Behavioural Brain Research 253 (2013) 54 59

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Behavioural Brain Research

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Research report

Freezin tr(TMT) utdeaffer

Luke W. y Ba Behavioral Ne SAb Department of Neurobiology, Evelyn F. McKnight Brain Institute, University of Alabama at Birmingham, Birmingham, AL, USA

h i g h l i g h t s

Rats were Subjects la Rats witho We conclu

a r t i c

Article history:Received 29 JaReceived in reAccepted 29 JuAvailable onlin

Keywords:Olfactory bulbTrigeminal neTMTButyric acidPredator odor

1. Introdu

Predatorlized to asseOdors derivshown to eincluding ament, and f

Corresponof Delaware, N

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0166-4328/$ http://dx.doi.oexposed to TMT following either olfactory bulb removal or trigeminal nerve transection.cking facial trigeminal innervation show normal fear behavior to TMT.ut olfactory bulbs fail to respond to TMT, though they can show fear to other stimuli.de that fear behavior to TMT is mediated by the olfactory systems.

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nuary 2013vised form 25 June 2013ne 2013e 4 July 2013

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a b s t r a c t

2,4,5 dihydro 2,5 trimethylthiazoline (TMT) is a synthesized component of red fox anal secretions thatreliably elicits defensive behaviors in rats and mice. TMT differs from other predator odors because itis a single molecule, it can be synthesized in large quantities, and the dose for exposure is highly con-trollable in an experimental setting. TMT has become a popular tool for studying the brain mechanismsthat mediate innate fear behavior to olfactory stimuli. However, this view of TMT as a biologically rele-vant olfactory stimulus has been challenged by suggestions that the odor elicits fear behavior due to itsirritating properties, presumably working through a nociceptive mechanism.

To address this criticism our lab measured freezing behavior in rats during exposures to 2 odors (TMTand butyric acid) and H2O (no odor control) following either surgical transection of the trigeminal nervesor ablation of the olfactory bulbs. Our ndings (Experiment 1) indicate that freezing behavior to TMTrequires an intact olfactory system, as indicated by the loss of freezing following olfactory bulb removal.Experiment 2 revealed that rats with trigeminal nerve transection freeze normally to TMT, suggestingthe olfactory system mediates this behavior to TMT. A replication of Experiment 1 that included con-textual fear conditioning revealed that the decreased freezing behavior was not due to an inability ofolfactory bulb ablated rats to freeze (Experiment 3). Taken together, these ndings support TMTs role asan ecologically relevant predator odor useful in experiments of unconditioned fear that is mediated viaolfaction and not nociception.

2013 Elsevier B.V. All rights reserved.

ction

s and predator odors have become increasingly uti-ss the neural mechanisms that mediate fear responses.ed from cats, ferrets, weasels, and foxes have been

licit a variety of innate defensive behaviors in rodents;voidance, reduction in locomotor activity, risk assess-reezing [2,8,15,21]. Until recently, most odors used in

ding author at: Department of Psychology, 108 Wolf Hall, Universityewark, DE 19716, USA. Tel.: +1 267 393 0150; fax: +1 302 831 3645.ress: layers@psych.udel.edu (L.W. Ayers).

these experiments have been obtained from fur, hair, saliva, urine,and feces samples. However, synthetic predator odors originallyisolated from bodily secretions can now be chemically synthe-sized for laboratory use. One odor in particular, 2,4,5 dihydro 2,5trimethylthiazoline (TMT), a volatile compound originally isolatedfrom the anal secretions of the red fox, induces robust freezingbehavior in rats [9,17,26,31,33] and mice [7,13,14]. In an experi-mental setting TMT offers advantages over natural predator odorsbecause it is a single molecule, its concentration can be preciselycontrolled, and it induces freezing in rats and mice in a dose-dependent manner [10,26,33].

Despite these advantages, TMT has received considerable crit-icism as to whether its behavioral effects are due to its olfactory

see front matter 2013 Elsevier B.V. All rights reserved.rg/10.1016/j.bbr.2013.06.034g to the predator odor 2,4,5 dihydro 2,5is disrupted by olfactory bulb removal bentation

Ayersa,, Arun Asoka, Frankie D. Heywarda,b, Jeffreuroscience Program, Department of Psychology, University of Delaware, Newark, DE, Um/locate /bbr

imethylthiazoline not trigeminal

. Rosena

L.W. Ayers et al. / Behavioural Brain Research 253 (2013) 54 59 55

properties. More specically, it has been postulated that TMTinduces fear behavior simply because it is a noxious, irritatingsubstance [2,4,11,23]. It is also possible that it has both olfactoryand noxious properties, since the olfactory system and the trigem-inal nocicepperception

Predatorthrough thting from tsecond origolfactory btion from tnose is relopthalamicbranch procavity, wheexternal sumeminal nethe mouth then projecto the trigparts of thebrainstem [

If TMT elfore requirefreezing bestimulus iting responsby selectiveor portionsjects on thebutyric acidan unpleasalus since rotrigeminal n

2. Methods

2.1. Subjects

A total of (Wilmington, iment subjectwere pair-houa 07001900 limental sessioAll proceduresfor the Care anof Delawares

2.2. Apparatu

All experimconducted in with metal griapart). Each bomination provfrom the chammitted the sigusing FreezeFtication of thsoftware.

2.3. Surgical p

2.3.1. OlfactorThe target

and accessoryinjection of astereotaxic surments, Tujung

was exposed, and a 5 mm2 window was drilled through the bone between 5and 10 mm anterior to Bregma. After clearing of the meninges a bilateral aspira-tion of the olfactory bulbs was performed using a Pasteur pipette (Fisherbrand)attached to a trap and vacuum pump. Following this aspiration sterile Gelfoamwas inserted into the cavity and the wound closed with VetBond. Subjects were

to heal for 7 days following surgery and their weights were consistentlyed. Shand ciniste.

ransec weeke and ia). The wass were

bone. The fed durd isolre the(Ethicn conhe sam.01 m

givenen ib

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lfactorwingbrain

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includ

rigemiwinge/xylaine. Val ner

and vwith as was tive system are often both involved in the qualia of odor[4,10].

odors are thought to induce behavioral responsese activation of two olfactory pathways: one projec-he nasal epithelium to the main olfactory bulb, and ainating from the vomeronasal organ to the accessoryulb [19,20,22,28]. Alternatively, nociceptive informa-he intranasal muscosa and the dorsum and tip of theayed via the frontal and ethmoidal branches of the

division of the trigeminal nerve [32]. The ethmodialvides central afferents for nociception from the nasalreas the frontal branch provides afferents from therface of the snout. The infraorbital branch of the trig-rve also is responsible for sensory information aroundregion and whiskers [32]. These trigeminal pathwayst from the nasal cavity and frontal part of the faceeminal sensory nucleus and subsequently innervate

subnucleus caudalis and subnucleus interpolaris in the1,32].icits fear responses through nociception it would there-

a functional trigeminal pathway in order to producehavior. Conversely, if TMT is operating as an olfactory

would require intact olfactory bulbs to elicit freez-es. The present study aimed to answer this questionly removing the main and accessory olfactory bulbs

of the facial trigeminal nerves and then testing sub-ir freezing behavior during exposure to H2O, TMT and

(BA). Butyric acid, a component of rancid butter withnt and acrid smell, was chosen as a comparison stimu-dents avoid the compound, suggesting it stimulates theerve [9,13,33].

60 male Sprague-Dawley rats obtained from Charles River breedersMA) were used in the following studies. At the start of each exper-s were 90120 days old and weighed between 300350 g. All ratssed in opaque shoebox cages (20 cm 46 cm 23 cm), maintained onight/dark cycle, and given access to food and water ad libitum. Exper-ns were conducted during the light phase, between 0800 and 1700 h.

were in accordance with the US National Institutes of Health Guided Use of Experimental Animals and were approved by the UniversityIACUC.

s

ents measuring freezing behavior during odorant presentation werefour rectangular Plexiglas chambers (16.5 cm 12.1 cm 21.6 cm)d ooring (nine stainless steel bars 4 mm in diameter, spaced 1.0 cmx was positioned on a Plexiglas frame inside a fume hood with illu-ided by an overhead light. A camera positioned approximately 2 ftbers simultaneously recorded activity in each chamber and trans-nal to a nearby Dell Computer. Freezing behavior was recorded

rame (Actimetrics Software) set to 4 chamber/1 mode and quan-e behavior utilized the partner FreezeView (Actimetrics Software)

rocedures

y bulb removal of the ablation procedure was complete removal of both the main

olfactory bulbs. Each rat was anesthetized via an intraperitoneal ketamine/xylazine cocktail (87 mg/13 mg/kg) and positioned in agical apparatus to ensure stability during the operation (Kopf Instru-a, California). Incisions were made in the scalp, the skull surface

allowedmonitorsurface, was adminfection

2.3.2. TOne

as abovCalifornnal nervincisionfrom thelaterallytransectreach anthese wesutures conditioskin in tphine (0s.c.) waswere givdays.

2.4. Beh

Odoto all exper day two piecprior to immediaants and(300 meach strchosen bSince TMwas use

In Ex1-shockjects we1 s, 1.5 mof freeziwere retbutyric a

Follochambenature oto ensurbehavio[3]. In althresholwas anausing SP

2.5. His

2.5.1. OFollo

and the stored invisually ness of ablation

2.5.2. TFollo

ketamin0.9% saltrigeminthe orbitsubject brancheam surgeries consisted of making scalp incisions, exposing the skulllosing the wound with VetBond. Buprenorphine (0.01 mg/kg, s.c.)red to manage pain and baytril (5.0 mg/kg, sic) was given to inhibit

tion of the trigeminal nerve following arrival in the animal colony each rat was anesthetizedpositioned in a stereotaxic apparatus (Kopf Instruments, Tujunga,e surgical procedure for the transection of the infraorbital trigemi-

adapted from Kernisant et al. [18]. Bilateral 7 mm anteriorposterior made 2 mm above each eye. Fascia and muscle were moved away

using a periosteal elevator and the contents of the orbit retractedrontal branch of the ophthalmic branch of the trigeminal nerve wasing this procedure. Once retracted, a small surgical hook was used toate the infraorbital and ethmoidal branches of the trigeminal nerve;n transected with a scissors. The wound was then sutured with Vicrylon, Inc.) and allowed to heal for 5 days. The procedure for the shamsisted of making the incisions, exposing the orbit, and suturing the

e manner as was done for the transection of the nerve. Buprenor-g/kg, s.c.) was administered to manage pain and baytril (5.0 mg/kg,

to inhibit infection. To reduce post-surgery inammation all subjectsuprofen (0.2 mg/mL) in their drinking water during the 5 recovery

l protocols

sure was conducted in an identical manner for all experiments. Priores subjects were acclimated to the testing environment for 10 mino days. Odor exposure was performed by adding liquid odorant tolter paper (2 cm2) afxed to the left and right test chamber wallsg the subject into the chamber. Recording of freezing behavior begannd continued for a total of 10 min per exposure session. The odor-entrations, and volume applied per exposure were as follows: TMT9.4 l on each strip of paper), Butyric Acid (900 mole, 39.6 l onaper), and H2O (30 l on each strip of paper). 300 mole of TMT wase this amount produces asymptotic levels of freezing behavior [33].ree times as volatile as butyric acid, three times as much butyric acidoduce equivalent levels of TMT and butyric acid exposure [17].ent 3 evaluation of shock-induced freezing was conducted through axtual fear conditioning design. One day following TMT exposure sub-wed to acclimate in the testing chambers for 3 min before receiving aambled foot shock delivered through the metal grid oor. Recordinghavior continued for 4 min following shock delivery before subjects

to their home cages. Subjects in Experiment 3 were not exposure to water.

each test, subjects were returned to their home cage and the teste cleaned with a 5% ammonium hydroxide solution. Due to the volatiledorants a minimum interval of 15 min occurred between each sessionilation of the chambers and testing room. For all experiments, freezingdened as the cessation of all bodily movement except for breathingses freezing bout length was set to a minimum of 0.75 s and freezing

set based upon the instructions of the FreezeFrame software. Behaviorwith mix-model analysis of variance and Bonferroni corrected t-teststware (version 16).

y bulb removal all behavioral tests subjects were euthanized via rapid decapitation

was harvested, ash frozen in dry ice chilled 2-methylbutane, and0 C freezer. Evaluation of the olfactory bulb lesion was performedotographing each subjects brain and inspecting them for complete-moval. Subjects with incomplete olfactory bulb removal or whoseed frontal cortical tissue were excluded for the analyses.

nal transection all testing procedures all subjects were overdosed with azine cocktail (87 mg/13 mg/kg, i.p.) and perfused intracardially witherication of transected ethmoidal and infraorbital branches of theve was performed visually by removing the eye, skin and tissue aroundisually inspecting whether the nerve was cut or remained intact. Anyn intact or partial transection of infraorbital and ethmoidal nerveeliminated from the data pool.

56 L.W. Ayers et al. / Behavioural Brain Research 253 (2013) 54 59

Fig. 1. Representative images of brains from sham (top) and olfactory bulb aspira-tion (bottom) procedures. The black arrow indicates an intact olfactory bulb; thegray indicates the site of a successful ablation.

3. Results

3.1. Experiment 1: Ablation of the olfactory bulbs reducesfreezing to TMT

Post-mortem examination of the olfactory bulb lesions revealedsuccessful bilateral aspiration of the olfactory bulbs in 6 of 9 sub-jects. Subjects with incomplete aspiration of the olfactory bulbswere remoremained, 6depictions o

Subjects(BA) in indmodel Anasurgery conicant main of conditioncondition in

Bonferrolevel of odo

Fig. 2. Freezing to TMT is reduced following ablation of the olfactory bulbs. Removalof the olfactory bulbs signicantly reduces freezing behavior during a 10 min expo-sure to TMT. Freezing to H2O and butyric acid are unaffected.

analyses revealed that Sham rats froze more to TMT than to H2O(p < 0.001) and BA (p < 0.001), but freezing to BA and H2O did notdiffer from each other. Freezing to TMT in olfactory bulb lesionedrats (OB-lesion) was signicantly reduced compared to Sham rats(p < 0.001), but freezing to H2O and BA did not differ between OB-lesion and Sham rats.

perimg to T

t-mosful es in

the8 su

andcted jectsnden

Fig. 3. Represtrigeminal nera transected suved from the analyses so that a total of 15 subjects OB-lesioned and 9 shams. See Fig. 1 for representativef Sham and OB-lesioned brains.

were tested on freezing to H2O, TMT and Butyric Acidependent 10 min exposure sessions (Fig. 2). A mixedlysis of Variance comparing odor (H2O, TMT, BA) andditions (Olfactory bulb lesion and Sham) reveal a signif-effect of odor (F1,13 = 28.01; p < 0.001), no main effect

(F1,13 = 2.55; n.s.), yet a signicant odor by surgeryteraction effect (F1,13 = 21.81; p < 0.001).ni corrected t-tests were then used to examine eachr both within and between the surgery conditions. These

3.2. Exfreezin

Possuccesbranchtion oftotal, 2sectiontranse

Subindepeentative image of the trigeminal branches cut. (Left) Sample image showing the intact inve in a sham subject. (Right) Sample image showing the loss of the infraorbital (black arbject.ent 2: Trigeminal nerve transection does not affectMT

rtem verication of the trigeminal nerve lesion revealedbilateral transection of the infraorbital and ethmoidal

14 of 20 subjects. Subjects without complete transec- trigeminal nerve were removed from all analyses. Inbjects were used in the analyses, 14 trigeminal tran-

14 shams. Fig. 3 shows examples of the intact andnerves observed during the visual inspection process.

were tested on freezing to H2O, TMT, and BA int 10 min exposure sessions (Fig. 4). A mixed modelfraorbital (black arrow) and ethmoidal (gray arrow) branches of therow) and ethmoidal (gray arrow) branches of the trigeminal nerve in

L.W. Ayers et al. / Behavioural Brain Research 253 (2013) 54 59 57

Fig. 4. Freezinthe trigeminal

analysis of conditions cant main emain effectnicant intp < 0.05).

Bonferrotrigeminal (both p < 0.0inal transecTMT or H2Oicantly morand also siganalyses revon freezinging behavio

3.3. Experimbulb remova

The reduto questionaltered a sudisplay incrLeonard [2ing behaviohypothesis subjects lacfollowing a conditionin

Olfactorwith 2 sucses. A totawere testedand followiVariance codition (olfamain effect(F1,4 = 1.122(F1,4 = 31.91olfactory buto TMT (p