PAG had no effect upon defen-sive rage behavior, it is likely that such effects are phasic ratherthan tonic in nature.

Immunocytochemical labeling for IL-2 receptors in the PAGprovides further support for a functional role of IL-2 in thisregion. The pattern of IL-2 receptor labeling, observed in thepresent study, indicates that IL-2 receptors are densely dis-tributed in the dorsal part of the PAG, in contrast to the ventralpart of the PAG that displays sparse labeling of IL-2 receptors. Its e pri-m asso-c st pinal

hat IL-2 receptors are distributed widely and in a relativelyorm manner throughout the dorsal half of the PAG (Fig. 7B).lthough IL-2 receptors could also be detected withinentral aspect of the PAG, a much weaker pattern of la
-
hould be pointed out that the dorsal aspect of the PAG is thary receiving area for axons of the medial hypothalamus

iated with defensive rage behavior[20,40,59]. IL-2 can pashrough the blood–brain barrier, it is present in cerebros


fluid [17,44]; and it is also synthesized in neurons[27]; thus, itis also possible that the effects of IL-2 upon defensive rage maybe mediated through its release from T-cells in peripheral bloodin addition to its release from nerve terminals in the milieu ofthe PAG.

In the present study, the facilitative effects of IL-2 in thePAG are manifested via an NK1 receptor mechanism. Thisconclusion is supported by the present observation that NK1receptor-blockade in the PAG blocks the potentiating effects ofIL-2 upon defensive rage. This finding is consistent with pre-vious studies in our laboratory which have demonstrated thatactivation of NK1 receptors in the medial hypothalamus andPAG potentiates defensive rage, an effect that can be blocked bypre-treatment with an NK1 receptor antagonist[8,23].

At this point, it is only possible to speculate about possiblemechanisms underlying the nature of the interaction betweenIL-2 and NK1 receptors in potentiating defensive rage behav-ior. It should be noted that the relationship between IL-2 andNK1 receptors is established within the immune system; how-ever, very little is known about such possible interactions in thebrain. In the immune system, activation of substance P has beenshown to increase IL-2 receptor expression[56,57]. Regardingits role in the nervous system, one report has shown that whenIL-2 was injected into the hippocampus, it increased substanceP like reactivity in the PAG[78]. Collectively, these data sug-gest that IL-2 and NK receptors act in a synergistic manner,a press tionb ficr roin-j largeq ani us,t iatet

I ectso -15r hatI es.T thati td ls bytbd ratet cifict cil-i ctso torm

jec-t ofd effewr kinea ions

can differentially affect the same behavioral response. Whencomparing the effects of IL-2 administration into the PAG andmedial hypothalamus, it should be clearly noted that both regionsshare significant features relevant to the present study. Thefirst is that both regions constitute primary sites from whichdefensive rage behavior is elicited[67,68]and, as noted above,are intimately linked through reciprocal anatomical connections[19,20,61,67,68]. The second is that both regions contain sim-ilar receptor populations. These include, in part, the presenceof extensive quantities of NK1, GABAA, 5-HT1A, 5-HT2, IL-1and IL-2 receptors[8,9,23,29–31,62,64,66,79,80]. Accordingly,it would be difficult to attribute the differential effects observedwith administration of IL-2 to the presence or absence of NK1 orGABAA receptor populations in one region relative to the other.

There are several possible ways to account for this phe-nomenon. One possibility is that there is a difference in IL-2signaling in the PAG and medial hypothalamus. The IL-2 recep-tor is a multi-subunit cellular receptor consisting of three sub-units:�, � and� [45,72]. Various combinations of these receptorsubunits are known to occur, resulting in varying signal trans-duction capabilities. If the IL-2 receptor consists of all threesubunits, it is considered a high affinity signal transducing recep-tor [72]. In contrast, if it contains only the� and� subunits,little or no activity may occur[53]. Therefore, it is possible thatthe specific nature of the IL-2 receptor type distribution in thePAG or hypothalamus will govern which signaling pathway willb stud-i n oft ions.T g oft tiono pond

ed Krw tion[ ionsoI theN fectsu andN shipw IL-2a d, it isl ithG

uced neu-r pro-v onset veb ntd cleuso het s inn ei by

1nd that the activation of one receptor modulates the exion of the other in a positive manner. However, the interacetween IL-2 and NK1 receptors may be limited to speciegions of the brain, such as the PAG, since IL-2, when micected into the medial hypothalamus (which also containsuantities of NK1 receptors[79,80], has been shown to have

nhibitory effect upon defensive rage. Within the hypothalamhe suppressive effects of IL-2 upon defensive rage are medhrough a GABAA receptor mechanism[9].

Since the IL-2 receptor shares its� and� subunits with theL-15 receptor, one might alternatively argue that the efff IL-2 are mediated through a mechanism involving an ILeceptor[21,22,49]. However, recent findings have shown tL-15 functions in a manner that differs from other cytokinhe IL-15 receptor� subunit shows an expression pattern

nvolves cells that lack IL-2/IL-15R� and/or� subunits. Recenata suggest that IL-15 exerts its effect on opposing cel

he process oftrans-presentation in which the IL-15R� subunitinds to intracellular IL-15 and presents it to�� subunits onifferent cell types[58]. Since the present findings demonst

hat pre-treatment with an anti-IL-2 receptor antibody speo the�-subunit of the IL-2 receptor completely blocks the fatative effects of IL-2, it is not likely that the behavioral effef IL-2 can be attributed to a linkage with an IL-15 recepechanism.A striking feature of the present findings is that microin

ions of a dose of IL-2 into the PAG results in potentiationefensive rage, while the same dose has a suppressivehen administered into the medial hypothalamus[9]. In this

espect, it is the first series of reports showing that a cytodministered at a given dose into two different brain reg

-

d

ct

ecome activated. Our laboratory is presently conductinges directed at identifying the nature of the specific patterhe subunit expression of the IL-2 receptor in these two reghis would serve to provide new clues into our understandin

he underlying signaling pathways activated by the combinaf each of these subunits in relationship to their effects uefensive rage behavior.

A second possibility is that the NK1 receptor isoforms arifferentially expressed in the two regions of the brain. The N1eceptor has two isoforms, a short and a long isoform[38,43],here only the long isoform is capable of signal transduc

38]. In the human brain, it has been shown that different regf the brain express different isoforms of the NK1 receptor[11].

t is possible that the PAG contains only the long isoform ofK1 receptor and is thus capable of inducing potentiating efpon defensive rage related to the interaction between IL-2K1. One could presume that the same signaling relationould not be present in the medial hypothalamus wheredministration suppressed defensive rage behavior. Instea

ikely that a different mechanism linked to a relationship wABA receptors is present[9].The literature has provided evidence that IL-2 can ind

epressive, mixed or potentiating effects upon a variety ofal and behavioral responses. Depressive effects includeoked ptosis, social interaction, sexual activity and respo food reward[4,5,77]. Evidence of opposing effects haeen reported by Bindoni et al.[10], who described significaecreases in the neuronal activity in the ventromedial nuf the hypothalamus following administration of IL-2 into t

hird ventricle of the rat while observing marked increaseeuronal activity in the supraoptic and paraventricular nucl


this procedure. Excitatory effects of IL-2 have been describedby several investigators. These include studies by Pawlak et al.[48], who showed that increased IL-2 levels into the stratumresulted into enhanced plus-maze behavior in rats, and by Zal-cman[83], who observed that climbing behavior was increasedfollowing systemic administration of IL-2. The results of thepresent study are thus consistent with the findings demonstrat-ing excitation, suggesting that IL-2 can have a facilitating effecton specific forms of behavior, although such effects are likely todepend upon the signaling mechanisms present in regions whereinteractions of IL-2 occur.

The direct central effects of IL-2 upon defensive rage behav-ior demonstrated in the present study, however, do not precludean adaptive role for cytokines in influencing the expressionof aggressive behavior during an orchestrated or subclinicalimmune response[84]. Such a role for IL-2 responses maymanifest during its expression in either socially aggressive orsubmissive animals where these responses are influenced by psy-chological and physical stress[18,28] and genetic differences[51].

In conclusion, the present findings demonstrate that IL-2 inthe PAG selectively potentiates defensive rage behavior (but notpredatory attack) and that the effects of IL-2 upon a given behav-ioral process are regionally specific. The specificity of IL-2 maybe linked to a distinct interaction between IL-2 and NK1 recep-tors in the PAG.

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[ fen-

cknowledgements

The study was supported by NIH grant NS 07941-33 fhe National Institutes of Neurological Diseases and Strokeuthors want to thank Dr. Rekha Bhatt and Ms. Olga Ras

heir critical review of the manuscript and Mr. Markus Meyoffer for his excellent assistant in the histological preparaf the tissues.

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