Ch

CAa

Bb

B

a

ARR2AA

KGRNNHT

1

fidawelmMt(h

pPf

0h

Neuroscience Research 77 (2013) 187–201

Contents lists available at ScienceDirect

Neuroscience Research

jo ur nal homepage: www.elsev ier .com/ locate /neures

ontext-dependent differences in grooming behavior among the NIHeterogeneous stock and the Roman high- and low-avoidance rats

. Estanislaua,b,∗, S. Díaz-Morána, T. Canetea, G. Blázqueza, A. Tobenaa,

. Fernández-Teruela

Unitat de Psicologia Mèdica, Departament de Psiquiatria i de Medicina Legal, Institut de Neurociències, Universitat Autònoma de Barcelona, Bellaterra,arcelona, SpainGrupo de Pesquisa em Psicobiologia, Departamento de Psicologia Geral e Análise do Comportamento, Universidade Estadual de Londrina, Londrina, PR,razil1

r t i c l e i n f o

rticle history:eceived 26 July 2013eceived in revised form5 September 2013ccepted 30 September 2013vailable online 11 October 2013

eywords:rooming

a b s t r a c t

Grooming occurs during/after stress and seems to accompany dearousal. Here, grooming was investigatedunder testing situations involving different levels of aversiveness, taking advantage of differences amongthree rat strains in fearfulness/anxiety. Inbred Roman High Avoidance (RHA-I) rats are less anxious/fearfulthan inbred Roman Low Avoidance (RLA-I). The outbred genetically heterogeneous stock of rats (NIH-HS),which resembles the RLA-I in many behavioral traits, was also studied. Adult male rats (RLA-I: n = 9, RHA-I: n = 10, NIH-HS: n = 12) were observed for 30 min in: a novel open-field, a novel hole-board and in thehome-cage. They were also observed during two-way active avoidance training. Differences in groomingdepended on test situation: (a) No differences were found in the home-cage. (b) While tested in a novel

oman strainsIH heterogeneous rat stockoveltyome-cagewo-way active avoidance

environment, RHA-I showed less grooming activity than the other rats. (c) After avoidance responsesappeared, differences among the strains were opposite to the observed in novelty tests. Furthermore,results suggest that (i) grooming is mostly suppressed when assured aversive experience is under way; (ii)rostral grooming prevails when experience with aversive stimuli is unpredictable (novelty) or potential(avoidance training); (iii) body grooming increases for a period in novel environments. In general, our

ming13 El

results support that groo© 20

. Introduction

Self-grooming behavior has the essential function of caringor and protecting the surface of the body, but its occurrences influenced by emotional factors. Indeed, grooming is observeduring (and following) exposure to different types of stressful situ-tions/stimuli (Spruijt et al., 1992; van Erp et al., 1994). Consistentith that, small doses of adrenocorticotropic hormone (Gispen

t al., 1975) and other agents are able to induce grooming (pro-actin: Drago et al., 1980; endorphins: De Wied and Jolles, 1982;

elanocyte-stimulating hormone: Spruijt et al., 1985; vasopressin:eisenberg, 1988). These observations have led to the suggestion

hat grooming is a behavioral expression of a dearousal processSpruijt et al., 1992). In this connection, several recent studiesave suggested the putative utility of some grooming measures

∗ Corresponding author at: Departamento de Psicologia Geral e Análise do Com-ortamento, Centro de Ciências Biológicas, Universidade Estadual de Londrina,R445, Km 380, 86051-990 Londrina, PR, Brazil. Tel.: +55 4333714261;ax: +55 4333714227.

E-mail addresses: estanislau@uel.br, celio.estanislau@gmail.com (C. Estanislau).1 Permanent address.

168-0102/$ – see front matter © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Sttp://dx.doi.org/10.1016/j.neures.2013.09.012

takes place during dearousal.sevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

for the evaluation of stress or anxiety in rodents (see, for example:Komorowska and Pisula, 2003; Steimer and Driscoll, 2003; Kalueffand Tuohimaa, 2005a,b; Estanislau, 2012).

Opposite grooming amounts can be seen in two rat lines/strains,the Roman High-avoidance (RHA) displaying much less groomingthan the Roman Low-Avoidance (RLA) rats when exposed to a vari-ety of anxiety/fearfulness tests involving novelty, such as timidity(measured in a novel cage) tests, different types of open-field tests,the elevated plus-maze test, the hole-board test, and others (e.g.Ferré et al., 1995; Steimer et al., 1997, 1998; Escorihuela et al., 1999;Steimer and Driscoll, 2003; Guitart-Masip et al., 2006). Importantly,these lines/strains, psychogenetically selected for rapid (RHA) vsextremely poor (RLA) acquisition of the two-way active avoidanceresponse (e.g. Escorihuela et al., 1999; Steimer and Driscoll, 2003;Driscoll et al., 2009), also present opposite profiles in unconditionedand conditioned anxiety/fearfulness tests, as well as in tests of frus-tration and in stress hormone reactivity, the RLA being much moreanxious/fearful, frustration-prone and stress-prone than the RHA

(e.g. Steimer et al., 1997, 1998; Escorihuela et al., 1999; Steimerand Driscoll, 2003; Rosas et al., 2007; Carrasco et al., 2008; Driscollet al., 2009; López-Aumatell et al., 2009a,b; Cuenya et al., 2012;Díaz-Morán et al., 2012, 2013).

ociety. All rights reserved.

1 ence R

acobEbiia

buiFgewFwtotsbrt2tHirrto

2

2

cwNss

tt(WsaoaaasC

e(a((

88 C. Estanislau et al. / Neurosci

Rodent grooming behavior is a heterogeneous collection ofctivities that in some situations tends to be organized in cephalo-audal sequence (Berridge et al., 1987) and, depending on the typef test condition, is differentially devoted to different parts of theody (regional distribution) and follows specific time courses (vanrp et al., 1994). Accordingly, recent claims that grooming coulde useful in the evaluation of stress/anxiety have emphasized that

t can be much more heuristic if the researcher takes into accountts sequencing, regional distribution and time course (Komorowskand Pisula, 2003; Kalueff and Tuohimaa, 2005a; Estanislau, 2012).

As the differences in anxiety/fearfulness and stress responsesetween the Roman strains are well known, these strains weresed in the present study for an evaluation of how grooming activ-

ty is able to distinguish high versus low anxious/stress profiles.or comparison purposes, a third strain was studied, the outbredenetically heterogeneous stock of rats (i.e. NIH-HS rats), originallystablished in the National Institutes of Health. More specifically,e have used the heterogeneous NIH-HS rats for several reasons.

irst, they were derived from eight inbred strains, at least three ofhich originated from Wistar stocks (see “Section 2.1” below), and

he RHA-I/RLA-I rat strains were also derived from Wistar. Second,ur previous characterization of the NIH-HS rat stock has revealedhat these heterogeneous rats show anxiety responses and a (pas-ive) coping style which are very similar to the profiles showny the RLA-I strain, although in some fear measures (e.g. startleesponses) the profiles of NIH-HS rats are intermediate betweenhose of RLA-I and RHA-I rats (e.g. López-Aumatell et al., 2008,009b; Díaz-Morán et al., 2012, 2013). Third, while genetic drift inhe inbred (Roman) strains might influence phenotypes, the NIH-S stock is the most (genetically) heterogeneous laboratory rat

n existence (see review by Díaz-Morán et al., 2013), and for thiseason it is to be expected that the phenotypic (behavioral and neu-obiological) results obtained with the NIH-HS rat stock could leado more generalizable conclusions than the results obtained withther specific rat strains.

. Methods

.1. Subjects

Adult male rats from three strains/stocks of our permanentolonies maintained at the Autonomous University of Barcelonaere used: 9 from the RLA-I, 10 from the RHA-I, and 12 from theIH-HS strains/stock. The rats from these groups were randomly

elected from the 20 breeding families of each the RHA-I and RLA-Itrains, and from the 40 breeding families of NIH-HS.

The genetically heterogeneous NIH-HS rat stock (“National Insti-utes of Health Genetically Heterogeneous Rat Stock”) was formedhrough an eight-way cross among the following 8 inbred rat strainsHansen and Spuhler, 1984): the MR/N (Maudsley Reactive), the

N/N (Wistar Nettleship) and WKY/N (Wistar Kyoto; these threetrains trace their ancestry to the original Wistar stock), the M520/Nnd F344/N (Fischer 344; both strains established in the 1920s, butf unknown origin), the ACI/N (Agouti; hybrid between the Augustnd Copenhagen strains), the BN/SsN (Brown Norway; derived from

color mutant from a stock of wild rats kept at the Wistar Institute)nd the BUF/N (Buffalo) strain. The progenitors of our NIH-HS rattock were kindly provided by Dr. Eva Redei in 2004 (Center foromparative Medicine, Northwestern University, Chicago, USA).

The rats were 2-months old at the beginning of thexperiment. They were housed in pairs in macrolon cages

50 cm × 25 cm × 14 cm), maintained with food and tap watervailable ad libitum, under conditions of controlled temperature22 ± 2◦ C; 50–70% humidity) and a 12-h light/12-h dark cyclelights on at 08:00 h). All the procedures were in accordance

esearch 77 (2013) 187–201

with the Spanish legislation on “Protection of Animals Used forExperimental and Other Scientific Purposes” and the EuropeanCommunities Council Directive (86/609/EEC) on this subject.

2.2. Equipment

Open-field: The apparatus was a beige circular arena (diameter,83 cm) enclosed by white walls (height, 34 cm) and divided into 19equal sectors by lines drawn on the floor.

Hole-board: Made of white wood (66 cm × 66 cm × 47 cm) andcontaining four equidistant holes (diameter: 3.7 cm) on the floor.For purposes of recording locomotion, the apparatus was dividedinto four equal quadrants. A novel object (rubber sphere, diameter:3.5 cm) was placed in a container bellow each hole.

Two-way active avoidance: A shuttle-box (Letica, Panlab,Barcelona, Spain) was used. It consists of two equally sizedcompartments (25 cm × 25 cm × 28 cm), connected by an opening(8 cm × 10 cm). A 2400-Hz, 63-dB tone plus a light (from a small,7-W lamp) functioned as the conditioned stimuli (CS). The uncon-ditioned stimulus (US), which commenced at the end of the CS, wasa scrambled electric shock of 0.7 mA delivered through a grid floor.The delivery of CS and US was controlled by a computer.

Open-field and hole-board tests were conducted in a black roomwhich was illuminated with white fluorescent lights. Previously tothe session, the apparatus was always cleaned with paper tow-els and an ethanol solution (5%). The home-cage activity recordingwas performed in the same room (see procedures below). Anotherroom was used for the shuttle-box sessions; the apparatus was illu-minated by a 60-W bulb placed one meter in front of it. A highresolution video-camera placed one meter in front of the shuttle-box, or two meters above the other apparatuses, was used for allbehavioral recordings.

2.3. Procedure

Behavioral tests were performed in the sequence below. At leastone week elapsed between two tests, with the exception of the twosessions of two-way active avoidance training, which were sep-arated by approximately 24 h. All tests were performed between2:00 PM and 6:00 PM, with the exception of the two-way activeavoidance sessions, which occurred between 9:00 AM and 6:00 PM.

Open-field: Each rat was gently placed near the wall and allowedto freely explore the apparatus for 30 min. The following behav-iors were scored: (1) Crossings: recorded whenever the rat steppedthe fourth paw into a sector. (2) Time in the center area: recordedwhen the rat was in the areas which were not in contact withthe wall. (3) Rearing: recorded (frequency and duration) when therat suspended its forepaws (rearings were discriminated betweenwall-rearing and free-rearing, i.e. whether or not the animaltouched the wall with the forepaws). (4) Stretched attend posture(SAP): recorded (frequency and duration) when the rat exhibitedelongation of the body maintaining the hind paws fixed. In addi-tion to these behaviors, grooming activities were recorded (see thescoring procedure below).

Hole-board: Each rat was gently placed near the wall and allowedto freely explore the apparatus for 30 min. Crossings, rearings, SAPs,and grooming were recorded as for the open-field test. Additionally,head dipping (frequency and duration) was recorded when a ratinserted the head, at least to the level of the eyes, into a hole.

Home-cage activity: For the investigation of the behavior inthe home-cage, each home-cage containing each pair of rats fromthe same strain was placed in the observation room at least one

hour before recording began and no challenge (i.e. environmentalchange) was then presented. In order to allow a complete view ofthe cage, the water bottle and the chow were removed from thegrid cover, some pellets being put inside the cage. At the end of the

ence R

ovi(aiclwat(otsosap

itlcba(doeetEfttseti

potltFdiogowpstugteA(ocrb

C. Estanislau et al. / Neurosci

ne-hour period, the room door was open for some seconds and theideo-camera was turned on by means of a remote control. Behav-oral recording lasted 30 min. The following behaviors were scored:1) Crossings: the cage floor was divided into four equal sectors andn entry was recorded whenever the rat stepped the fourth pawnto one of them. (2) Eating duration: recorded if the mouth was inontact or forepaws were grasping a food pellet. (3) Time motion-ess: recorded when the animal was in a relaxed still posture, but

ith open eyes. An animal was assumed as sleeping if motionlessnd with closed eyes. Those which slept were observed longer soo ensure the completion of a 30-min recording of awake activity.4) Allogrooming duration: recorded when a rat exhibited frictionf the mouth against the skin/fur of its cage partner. In additiono these behaviors, self-grooming activities were recorded (see thecoring procedure below). Each rat seemed to follow cyclic patternsf activity/inactivity of variable durations; however, there was noynchrony among the animals in these cycles. For these reasons nottempt of time course investigation was tried and only the wholeeriod scores were compared among the groups.

Two-way active avoidance acquisition: Once the rat was placednto the shuttle box, a 4-min familiarization period elapsed beforeraining commenced. Each training trial consisted of a 10-s CS, fol-owed by a 20-s US. The CS or US was terminated when the animalrossed to the other compartment, with crossing during the CSeing considered as an avoidance response and during the US asn escape response. Once a crossing had been made or the shockUS) discontinued, a 60-s inter-trial interval (ITI) was presenteduring which crossings (ITC) were also scored. Training consistedf two 50-trial sessions. The achievement of three consecutivescapes with latency shorter than 1 s was used as the criterion forscape acquisition. Similarly, the achievement of three consecu-ive avoidances was used as the criterion for avoidance acquisition.scape and avoidance acquisitions were assumed as key momentsor changes in arousal. Therefore, observations of grooming duringhe two-way active avoidance training were conducted focusing onhree different phases: (1) the final 10-min period before the acqui-ition of the escape response; (2) the first 10 min period when thescape response was established; (3) the first 20 min period whenhe avoidance response was established. These data were convertedn score/min values.

Grooming scoring: Previous descriptions of grooming scoringrocedures (Berridge et al., 1987, 2005) were used as a basis forur modified procedure. Grooming activity was classified into threeypes of components. (1) Fore paw/nose grooming: When the raticks the forepaws or performs rhythmic friction movements ofhese paws with the mouth and nose region. (2) Head grooming:riction movements in the form of unilateral or bilateral strokesirected to the ears region. A pattern of rhythmic alternation typ-

cally involving one stroke directed to the ears region followed byne-two directed to the rostral region was also recorded as “headrooming”. (3) Body grooming: Recorded when oral friction, licksr bites directed to the trunk, rear paws, anogenital region or tailere observed. It is characterized by rhythmic movements com-osed of a larger movement followed by 2–3 smaller ones. Rear pawcratching was also included in the ‘body grooming’ category. Theransitions between the different elements were recorded and eval-ated whether followed a strictly cephalocaudal sequence, i.e. norooming → fore paws/nose → head → body → no grooming. Anyransitions different from this (even the skipping of components;.g. fore paws/nose → body) were recorded as ‘noncephalocaudal’.

bout was assigned as interrupted if provided of one or more shortup to 5 s) pauses between two presentations of the same element

r between two different elements. Intervals greater than 5 s wereonsidered to separate two different bouts. A stereotyped chain wasecorded whenever 6–7-Hz elliptical rostral strokes were followedy a short series of slower strokes directed to the head and then

esearch 77 (2013) 187–201 189

by licking of the ventrolateral body surface (see detailed descrip-tions of the stereotyped chains at Berridge et al., 1987, 2005).Comparisons were performed in respect to the number of bouts,total duration of grooming, duration of the rostral (fore paws/noseplus head) and the body grooming components, the number oftransitions between elements, the number of stereotyped chains,the percentage of noncephalocaudal transitions, mean number ofinterruptions per minute of grooming, and the percentage of inter-rupted bouts. These three later measures were not used for studyinggrooming in the two-way active avoidance training, since the pre-sentation of the CS and the US clearly leaded to interruptions orsuspension of grooming, thus interfering in these measures.

2.4. Data analyses

For the open-field and hole-board data, behavioral changesalong the 30-min sessions were explored by dividing them in six5-min blocks. The three groups were compared by means of two-way analyses of variance (ANOVA) for repeated measures with thestrain as one factor (three levels: RHA-I, RLA-I and NIH-HS) and thetime block in the session as the repeated measure factor (six levels:1–5, 6–10, 11–15, 16–20, 21–25 and 26–30 min time blocks).

In some cases, in which a clear a priori hypothesis (based onresults of previous studies) of difference between the Roman strainsdeserved evaluation, Student t-tests for unpaired samples wereapplied.

Home-cage activity data were studied by comparing the threestrains by means of one-way ANOVA. The same was applied tothe two-way active avoidance training. In addition, the number ofavoidances per block of 10 trials was analyzed by means of two-way ANOVAs for repeated measures with the strain as one factor(three levels: RHA-I, RLA-I and NIH-HS) and the 10-trial blocks inthe session as the repeated measure factor (ten levels: blocks 1–10).

Whenever necessary, the Duncan’s post hoc test was used. In allcases, the significance level was set at p < 0.05.

3. Results

3.1. Open-field, hole-board and home-cage tests

3.1.1. Non-grooming behaviorsNon-grooming behaviors measures recorded in the open-field

are shown in Fig. 1 and Table 1. Crossings performed during the testshowed main effects of the strain (F[2,28] = 6.31, p < 0.001) and thetime block (F[5,140] = 45.73, p < 0.001). Post hoc comparisons showedthat RHA-I rats made more crossings during the 1–5 block than boththe other groups. Additionally, the NIH-HS rats made less crossingsthan both the other strains during the last 20 min. The significanteffect of time block and the absence of interaction with strain, indi-cate significant habituation across time blocks as well as similarhabituation patterns for the three groups (Fig. 1).

Concerning “crossings” results in the hole-board test (Table 2),they were similar to those from the open field test. Thus, the ANOVAapplied to “crossings” showed main effects of the strain and thetime block, as well as an interaction between both factors (seeANOVAs in Table 2). Post hoc comparisons showed that the RHA-Imade significantly more crossings than the NIH-HS group dur-ing the 1st block (there was also a nonsignificant trend – p = 0.10,Duncan’s test – between RHA-I and RLA-I rats in the same block).Habituation (i.e. decrease) of crossings across time blocks was very

pronounced in the three groups (thus the “time block” effect), butit was relatively more marked in RHA-I rats (i.e. from a mean valueof 23.4 crossings in the first to 9.4 in the last time block), which ledto a significant “strain x time block” effect (Table 2).

190

C. Estanislau

et al.

/ N

euroscience R

esearch 77

(2013) 187–201

Table 1Some non-grooming behavioral measures obtained in the open-field test and their corresponding two-way ANOVA for repeated measures results.

Measure Strain Mean ± SEM 2-Way ANOVA for repeated measures

Time block (min) Strain Time block Interaction

1–5 6–10 11–15 16–20 21–25 26–30 F[2,28] p F[5,140] p F[10,140] p

Time spent in thecenter (s)

NIH-HS 11.1 ± 7.2 19.6 ± 9.3 4.7 ± 3.0 6.9 ± 2.5 4.1 ± 2.5 4.7 ± 3.0 1.06 ns 2.10 ns 1.03 nsRLA 9.1 ± 2.2 23.1 ± 8.6 10.4 ± 6.4 16.4 ± 6.9 12.0 ± 5.4 10.4 ± 6.4RHA 12.8 ± 4.6 6.8 ± 1.9 8.0 ± 3.6 7.7 ± 2.8 4.0 ± 1.5 8.0 ± 3.6

Wall-rearing duration (s) NIH-HS 13.4 ± 2.0** 13.4 ± 2.8*,** 11.3 ± 3.1*,** 8.5 ± 2.6*,** 5.0 ± 1.3*,** 4.9 ± 2.1*,** 17.87 <0.01 2.47 <0.05 1.87 nsRLA 29.4 ± 3.5* 31.4 ± 4.2 37.9 ± 5.1 39.3 ± 8.4* 24.9 ± 6.3 41.3 ± 6.2RHA 14.9 ± 2.1 27.1 ± 4.0 26.6 ± 4.2 22.8 ± 4.1 21.9 ± 4.1 30.3 ± 10.2

Free-rearing frequency NIH-HS 5.3 ± 1.3 6.3 ± 2.0 2.6 ± 1.0 5.5 ± 1.5 2.3 ± 0.7 2.6 ± 1.0 0.52 ns 4.88 <0.01 0.61 nsRLA 5.9 ± 2.1 7.4 ± 1.0 5.2 ± 1.8 5.8 ± 2.1 3.9 ± 0.9 5.2 ± 1.8RHA 7.0 ± 1.8 10.1 ± 3.2 5.6 ± 1.9 4.3 ± 1.5 5.8 ± 1.9 5.6 ± 1.9

Free-rearing duration (s) NIH-HS 3.7 ± 0.9 8.0 ± 3.1 5.1 ± 2.2 11.0 ± 4.8 6.1 ± 2.4 5.1 ± 2.2 0.22 ns 3.70 <0.01 0.99 nsRLA 3.5 ± 1.2 5.6 ± 0.7 4.6 ± 1.5 6.0 ± 2.5 4.8 ± 2.2 4.6 ± 1.5RHA 3.6 ± 0.9 9.9 ± 3.8 6.2 ± 2.7 3.0 ± 1.1 8.2 ± 3.0 6.2 ± 2.7

SAP frequency NIH-HS 4.2 ± 0.9 2.5 ± 0.8 1.2 ± 0.5 1.5 ± 0.6 0.8 ± 0.3 1.2 ± 0.5 1.32 ns 23.91 <0.01 0.89 nsRLA 5.7 ± 1.1 3.1 ± 1.1 0.3 ± 0.2 0.9 ± 0.4 0.7 ± 0.4 0.3 ± 0.2RHA 6.9 ± 1.5 3.6 ± 1.1 1.2 ± 0.4 1.4 ± 0.5 2.1 ± 0.7 1.2 ± 0.4

SAP duration (s) NIH-HS 5.5 ± 1.9 2.0 ± 0.6 1.5 ± 0.6 2.1 ± 1.0 1.4 ± 0.6 1.5 ± 0.6 0.00 ns 11.05 <0.01 0.59 nsRLA 6.7 ± 2.0 3.9 ± 2.0 0.4 ± 0.2 1.2 ± 0.6 1.1 ± 0.9 0.4 ± 0.2RHA 5.1 ± 1.4 2.7 ± 1.0 1.1 ± 0.4 2.0 ± 0.6 1.8 ± 0.7 1.1 ± 0.4

ns, non-significant.* p < 0.05 as compared with the RHA during the same time block (Duncan).

** p < 0.05 as compared with the RLA during the same time block (Duncan).

C. Estanislau

et al.

/ N

euroscience R

esearch 77

(2013) 187–201

191

Table 2Behavioral measures obtained in the hole-board test and their corresponding two-way ANOVA for repeated measures results.

Measure Strain Mean ± SEM 2-Way ANOVA for repeated measures

Time block (min) Strain Time block Interaction

1–5 6–10 11–15 16–20 21–25 26–30 F[2,28] p F[5,140] p F[10,140] p

Crossings NIH-HS 8.5 ± 1.7** 8.7 ± 2.3 5.0 ± 1.9 1.9 ± 0.5 0.8 ± 0.5 0.4 ± 0.3 10.36 <0.01 34.75 <0.01 2.08 <0.05RLA-I 13.9 ± 2.0 14.6 ± 2.4 10.9 ± 2.8 8.3 ± 2.4 6.6 ± 1.9 5.4 ± 1.9RHA-I 23.4 ± 2.2 17.2 ± 2.5 12.7 ± 2.6 7.8 ± 1.8 9.1 ± 1.5 9.4 ± 1.6

Dipping frequency NIH-HS 4.2 ± 1.2** 4.5 ± 1.5 3.0 ± 1.2 1.2 ± 0.4 0.7 ± 0.4 0.3 ± 0.2 7.38 <0.01 11.90 <0.01 1.08 nsRLA-I 6.3 ± 1.0 6.8 ± 0.7 3.9 ± 1.0 3.2 ± 1.0 2.2 ± 0.7 1.2 ± 0.5RHA-I 10.2 ± 1.6* 6.1 ± 1.5 6.1 ± 2.2 2.5 ± 0.9 2.7 ± 0.7 4.5 ± 1.5

Dipping duration (s) NIH-HS 9.4 ± 3.9 11.4 ± 4.7 6.3 ± 2.1 3.1 ± 1.3 1.9 ± 1.4 0.8 ± 0.5 3.33 = 0.05 4.56 <0.01 1.34 nsRLA-I 9.7 ± 1.7 10.8 ± 1.0 9.2 ± 2.4 7.3 ± 2.6 8.4 ± 2.3 3.6 ± 1.8RHA-I 21.0 ± 3.7* 9.3 ± 2.5 11.3 ± 5.1 7.3 ± 2.8 7.6 ± 2.0 10.7 ± 3.7

Wall-rearing frequency NIH-HS 6.1 ± 1.7 8.4 ± 2.6 4.5 ± 1.4 1.3 ± 0.5 1.1 ± 0.4 0.9 ± 0.5 12.84 <0.01 8.21 <0.01 1.80 nsRLA-I 12.9 ± 1.9 19.9 ± 3.6 18.2 ± 3.6 16.8 ± 4.1 12.0 ± 2.9 11.8 ± 3.6RHA-I 16.3 ± 1.4 17.0 ± 1.8 13.1 ± 2.0 12.9 ± 2.3 13.1 ± 2.8 13.6 ± 2.2

Wall-rearing duration (s) NIH-HS 7.7 ± 2.6 12.1 ± 4.4 8.4 ± 3.3 2.3 ± 1.1 2.4 ± 1.1 2.8 ± 1.7 11.58 <0.01 4.02 <0.01 3.08 <0.01RLA-I 24.3 ± 4.7 39.2 ± 7.9 50.1 ± 8.6 53.2 ± 13.6*** 43.9 ± 9.7 46.5 ± 12.6RHA-I 19.8 ± 3.1 28.0 ± 5.6 27.7 ± 6.2 38.2 ± 9.6 35.4 ± 9.5 41.0 ± 10.7

Free-rearing frequency NIH-HS 1.8 ± 0.9 3.1 ± 1.2 3.8 ± 1.4 2.3 ± 1.1 0.6 ± 0.4 0.4 ± 0.3 1.48 ns 3.27 <0.01 1.75 nsRLA-I 0.4 ± 0.2 1.1 ± 0.6 2.1 ± 1.0 2.2 ± 0.6 2.6 ± 1.5 1.1 ± 0.6RHA-I 0.9 ± 0.4 2.2 ± 0.6 4.5 ± 1.6 3.6 ± 1.1 4.5 ± 1.1 3.2 ± 1.0

Free-rearing duration (s) NIH-HS 1.6 ± 0.7 4.0 ± 2.0 7.8 ± 4.0 4.1 ± 2.4 0.8 ± 0.5 1.6 ± 1.2 0.70 ns 2.45 <0.05 1.48 nsRLA-I 0.3 ± 0.1 1.3 ± 0.9 1.4 ± 0.7 2.5 ± 0.7 3.2 ± 2.4 1.8 ± 1.4RHA-I 0.9 ± 0.4 1.8 ± 0.6 5.7 ± 2.4 3.9 ± 1.3 5.9 ± 1.3 3.8 ± 1.5

SAP frequency NIH-HS 3.6 ± 0.8 2.3 ± 0.6 2.5 ± 0.9 0.8 ± 0.3 0.4 ± 0.2 0.5 ± 0.2 0.04 ns 24.87 <0.01 1.15 nsRLA-I 4.4 ± 1.2 2.3 ± 0.9 1.0 ± 0.4 0.4 ± 0.2 0.3 ± 0.2 0.7 ± 0.3RHA-I 4.5 ± 0.7 1.5 ± 0.4 1.1 ± 0.3 0.7 ± 0.3 0.7 ± 0.3 1.0 ± 0.4

SAP duration (s) NIH-HS 5.1 ± 1.4 2.9 ± 1.1 4.3 ± 1.6 1.7 ± 0.8 0.7 ± 0.4 0.6 ± 0.3 1.60 ns 12.70 <0.01 1.41 nsRLA-I 5.4 ± 1.5 2.0 ± 0.9 0.8 ± 0.3 0.5 ± 0.3 0.6 ± 0.5 0.9 ± 0.5RHA-I 3.6 ± 0.8 1.1 ± 0.3 1.0 ± 0.4 0.4 ± 0.1 0.6 ± 0.3 1.1 ± 0.5

ns, non-significant.* p < 0.05 as compared with the RLA-I during the same time block (t-test).

** p < 0.05 as compared with the RHA-I during the same time block (Duncan).*** p < 0.05 as compared with the NIH-HS during the same time block (Duncan).

192 C. Estanislau et al. / Neuroscience Research 77 (2013) 187–201

ab bab ab

bc

0

15

30

45

60

75

90Cr

ossin

gsNIH-HSRLA-IRHA-I

bab

ab abab ab

0

5

10

15

20

25

30

1-5 6-10 11 -15 16 -20 21 -25 26 -30

Wal

l-rea

ring

freq

uenc

y

Fig. 1. Crossings and wall-rearing (mean ± SEM) displayed during the open-fieldsession. Rats from the NIH-HS, RLA-I and RHA-I strains were tested for 30 min.ap < 0.05 as compared with the RHA-I during the same time block; bp < 0.05 as com-pN

tpfdpdt(v(irnRoti

ibat

((tTafsdRHo

Table 3Behavioral measures studied in the home-cage.

Measure NIH-HS RLA-I RHA-I

Grooming measuresNumber of bouts 6.7 ± 1.3 7.0 ± 0.6 7.1 ± 1.0Total duration (s) 85.7 ± 15.5 118.8 ± 21.4 125.0 ± 25.6Body component (s) 39.4 ± 7.6 50.2 ± 9.6 65.2 ± 13.6Rostral component (s) 39.0 ± 8.3 57.1 ± 11.4 50.6 ± 13.1Number of pattern transitions 20.8 ± 3.3 29.8 ± 4.4 20.8 ± 2.9Noncephalocaudal transitions

(%)47.7 ± 4.8 48.3 ± 2.5 43.1 ± 4.3

Number of stereotyped chains 0.7 ± 0.2 1.7 ± 0.4 1.0 ± 0.2Interruptions per min of

grooming1.4 ± 0.3 2.4 ± 0.4 2.2 ± 0.5

Interrupted bouts (%) 19.4 ± 5.1 37.9 ± 6.3 37.2 ± 9.1

Complementary measuresAllogrooming duration (s) 3.1 ± 1.6 0.7 ± 0.7 5.7 ± 1.9Feeding duration (s) 78.2 ± 27.8 44.7 ± 19.1 95.5 ± 15.6Time motionless (s) 690 ± 145 302 ± 96◦ 267 ± 124◦

Crossings 22.1 ± 4.8 44.1 ± 9.6 65.7 ± 14.2◦

ared with the RLA-I during the same time block; c, p < 0.05 as compared with the/Nih-HS during the same time block (Duncan).

Regarding vertical activity in the open field (Fig. 1 and Table 1),he ANOVAs showed main effects of the strain (F[2,28] = 16.95,

< 0.001) and time block (F[5,140] = 17.35, p < 0.001) on wall-rearingrequency (Fig. 1), and similar effects were found in wall-rearinguration (Table 1). Post hoc comparisons showed that RLA-I ratsresented higher wall-rearing frequency than the NIH-HS groupuring the 1–5 block (Fig. 1), as well as longer wall-rearing timehan the RHA-I group during the 1–5 and 16–20 time blocksTable 1). Moreover, NIH-HS rats presented lower levels of bothariables than RHA-I/RLA-I groups in all but the 1–5 time blockFig. 1 and Table 1). The time block effect (and the absence ofnteraction with “strain”) on both frequency and duration of wall-earing indicate patterns of variation across time blocks that doot differ statistically among the three groups (Fig. 1 and Table 1).egarding free-rearing behavior (frequency and duration) in thepen-field test (Table 1) the ANOVA only showed main effects ofhe time block, thus reflecting similar variation across time blocksn the three groups.

Open-field SAP results (frequency and duration) are also shownn Table 1. The ANOVAs showed only main effects of the timelock (Table 1), thus indicating similar variation across time blocksmong the three groups. No significant effects were found in theime spent in the center area of the open-field (Table 1).

Wall-rearing frequency and duration in the hole-board testTable 2) presented main effects of the strain and the time blocksee ANOVAs in Table 2), with NIH-HS rats presenting lower levelshan both the other strains, as it was seen in the open-field test.he effect of time block indicates that all three strains/stocks over-ll decreased wall-rearing frequency along the session, especiallyrom the second time block onwards (Table 2). The ANOVA alsohowed a significant “strain × time block” effect on wall-rearing

uration, due to the fact that wall-rearing duration increased inLA-I and RHA-I groups across time blocks and decreased in NIH-S rats across time (Table 2). Free-rearing frequency and durationnly showed main effects of the time block (see ANOVAs in Table 2),

◦p < 0.05 as compared to the NIH-HS strain (Duncan).

as was also seen in the open-field test, as all the groups showed aclear trend to display lower levels (of both variables) during thefirst than during the other time blocks.

As observed in the open-field test, SAP frequency and durationonly showed main effects of the time block (see ANOVAs in Table 2),due to the overall higher SAP values (in the three groups) duringthe first than during the last time blocks (Table 2).

Head-dipping frequency and duration, the most important vari-ables (related to novelty-seeking, File and Wardill, 1975) from thehole-board test, showed main effects of the strain and the timeblock (see ANOVAs in Table 2). The time block effects were due tothe overall decreases in head-dipping along the session. Post hoccomparisons showed that the RHA-I group made more head dipsthan the NIH-HS strain during the 1st block. As from the resultsof previous studies we had clear a priori hypotheses with respectto head-dipping differences between the Roman rats, i.e. the RHA-I should display more head-dipping than the RLA-I strain (e.g. asseen in Fernández-Teruel et al., 1992; Escorihuela et al., 1999;Guitart-Masip et al., 2006), Student’s t-tests were applied to com-pare head-dipping variables between both Roman rat strains. Theyshowed that RHA-I rats spent longer time head-dipping (t(17) = 2.67,p < 0.05, Table 2; a clear trend in the same direction was alsoobserved in the number of head dips, t(17) = 1.97, p = 0.07; Table 2)than their RLA-I counterparts, thus confirming our hypotheses (seeFernández-Teruel et al., 1992; Escorihuela et al., 1999; Guitart-Masip et al., 2006).

Finally, regarding the home-cage activity test, for the reasonssaid above (see “Sections 2.3 and 2.4”) no time course evaluationwas performed and thus the whole period scores were comparedamong the groups (Table 3). The one-way ANOVAs indicated differ-ences among the strains in the time spent motionless (F[2,28] = 3.54;p < 0.05; NIH-HS rats spent longer time motionless than both theother strains, see Duncan’s test in Table 3) and in the number ofcrossings (F[2,28] = 5.23; p < 0.05; NIH-HS rats made less crossingsthan RHA rats, the RLA’s values falling in between; see Duncan’stest in Table 3). Regarding other measures obtained in the home-cage recording, one-way ANOVAs showed no differences (Table 3)among groups neither in the time spent feeding (F[2,28] = 1.17;p > 0.05) nor in allogrooming (F[2,28] = 2.36; p > 0.05).

3.1.2. Grooming measures

The mean latencies to start grooming in the open field test pre-

sented a tendency to be different between the RLA-I (79.4 ± 6.3 s)and the RHA-I (143.9 ± 17.5 s) strains, with the NIH-HS group values

C. Estanislau et al. / Neuroscience Research 77 (2013) 187–201 193

bc

b b

bc0

1

2

3

4

5Bo

uts

NIH-HSRLA-IRHA-I

bcbc c

bc0

20

40

60

80

100

120

140

Tota

l dur

a�on

(s)

b

bc bc c c0

20

40

60

80

100

120

140

1-5 6-1 0 11-1 5 16-2 0 21-2 5 26-30

Body

com

pone

nt (s

)

Minut es

Fig. 2. Number of bouts, total duration of grooming and time devoted to its bodycfic

fw

toissoest

oreFAbs

Fig. 3. Time spent in the rostral component of grooming and percentage of non-cephalocaudal transitions between grooming elements. Rats from the NIH-HS, RLA-Iand RHA-I strains were tested in an open-field for 30 min. bp < 0.05 as compared with

c

omponent. Rats from the NIH-HS, RLA-I and RHA-I strains were tested in an open-eld for 30 min. bp < 0.05 as compared with the RLA-I during the same time block;p < 0.05 as compared with the N/Nih-HS during the same time block (Duncan).

alling in between (120.4 ± 25.7 s), although the one-way ANOVAas not significant (F[2,28] = 2.38, p > 0.05).

Fig. 2 shows the grooming episodes (bouts) and grooming dura-ion exhibited by the rats during the open-field test. Main effectsf the strain and the time block were found, as well as a significantnteraction between these factors (Table 4). Post hoc comparisonshowed that the RHA-I strain presented less grooming bouts and/orhorter duration than both the other strains in the 1–5, 6–10 andther time blocks (see Fig. 2 and Table 4). The “strain X time bock”ffect on grooming bouts (Table 4) is due to the fact that groominghowed a tendency to a progressive increase in RHA-I rats acrossime blocks, the other groups showed an opposite tendency (Fig. 2).

Time spent in the body component of grooming during thepen-field test is also shown in Fig. 2. Although this component cor-esponded only to nearly half of the total duration of grooming, theffects found in it mirrored those found in total duration (Table 4).

ig. 3 shows the time spent in the rostral component of grooming.s no effect of the repeated measure was found (nor an interactionetween strain and the repeated measure), scores from the wholeession were compared among the strains by means of a one-way

the RLA-I; p < 0.05 as compared with the N/Nih-HS (Duncan).

ANOVA. A significant effect was found (F[2,28] = 4.37; p < 0.05), withRHA-I rats showing decreased levels of rostral grooming (p < 0.05as compared to the NIH-HS and p = 0.10 as compared to the RLA-I,Duncan’s tests).

Fig. 4 shows the number of pattern transitions shown while therats were grooming in the open-field test. Main effects of the strainand the time block were found (Table 4). Post hoc comparisonsshowed that the RHA-I presented less transitions than both theother strains from the 1–5 to the 21–25 time blocks.

The strains differed regarding the percentage of noncephalocau-dal transitions during grooming in the open-field test. As no effectof the repeated measure was found, nor a significant interaction,the three strains were compared by means of a one-way ANOVAon data from the whole session, and a significant effect was found(F[2,28] = 5.00, p < 0.05). Post hoc comparisons showed that the RHA-I value was significantly (p < 0.01; Duncan’s test) lower than thatof the RLA-I strain, with the NIH-HS rats falling in between bothRoman rat groups (Fig. 3).

The number of stereotyped chains during grooming in the open-field test is shown in Fig. 4. Main effects of the strain and the timeblock were found (see ANOVA in Table 4). In general, it can be seenthat the RLA-I presented higher values than both the other groupsalong the session–the difference was found to be statistically signif-icant in the comparison with the RHA-I rats in the 6–10 time block(Fig. 4). The “time block” effect and the absence of interaction with“strain” reflect that curves across time are statistically significantand similar among the three strains/stocks (Fig. 4 and Table 4).

Fig. 4 also shows the rates of interruptions per min of groom-ing activity in the open-field test. Main effects of the strain andthe time block were found (Table 4). The post hoc comparisonsshowed that, in general, from the 6–10 to the 21–25 time block,the RLA-I strain presented significantly higher rates than both theother strains. Again, the “time block” effect and the absence of inter-action with “strain” indicate that the curves across time blocks are

statistically significant and similar among the three groups (Fig. 4and Table 4).

194 C. Estanislau et al. / Neuroscience Research 77 (2013) 187–201

Table 4Results of a two-way ANOVA for repeated measures applied to grooming measures studied in the open-field test.

Measure Strain Time block Interaction

F[2,28] p F[5,140] p F[10,140] p

Number of bouts 4.94 <0.05 3.49 <0.01 2.08 <0.05Total duration (s) 8.36 <0.01 4.64 <0.001 1.22 nsBody component (s) 9.25 <0.001 5.06 <0.001 1.50 nsRostral component (s) 4.37 <0.05 1.51 ns 0.81 nsNumber of pattern transitions 10.82 <0.001 4.83 <0.001 1.33 nsNon cephalocaudal transitions (%) 4.02 <0.05 1.29 ns 1.03 nsNumber of stereotyped chains 6.61 < 0.01 5.68 < 0.001 0.74 nsInterruptions per min of grooming 12.78 < 0.001 4.12 < 0.01 0.46 nsInterrupted bouts (%) 4.35 < 0.05 3.47 < 0.01 0.40 ns

n

tbgasttsb

FftsstaerX

totpepgNtpl2i–(dttlr

gstttng

s, non-significant.

Finally, the percentages of interrupted bouts in the open-fieldest can be seen in Fig. 4. Main effects of the strain and the timelock were found (see ANOVA in Table 4). In general, the RLA-Iroup presented higher percentages than both the other strainsnd the post hoc comparisons indicate that such a difference wasignificant in the comparison with the NIH-HS during the 11–15ime block. Evolution of the percentage of interrupted bouts acrossime blocks was statistically significant (i.e. time block effect) andimilar among the three groups, and thus the absence of interactionetween both factors.

Grooming results from the hole-board test are shown in Table 5.or the number of bouts performed in the test, the two-way ANOVAor repeated measures revealed main effects of the strain and theime block, as well as an interaction between both factors. Con-istent with the observations from the open field test, RHA-I ratshowed lower number of bouts than both the other strains duringhe 1–5 time block in the hole board test. Grooming bouts showed

progressive decrease along the session (i.e. ANOVA “time block”ffect), a trend that was clearly more marked in RLA-I and NIH-HSats than in the RHA-I group (see Table 5), and this is why the “strain

time block” effect appeared.Regarding the total time spent grooming during the hole-board

est, as well as its body and rostral components (Fig. 5), no effectsf the time block were found (nor interactions between strain andime block), and thus scores from the whole session were com-ared among the strains by means of a one-way ANOVA. Significantffects were found in each of the three measures (F[2,28] > 7.96;

< 0.01). Post hoc comparisons indicated that total duration ofrooming was shorter for the RHA-I strain, as compared with theIH-HS (the comparison with the RLA-I showed a nonsignificant

rend, p = 0.07). As clear a priori hypothesis could be drawn fromreviously published results, i.e. the RLA-I were expected to groom

onger in the hole-board than the RHA-I strain (Guitart-Masip et al.,006), a Student’s t-test was applied in order to compare groom-

ng duration between the Roman strains, showing – as expected higher grooming duration in the RLA-I than in the RHA-I straint(17) = 3.90, p < 0.01). Differences among the groups in the timeevoted to the body component of grooming roughly mirrored theotal duration results just described above. Differences in the ros-ral component were also similar, with RHA-I rats displaying lowerevels of this behavior than RLA-I and NIH-HS rats (Fig. 5), in a wayoughly similar to what was observed in the open field test.

The number of pattern transitions (Table 5) presented by theroups along the hole-board session revealed main effects of thetrain and the time block. The post hoc comparisons showed thathe RLA-I presented more transitions than the RHA-I strain during

he first two time blocks (see Duncan’s tests in Table 5). Moreover,here was an overall significant decrease along the session, i.e. sig-ificant “time block” effect, especially marked in RLA-I and NIH-HSroups (Table 5).

The ANOVA applied to the percentage of noncephalocaudal tran-sitions only revealed a main effect of the time block. Accordingly,in general, the groups seemed to present lower levels toward theend of the session (Table 5). Similarly, the number of stereotypedchains and the percentage of interrupted bouts only presented maineffects of the time block and, again, the last two time blocks seemedto have lower values than the initial time intervals (Table 5). Nomain effect or significant interaction was found in the number ofinterruptions/min of grooming (Table 5).

The grooming measures studied in the home-cage test areshown in Table 3. The one-way ANOVAs did not reveal significantdifferences among the groups in any of the grooming variables.

3.2. Two-way escape/avoidance acquisition

3.2.1. Performance measuresFig. 6 shows the results of the two-way escape/avoidance task.

For the escape response to be considered established, a criterion of3 consecutive responses shorter than 1 s should be achieved. OneNIH-HS and four RHA-I began to avoid the US before reaching thisescape acquisition criterion; these rats were not considered in thecomparison regarding the number of trials to reach that criterion. Inthis comparison, the one-way ANOVA indicated differences amongthe strains (F[2,23] = 11.79; p < 0.001). According to the post hoc com-parisons, the RLA-I needed more trials to reach the criterion thanboth the other groups (Fig. 6A).

The number of trials needed to reach the criterion of 3 consecu-tive avoidances is also shown in Fig. 6B. Three rats from the NIH-HSand four from the RLA-I strain failed to reach the criterion alongthe two sessions; thus the roof value of 100 trials was attributedto these subjects for the statistical comparisons. According to theone-way ANOVA, the number of trials needed to reach the crite-rion of 3 consecutive avoidances presented differences among thestrains (F[2,28] = 26.40; p < 0.001). Post hoc comparisons showed thatRLA-I rats needed more trials to reach the criterion than the NIH-HS group, which, in turn, needed more trials than the RHA-I strain(Fig. 6B).

Fig. 6B shows the number of successful avoidances per blockof 10 trials along the two sessions. A two-way ANOVA indicatedmain effects of the strain (F[2,28] = 32.55; p < 0.001) and the block oftrials (F[9,252] = 27.62; p < 0.001), as well as a significant interactionbetween these factors (F[18,252] = 3.18; p < 0.001). Post hoc compar-isons showed that the number of avoidances of RHA-I rats washigher than that of the RLA-I group from the 2nd to the 8th block.The number of avoidances per block was also higher in RHA-I thanin NIH-HS rats, but the significance level was only reached in the

6th block (see Fig. 6B, Duncan’s tests). In a within group approach,all the groups were found to present increases in avoidance perfor-mance across the two sessions (i.e. “trial block” effect). However,the “strain × trial block” interaction is likely due to the fact that

C. Estanislau

et al.

/ N

euroscience R

esearch 77

(2013) 187–201

195

Table 5Grooming measures studied in the hole-board test and their corresponding two-way ANOVA for repeated measures results.

Measure Strain Mean ± SEM 2-Way ANOVA for repeated measures

Time block (min) Strain Time block Interaction

1–5 6–10 11–15 16–20 21–25 26–30 F[2,28] p F[5,140] p F[10,140] p

Number of bouts NIH-HS 3.5 ± 0.5 2.3 ± 0.5 2.3 ± 0.3 2.3 ± 0.5 1.5 ± 0.4 2.1 ± 0.6 4.01 <0.05 6.59 <0.01 1.89 = 0.05RLA-I 4.0 ± 0.4 2.6 ± 0.4 1.6 ± 0.3 2.3 ± 0.4 1.8 ± 0.5 1.2 ± 0.4RHA-I 1.4 ± 0.3*,** 1.4 ± 0.3 1.1 ± 0.5 2.3 ± 0.4 1.3 ± 0.3 1.0 ± 0.4

Number of pattern transitions NIH-HS 14.1 ± 2.9 16.6 ± 3.5 16.4 ± 2.4 15.4 ± 3.2 9.5 ± 3.0 10.1 ± 3.0 9.36 <0.01 4.03 <0.01 1.52 nsRLA-I 20.0 ± 2.7 21.7 ± 3.3 11.3 ± 2.7 15.9 ± 3.7 12.8 ± 3.7 9.3 ± 2.9RHA-I 4.2 ± 1.0* 6.8 ± 1.5* 5.4 ± 1.5 9.2 ± 1.5 6.2 ± 2.0 4.7 ± 1.8

Noncephalocaudal transitions (%) NIH-HS 48.2 ± 2.3 37.4 ± 7.1 50.5 ± 3.2 39.9 ± 5.8 25.6 ± 8.1 26.6 ± 8.5 3.18 ns 4.51 <0.01 0.60 nsRLA-I 45.3 ± 3.3 48.3 ± 3.3 43.8 ± 6.1 49.9 ± 7.4 33.8 ± 9.3 30.3 ± 9.7RHA-I 28.2 ± 7.2 34.5 ± 4.8 32.8 ± 8.1 42.6 ± 2.4 20.2 ± 8.7 22.1 ± 9.1

Number of Stereotyped chains NIH-HS 0.8 ± 0.3 1.0 ± 0.3 1.0 ± 0.3 1.0 ± 0.2 0.5 ± 0.2 0.4 ± 0.2 2.58 ns 3.03 <0.05 1.04 nsRLA-I 1.4 ± 0.3 1.6 ± 0.2 0.8 ± 0.1 0.8 ± 0.2 0.8 ± 0.2 0.7 ± 0.3RHA-I 0.4 ± 0.2 0.9 ± 0.3 0.6 ± 0.2 0.9 ± 0.3 0.4 ± 0.2 0.5 ± 0.2

Interruptions per min of grooming NIH-HS 2.9 ± 0.8 2.5 ± 0.5 3.0 ± 0.7 2.3 ± 0.5 1.9 ± 0.6 1.4 ± 0.4 1.58 ns 2.14 ns 0.24 nsRLA-I 3.8 ± 0.7 4.3 ± 1.1 4.3 ± 1.1 2.3 ± 0.8 2.4 ± 1.1 2.6 ± 1.0RHA-I 3.2 ± 1.2 3.5 ± 0.7 3.2 ± 0.9 3.1 ± 0.7 1.9 ± 0.7 2.2 ± 1.2

Interrupted bouts (%) NIH-HS 44.9 ± 11.3 62.4 ± 12.1 54.2 ± 9.6 68.5 ± 12.4 30.6 ± 13.3 32.5 ± 11.3 0.32 ns 3.74 <0.01 0.62 nsRLA-I 60.7 ± 10.7 65.2 ± 9.6 44.4 ± 13.3 30.6 ± 10.4 25.9 ± 11.8 37.0 ± 13.8RHA-I 41.7 ± 14.8 66.7 ± 14.1 44.0 ± 15.7 55.7 ± 13.1 21.7 ± 11.7 30.0 ± 13.6

ns, non-significant.* p < 0.05 as compared with the RLA during the same time block (Duncan).

** p < 0.05 as compared with the NIH-HS during the same time block (Duncan).

196 C. Estanislau et al. / Neuroscience Research 77 (2013) 187–201

acac c

ac

0

1

2

3

4

5

6

7

8

1-5 6-10 11-15 16-2 0 21-2 5 26-30

Inte

rrup

�ons

/min

of g

room

ing

Minut es

a

0.0

0.5

1.0

1.5

2.0

2.5

Ster

eoty

ped

chai

ns

c

0

20

40

60

80

100

Inte

rrup

ted

bout

s (%

)

c

bcbc b bc

bc0

5

10

15

20

25

30

35Pa

�er

n tr

ansi�

ons

NIH-HSRLA-IRHA-I

Fig. 4. Qualitative aspects of grooming studied during the open-field session. Ratsfrom the NIH-HS, RLA-I and RHA-I strains were tested for 30 min. ap < 0.05 as com-pared with the RHA-I during the same time block; bp < 0.05 as compared with theRt

waIoCAs

Fig. 5. Time spent in grooming and in its body and rostral components during thehole-board test. Rats from the NIH-HS, RLA-I and RHA-I strains were tested for

LA-I during the same time block; cp < 0.05 as compared with the N/Nih-HS duringhe same time block (Duncan).

hile the RHA-I strain rapidly showed a high level of successfulvoidances (i.e. in the 2nd–3rd trial blocks; see Fig. 6B), the RLA-

performance increased very slowly, with the acquisition curvef the NIH-HS falling in-between that of the two Roman strains.

onfirming and extending the 10-trial block analyses, one-wayNOVAs of total number of avoidances/session (data not shown)howed significant differences among the three groups in both

30 min. bp < 0.05 as compared with the RLA-I; cp < 0.05 as compared with the NIH-HS(Duncan). *p < 0.05 as compared with the RLA-I (t-test).

shuttle box sessions (F[2,28] > 19.01; p < 0.001). Post hoc Duncan’scomparisons showed that the three groups were different (p < 0.05)in the first session (with RHA-I showing the highest and RLA-I thelowest number of avoidances), while RHA-I rats presented moreavoidances (p < 0.05) than the RLA-I and NIH-HS groups (which inturn were no different to each other) in the second training session.

A complementary analysis that could be helpful in the interpre-tation of the grooming results (see below) concerns the degree ofsuccess in escape and avoidance performance of the three strainsduring the periods in which grooming behavior was evaluated. Dur-ing the 10-min period after the escape acquisition criterion wasreached, the percentage of successful escapes (i.e. response in lessthan 1 s after the presentation of US) did not differ among thegroups (F[2,22] = 0.65; p > 0.05). On the other hand, during the 20-min period after the avoidance acquisition criterion was reached,the percentage of successful avoidances was different among thegroups (F[2,21] = 7.28; p < 0.01). Specifically, RLA-I rats showed sig-nificantly poorer performance than both the RHA-I and the NIH-HSgroups during that period (Fig. 6C).

3.2.2. Grooming measuresBecause of not fulfilling the corresponding learning criteria, sev-

eral subjects (from the different experimental groups) had to beexcluded from each of the three acquisition phases (see Table 6).Thus, we considered that one-way ANOVAs for each learning phase,rather than repeated measures ANOVA (i.e. across learning phases),were more appropriate to analyze the present grooming results.

Regarding the comparison of grooming displayed before theescape acquisition criterion was reached (Table 6A), one NIH-HSand five RHA-I already showed low escape latencies (<1 s) sincethe beginning of training, and therefore were not included in thisgrooming evaluation. Comparisons of grooming measures during

the period before escape acquisition showed no significant groupeffects (all F[2,22] < 2.63; p > 0.09). Remarkably, all three groupsshowed very low grooming frequency and duration (Table 6A).

C. Estanislau et al. / Neuroscience Research 77 (2013) 187–201 197

acab

a

0

20

40

60

80

100

0

20

40

60

80

100

NIH-HS RLA-I RHA-I

Tria

ls be

fore

av

oida

nces

Tria

ls be

fore

es

cape

s

Trials before 3escapesTrials before 3avoidances

A

bb b b bc

b b

02468

10

1 2 3 4 5 6 7 8 9 10

Avoi

danc

es

Blocks of 10 trials

NIH-HSRLA-IRHA-IB

ac

0

20

40

60

80

100

NIH-HS RLA-I RHA-I

Succ

essf

ul a

void

ance

s (%

)

C

Fig. 6. Results from the two-way (shuttle-box) active avoidance training. For theescape response to be considered established, a criterion of 3 consecutive responsesshorter than 1 s should be achieved. For the avoidance response to be consideredestablished, a criterion of 3 consecutive successful avoidances should be achieved.Training consisted of two 50-trial sessions. (A) Trials before the occurrence of threeconsecutive (acquisition criterion) escapes or avoidances. (B) Number of successfulavoidances per block of 10 trials along the two sessions. (C) Percentage of successfulavoidances during the 20-min period after the avoidance acquisition criterion wasaR

hocfeb(Rga

tfiatiFh

Table 6Grooming measures obtained during the two-way (shuttle-box) active avoidancetraining. Comparisons were conducted focusing on three different phases: (A) thefinal 10-min period before the acquisition of the escape response; (B) the first10 min period when the escape response was established; (C) the first 20 min periodwhen the avoidance response was established. Values are shown as frequency/min(number of bouts, pattern transitions and stereotyped chains) or seconds/min (totalduration of grooming and the time spent in each of its components).

Score/min NIH-HS RLA-I RHA-I

(A) Escape > 1 sN 11 9 5Bouts 0.01 ± 0.01 0.02 ± 0.01 0.04 ± 0.04Total duration (s) 0.07 ± 0.07 0.32 ± 0.27 0.86 ± 0.79Body component (s) 0.07 ± 0.07 0.07 ± 0.05 0.06 ± 0.06Rostral component (s) 0.00 ± 0.00 0.22 ± 0.21 0.80 ± 0.80Pattern transitions 0.02 ± 0.02 0.08 ± 0.05 0.48 ± 0.39Stereotyped chains 0.00 ± 0.00 0.01 ± 0.01 0.00 ± 0.00

(B) Escape < 1 sN 11 8 6Bouts 0.04 ± 0.04 0.06 ± 0.03 0.13 ± 0.06Total duration (s) 0.10 ± 0.10 0.29 ± 0.14 0.86 ± 0.41◦

Body component (s) 0.00 ± 0.00 0.05 ± 0.05 0.23 ± 0.13*

Rostral component (s) 0.10 ± 0.10 0.24 ± 0.10 0.58 ± 0.28Pattern transitions 0.07 ± 0.07 0.15 ± 0.07 0.43 ± 0.20Stereotyped chains 0.00 ± 0.00 0.01 ± 0.01 0.05 ± 0.02*

(C) AvoidancesN 9 5 10Bouts 0.07 ± 0.02 0.08 ± 0.03 0.20 ± 0.03*

Total duration (s) 0.45 ± 0.18 1.15 ± 0.46 2.87 ± 0.42*

Body component (s) 0.03 ± 0.03 0.23 ± 0.16 0.69 ± 0.32Rostral component (s) 0.42 ± 0.18 0.80 ± 0.29 1.91 ± 0.24*

Pattern transitions 0.16 ± 0.06 0.34 ± 0.12 0.63 ± 0.10*

Stereotyped chains 0.00 ± 0.00 0.05 ± 0.03 0.05 ± 0.02

chieved. ap < 0.05 as compared with the RHA-I; bp < 0.05 as compared with theLA-I; cp < 0.05 as compared with the NIH-HS (Duncan).

Regarding grooming displayed after escape acquisition criterionad been reached, but before achieving the avoidance criterion,ne NIH-HS skipped this phase while one RLA-I never reached theriterion during the two sessions. These two rats were excludedrom this grooming evaluation. The ANOVA showed significantffects in total duration of grooming and in the time devoted to itsody component, as well as in the number of stereotyped chainsall F[2,22] > 3.49; p < 0.05). Post hoc comparisons showed that theHA-I presented increased levels in each of these measures (whenrooming total duration was compared between RHA-I and RLA-I,

nonsignificant trend was found, p = 0.06) (Table 6B).Regarding the first 20-min period after the avoidance acquisi-

ion criterion was reached, three rats from the NIH-HS and fourrom the RLA-I strain failed to reach the criterion and were notncluded in the analyses. All the other rats were considered in thenalyses (Table 6C). During this period, main effects were found inhe number of bouts, total duration of grooming and time spent in

ts rostral component, as well as in the number of transitions (all[2,21] > 6.40; p < 0.01). In all these measures, the RHA-I presentedigher levels than both the other strains (Table 6C).

* p < 0.05 as compared to both the other strains (Duncan).◦

p < 0.05 as compared to the NIH-HS strain (Duncan).

4. Discussion

The objective of the present investigation was to evaluate thegrooming responses of rats with known differences in anxiety andstress reactivity when tested in several situations involving dif-ferent degrees of aversiveness: (1) in the home-cage (a minimallychallenging environment); (2) in a novel open-field and in a novelhole-board (novel/threatening environments); (3) during two-wayactive avoidance training. Since escape acquisition and avoidanceacquisition were assumed as key moments for changes in arousal(in parallel to the progressive establishment of successful copingresponses), grooming observations comprised different phases: (a)prior to the establishment of stable escape from the foot-shock; (b)when successful escape responses were established; and, (c) whensuccessful avoidance responses were established. As an additionalvalue of the present work it should be underlined that the presentis the first time that NIH-HS rats are characterized and comparedto other strains in the tests used in this study (with the exceptionthat NIH-HS rats have been compared to the Roman strains in asingle shuttle-box avoidance session, but without scoring groom-ing behavior; e.g. López-Aumatell et al., 2009b; Díaz-Morán et al.,2012, 2013).

Concerning the main results obtained, an outstanding observa-tion has been that differences among the groups were found todepend on the test situation. More specifically: (I) No differencesamong the strains were found in the home-cage (only the timespent immobile was increased in NIH-HS rats). (II) When tested ina novel environment (i.e. open-field, hole-board), the RLA-I and theNIH-HS strains (known as more anxiety/stress prone than the RHA-Istrain) showed quantitative and qualitative differences in groom-

ing behavior as compared to the RHA-I strain. In general, RLA-I andNIH-HS rats exhibited lower horizontal/vertical activity (crossingsand head-dipping) and higher grooming responses than the RHA-I

1 ence R

sbeS2WiewtsbtNcsMatRA

bWwrtntopotFidca

TibooRcTeDelute2v2sfi

iEdsca

98 C. Estanislau et al. / Neurosci

train, being these results consistent with the known differencesetween both Roman strains in those two tests (Fernández-Teruelt al., 1992, 2002; Steimer et al., 1997, 1998; Escorihuela et al., 1999;teimer and Driscoll, 2003; Guitart-Masip et al., 2006; Driscoll et al.,009), but being reported for the first time in NIH-HS rats. (III)hen the animals learned to deal with the aversive stimulation

n the two-way active avoidance training, by performing correctscape or avoidance responses, the differences among the strainsere opposite to those observed in the novel environment tests:

he RHA-I showed more grooming responses than both the othertrains. (IV) During the first five minutes of the open-field and hole-oard tests, the response profiles (e.g. crossings and grooming inhe open field, grooming and head-dipping in the hole-board) ofIH-HS rats are much closer to RLA-I than to RHA-I rats, which isonsistent with previously reported findings in other anxiety- andtress-related responses (e.g. López-Aumatell et al., 2009b; Díaz-orán et al., 2012, 2013). Something similar occurs in the two-way

voidance task (both in avoidances and in grooming), although inhis test the NIH-HS show significantly better performance than theLA-I strain in some acquisition variables (see Fig. 6; and López-umatell et al., 2009b; Díaz-Morán et al., 2013).

It worth to highlight that our findings have implications forehavioral evaluations in areas such as stress, anxiety and fear.e found that grooming response to novelty was greater in ratsith a more fearful profile (i.e. RLA-I and NIH-HS). However, our

esults (and others that will be timely referred to) also suggestedhat one cannot expect a linear relationship between aversive-ess and grooming: while an aversive situation such as exposureo novelty could be followed by increases in grooming, potentialccurrence of a painful experience (electric foot shock) mostly sup-ressed it. While most of the studies show only gross measuresf grooming (i.e. frequency and duration), present results supporthat different grooming components can be separately modulated.inally, present results also suggest that some alterations in groom-ng could only become robust after many minutes (for example,uring exposure to novelty), what imply that short test durationan mask effects in this behavior. In the following paragraphs thesend some other issues are addressed.

Let us discuss the results of the different tests with some detail.hus, regarding the most commonly studied behavioral measuresn the open-field and hole-board tests (i.e. especially crossings inoth tests and head-dipping in the hole-board test), the resultsf RHA-I and RLA-I rats are generally in agreement with previ-us studies by showing decreased ambulation (i.e. crossings) inLA-I rats during the first 5 min of the open-field test (as well as alear trend in the first 5 min of the hole-board test; see Fernández-eruel et al., 1992, 2002; Steimer et al., 1997, 1998; Escorihuelat al., 1999; Steimer and Driscoll, 2003; Guitart-Masip et al., 2006;riscoll et al., 2009; López-Aumatell et al., 2009b). Such a differ-nce in ambulation between both Roman rat strains disappearedater in the session, as it has been observed in previous studiessing an “open field-like activity test”, i.e. in a 5-min session in thisest, RLA-I rats ambulate less than the RHA-I strain (López-Aumatellt al., 2009b), although no significant difference is reported in a0-min session (Díaz-Morán et al., 2012). Consistent with our pre-ious results (López-Aumatell et al., 2009b; Díaz-Morán et al., 2012,013), in the present study NIH-HS rats showed ambulation levelsimilar to those displayed by the RLA-I strain, especially during therst 5 min of the open-field and hole-board tests.

On the other hand, and also in agreement with previous stud-es (Fernández-Teruel et al., 1992, 2002; Steimer et al., 1998;scorihuela et al., 1999; Guitart-Masip et al., 2006), RHA-I rats

isplayed significantly higher levels of head-dipping (a novelty-eeking related response, File and Wardill, 1975) than their RLA-Iounterparts during the first 5 min of the hole-board test, which isgain in support to the contention that RHA-I rats are characterized

esearch 77 (2013) 187–201

by increased novelty-seeking (e.g. Fernández-Teruel et al., 1992,2002; Escorihuela et al., 1999; Driscoll et al., 2009). During the restof the session the differences disappeared, as also occurred withcrossings (both in the open-field and hole-board tests; see above),thus suggesting that the initial 5–10 min of exposure to those twotests (i.e. open-field and hole-board) constitute the critical inter-val to observe most strain-related differences in non-groomingresponses to a novel environment. Again, concerning head-dippingbehavior, the NIH-HS rats appeared to be very close to the (lownovelty-seeking) profile of RLA-I rats.

In summary, regarding non-grooming behaviors in both noveltytests (i.e. open-field and hole-board), in agreement with previ-ous results the RHA-I strain displays increased levels of activity(crossings) and head-dipping during the first 5–10 min of testing, ascompared to RLA-I and NIH-HS rats. The latter two groups presentbehavioral profiles – of lowered activity and head-dipping – that aregenerally similar to each other (see Fernández-Teruel et al., 1992,2002; Steimer et al., 1997, 1998; Escorihuela et al., 1999; Steimerand Driscoll, 2003; Guitart-Masip et al., 2006; Driscoll et al., 2009;López-Aumatell et al., 2009b; Díaz-Morán et al., 2012, 2013).

Grooming in the open-field test revealed that the RHA-I dis-played lower levels than both the other strains during most of thesession, with the exception of the 1st and the last 5-min block.In fact, for the RLA-I and the NIH-HS strains, it seems that thistest elicited grooming responses that achieved their peak nearly10–15 min after the session beginning and then fell toward the ini-tial levels by the end of the 30-min period. In contrast, the RHA-Istrain presented low levels of grooming during the 1st 5-min periodand gradually increased it during the next 10 min, although theirlevels at 10–15 min after beginning the session were far lower thanthose of the other two strains. These differences among the strainslend further support to the suggestion that novelty-induced groom-ing can be a further behavioral marker of the low-anxiety profileof RHA-I rats, as compared to the RLA-I and the NIH-HS strains(Ferré et al., 1995; Steimer et al., 1997; Escorihuela et al., 1999;Steimer and Driscoll, 2003; Driscoll et al., 2009; López-Aumatellet al., 2009a,b; Díaz-Morán et al., 2012, 2013). In addition, thepresent results for the first time show that NIH-HS rats present aprofile of novelty-induced grooming which is quite close to thatof the “high anxious” RLA-I rats. In this connection, in supportof the view that novelty-induced grooming is an anxiety-relatedresponse, rats administered with the anxiolytic diazepam (1 mg/kg)have been reported to show decreased levels of grooming whiletested for 30 min in a small Plexiglas box (Consoli et al., 2007). Inanother experiment, in which the anxiety levels of outbred ratswere manipulated by confining them to different aversive contextsprior to an elevated plus-maze session (Estanislau, 2012), the ratsexposed to the most aversive condition showed delayed increasesin grooming (during the plus-maze session) that can be seen assimilar to those observed in the RLA-I and the NIH-HS strains in thepresent study (see Fig. 2).

When the grooming components were separately evaluated,it was found that the effects in the body component durationmirrored those found in total duration. It seems therefore thattotal grooming duration results could mostly be accounted by theeffects influencing its body component. On the other hand, no evi-dence for a time-course in the rostral component response wasfound, although it is still remarkable that this grooming componentwas also able to distinguish the RHA-I from the other strains (seeFigs. 3 and 5). Rats stressed with light for 5 min and then observedfor 5 min during an “actimeter test” have also been reported to showincreases in rostral grooming (Kalueff and Tuohimaa, 2005a). In a

10-min long session, open space-induced anxiety was found to alsolead to increases in rostral grooming (the body component showinga nonsignificant trend) (Estanislau, 2012). While the rostral compo-nent seems to express anxiogenic effects even when the session is

ence R

onTscntP

weFta

wwsoimgenibsob2‘sts

ttsrebsrotatcet

thriAip

wdintit

C. Estanislau et al. / Neurosci

nly 5-min long, short sessions like that of the above studies seemot to be lengthy enough to present effects in the body component.his conclusion is indirectly supported by a study using a longeression duration (15 min) in which it is reported that the ‘rostralontent’ (i.e. grooming not devoted to the body) is high at the begin-ing and gradually decreases along the exposure to a novel arena,hen letting the body component to fully appear (Komorowska andisula, 2003).

In short, our suggestion about the grooming components is thathile the body component gradually increases along a novelty

xposure and depending on the anxiety level (see an example inig. 2), the rostral component would be sensitive to variations inhe anxiety level even in shorter periods and in a manner that ispparently not time dependent (see “Section 3” above).

The number of pattern transitions seemed to present results thatere similar to those of the total duration of grooming. Indeed, asas the case of grooming duration, the number of pattern tran-

itions was able to distinguish the RHA-I strain from both thether strains during most of the session. This result is not surpris-ng, since it is reasonable that with longer durations of grooming,

ore opportunities of pattern transitions would occur. Some otherrooming measures were also able to distinguish the strains; how-ver, they were not so sharply sensitive. It is the case of theumber of grooming bouts, the number of interruptions per min

n grooming (which revealed greater levels for the RLA-I than foroth the other strains during most of the session), the number oftereotyped chains, and the percentages of interrupted bouts andf noncephalocaudal transitions. These two latter measures haveeen suggested to be good anxiety indexes (Kalueff and Tuohimaa,005a,b; these studies label the noncephalocaudal transitions as

incorrect transitions’). However, it is important to note that thosetudies were performed using sessions of short duration. It seemshat the sensitivity of these measures is partially lost in longeressions.

In the hole-board test, grooming duration was different amonghe groups, with RHA-I rats displaying reduced levels with respecto the other two groups. The body component of grooming pre-ented effects much similar to those found in its total duration and,emarkably, the rostral component was also able to discriminateach strain from the others (NIH-HS > RLA-I > RHA-I). The num-er of grooming bouts and of pattern transitions again showedome ability in discriminating the strains. To sum up, groomingesults from the hole-board test are mostly comparable to thosef the open-field, although somewhat fewer differences amonghe strains were found. The fact that the hole-board test allows

wider range of behavioral responses than the open-field test (i.e.he hole-board test allows head-dipping through the holes, besidesrossings, rearings and grooming) may in part underlie these differ-nces between both tests as concerns to their relative sensitivitieso detect group/strain effects.

To sum up, the differences in grooming behavior among thehree strains are generally consistent between the open-field andole-board tests. As said earlier, this is the first time that NIH-HSats are characterized for grooming and non-grooming responsesn these two novelty tests and compared to the Roman rat strains.s expected, RHA-I rats generally showed lower scores of groom-

ng behavior than RLA-I and NIH-HS rats, while these two strainsresented grooming levels similar to each other.

When the behavior of the rats from the three strains was studiedhile they were in their home-cage in an undisturbed condition, noifference among the strains was found, except in the time spent

mmobile, which was increased in NIH-HS rats. It is important to

ote that the home-cage test was the less threatening condition inhe present study. Since no differences in grooming were found int, this strengthens the notion that the differences found in the otherests resulted from the aversiveness (due to novelty) they involve.

esearch 77 (2013) 187–201 199

It is remarkable that the time spent grooming by the three strainsduring the home-cage observation was low as compared to the lev-els found during the open-field or hole-board tests. Grooming is abehavior that, in a home-cage condition, is expected to occur regu-larly and paralleling the circadian sleep-wake cycle (Bolles, 1960).The home-cage levels of the three groups were comparable to thosepresented by the RHA-I strain during the open-field or hole-boardtests. The other two groups groomed at least twice the RHA-I lev-els during these tests. These comparisons suggest that while theother strains – RLA-I and NIH-HS – presented a novelty-inducedincrease in grooming (Bindra and Spinner, 1958; Jolles et al., 1979),the RHA-I seemed to lack it.

The performance of the Roman strains in the two-way activeavoidance training closely matched what could be expected on thebasis of previous studies. The RHA-I rats presented faster avoid-ance acquisition, contrasting with the markedly poor performanceof RLA-I rats (e.g. Escorihuela et al., 1999; Aguilar et al., 2004; López-Aumatell et al., 2009b; Díaz-Morán et al., 2012, 2013). The NIH-HS,on average, presented in-between performances closer to the RLA-Ilevels than to RHA-I scores, in accordance with what has previouslybeen reported (López-Aumatell et al., 2008, 2009b, 2011; Vicens-Costa et al., 2011; Díaz-Morán et al., 2012, 2013).

As at least part of the animals from each strain was able toachieve the acquisition criteria during the two-session procedure,grooming along the different stages of acquisition was evaluated.Interestingly, nearly no grooming was seen in the initial phase,when the animals were not yet even able to display short escapelatencies, i.e. when the situation was the most stressful/anxiogenicand uncontrollable for all three groups. As they became able topresent faster escape responses (so, when rats begin to gain con-trollability over the stressor) and, more clearly after they achievedthe avoidance criterion, grooming responses raised. In fact, such anincrease was more clearly seen in the RHA-I strain, which groomedlonger than the other two groups. This result is somewhat puz-zling, taking into account the opposite differences found amongthe strains in the grooming induced by the novelty exposure tests(open-field and hole-board). Therefore, the fact that the RHA-I ratsgroomed longer than the other strains after achieving the avoidanceacquisition criterion remains to be explained.

In this connection, it is important to keep in mind the sugges-tion that “grooming coincides more with the period after arousaland rather reflects the processes of dearousal due to the termina-tion of (or habituation to) a stressful situation than to enhancedfear” (Spruijt et al., 1992, p. 828). In this regard, it is noteworthythat the RHA-I strain, besides showing increased grooming duringthe period just after the acquisition of the avoidance response, alsoachieved the highest rates of successful avoidances in that period.These rates indicate better coping with the task. In better coping (i.e.increased “controllability”) the RHA-I rats could be able to begin todearouse. The rats from the RLA-I strain, which, in spite of beingable (some of them) to achieve the avoidance acquisition criterion,had lower rates of successful avoidances, could not dearouse dur-ing the period just after the acquisition of the avoidance responseand, accordingly, showed decreased grooming activity.

The fact that low grooming activity was found during the initialphase of the avoidance training (when the avoidance responseswere not yet established) is coherent with the suggestion thatthis behavior is rather suppressed during moments of enhancedfear, conflict and/or uncontrollability (Estanislau, 2012), when riskassessment (or even freezing; e.g. see Aguilar et al., 2004; López-Aumatell et al., 2011; Vicens-Costa et al., 2011; Díaz-Morán et al.,2012) is expected to prevail. Related to that contention, low groom-

ing activity has been reported while rats are under an intenseaversive experience, such as during exposure to a novel and highlyilluminated open-field (Bouwknecht et al., 2007) or during con-finement to an open arm of a plus-maze (Mairesse et al., 2007). The

2 ence R

gIannmteitssott(sidt

5

avgoanirssrartiaytc

tt1tagalpo

reepe2OuiR

00 C. Estanislau et al. / Neurosci

rooming component that most clearly was increased in the RHA- strain during the period after the achievement of the avoidancecquisition criterion was rostral. It is difficult to imagine an expla-ation for that somewhat selective increase. Some data support theotion that the rostral component can express the anxiety level, asentioned earlier in this discussion. On the other hand, the sugges-

ion that grooming can be increased in dearousal situations (Spruijtt al., 1992) is not specific for the particular grooming componentsmplied. Nevertheless, the results from the present work suggesthat the body component could only be expected to increase afterome habituation to a mild stressor (novel environment), i.e. afterome time of exposure to the situation. It is also worth to pointut that, in contrast to the experience of being in an environmento which the subject is already habituated, the two-way avoidancerials, even after the task is learned, require a great deal of attentionsee discussion in López-Aumatell et al., 2008). Therefore, the ten-ion involved in successfully behaving in the task could be involvedn the predominance of the rostral component in the groomingisplayed by the RHA-I rats during the two-way active avoidanceraining.

. Conclusion

In the present study, rats from strains/stocks which differ innxiety/stress responsiveness and coping styles were studied inarious (unconditioned and conditioned) situations. Differences inrooming among the strains were found to be specific to eachf these situations. No differences were found in the home-cage,

situation that can be seen as calm and unthreatening. Underovelty (i.e. open-field and hole-board tests), a situation of declin-

ng aversiveness along the time (because of habituation), RHA-Iats showed lower grooming activity than the other strains, whichhowed gradual increases until around half of the length of the ses-ion. A difference in the opposite direction was found when theats seemed to become able to avoid foot-shocks in the two-wayctive – shuttle-box – avoidance task, a learning stage in which theats can experience some degree of controllability (it is remarkablehat RHA-I rats showed higher rates of successful avoidances dur-ng that period). Outstandingly, no differences among the strainsnd poor grooming activity was observed when the rats did notet show quick escape responses in the shuttle-box task, a stage ofhe aversive learning task that is likely to be experienced as poorlyontrollable.

The absence of differences in the home-cage is suggestive thathe differences observed in the other situations were related toheir aversive/stressful nature. The increases in grooming found0–15 min after the beginning of the exposure to novelty (i.e. whenhe animal faced a potentially risky environment, investigated itnd became habituated to it) are coherent with the notion thatrooming takes place during the process of dearousal. Addition-lly, the differences found among the strains (the RHA-I showingow grooming activity) suggest that the grooming response can beroportional to the arousal elicited and then can be seen as an indexf the degree of that arousal.

Different pictures emerged when assured or potential expe-ience with aversive stimuli were under study. Assured aversivexperience (i.e. certainty of a given aversive event), such as the ‘poorscape’ phase of the avoidance training, resulted in grooming sup-ression. Instead, defensive reactions (freezing, risk assessment,tc.) are expected, as previously suggested elsewhere (Estanislau,012, see also Vicens-Costa et al., 2011; Díaz-Morán et al., 2012).

n the other hand, when potential experience with aversive stim-li was under way, some grooming could occur. In this situation,

nitially, grooming is predominantly rostral (as indicated by theHA-I performance in the ‘successful avoidances’ phase of the

esearch 77 (2013) 187–201

avoidance training). Furthermore, as already discussed, if no dan-ger was perceived by the subject after some time in a potentiallyrisky environment (novelty), increased grooming (mainly due tochanges in its body component) was observed and it is supposedto accompany a dearousal process.

Grooming is an activity that is neglected in many rodent stud-ies in research fields such as stress, anxiety, fear, etc. This occursdespite the vast literature implying grooming with stress and(de)arousal and the fact that this is one the behaviors most regu-larly seen in test situations important for these fields. Nevertheless,some reports have indicated grooming as a behavioral index thatcan be useful in those fields. Further characterization of the groom-ing response (including its regional distribution and time course)under situations differentially related to arousal, dearousal, poten-tial or assured aversive conditions, conflict, etc. are still needed.That characterization could be heuristic about the usefulness ofgrooming in the study of the processes involved in different behav-ioral disorders and enlarge the avenue toward the investigation oftheir pharmacological modulation and neurobiology.

Acknowledgments

Supported by grants for the MICINN (PSI2009-10532), “FundacióLa Maratò TV3” (ref. 092630/31), 2009SGR-0051 and the Europeanproject/consortium “EURATRANS” (grant agreement HEALTH-F4-2010-241504). C.E. was recipient of a postdoctoral fellowship fromCNPq (201456/2011-7) while the experiments were carried out andnow is recipient of a research fellowship from Fundac ão Araucária(817/2012).

References

Aguilar, R., Gil, L., Fernández-Teruel, A., Tobena, A., 2004. Genetically-based behav-ioral traits influence the effects of shuttle box avoidance overtraining andextinction upon intertrial responding: a study with the Roman rat strains. Behav.Processes 66, 63–72.

Berridge, K.C., Aldridge, J.W., Houchard, K.R., Zhuang, X., 2005. Sequential super-stereotypy of an instinctive fixed action pattern in hyper-dopaminergic mutantmice: a model of obsessive compulsive disorder and Tourette’s. BMC Biol. 3, 4.

Berridge, K., Fentress, C., Parr, J.C.H., 1987. Natural syntax rules control actionsequence of rats. Behav. Brain Res. 23, 59–68.

Bindra, D., Spinner, N., 1958. Response to different degrees of novelty: the incidenceof various activities. J. Exp. Anal. Behav. 1, 341–350.

Bolles, R.C., 1960. Grooming behavior in the rat. J. Comp. Physiol. Psychol. 53,306–310.

Bouwknecht, J.A., Spiga, F., Staub, D.R., Hale, M.W., Shekhar, A., Lowry, C.A., 2007.Differential effects of exposure to low-light or high-light open-field on anxiety-related behaviors: relationship to c-Fos expression in serotonergic and non-serotonergic neurons in the dorsal raphe nucleus. Brain Res. Bull. 72, 32–43.

Carrasco, J., Márquez, C., Nadal, R., Tobena, A., Fernández-Teruel, A., Armario,A., 2008. Characterization of central and peripheral components of thehypothalamus–pituitary–adrenal axis in the inbred Roman rat strains. Psy-choneuroendocrinology 33, 437–445.

Consoli, D., Leggio, G.M., Mazzola, C., Micale, V., Drago, F., 2007. Behavioral effectsof the �3 adrenoceptor agonist SR58611A: is it the putative prototype of a newclass of antidepressant/anxiolytic drugs? Eur. J. Pharmacol. 573, 139–147.

Cuenya, L., Sabariego, M., Donaire, R., Fernández-Teruel, A., Toneba, A., Mustaca, A.,Torres, C., 2012. The effect of partial reinforcement on instrumental successivenegative contrast in inbred Roman high- (RHA-I) and low-(RLA-I) avoidance rats.Physiol. Behav. 105, 1112–1116.

De Wied, D., Jolles, J., 1982. Neuropeptides derived from pro-opiocortin: behavioral,physiological, and neurochemical effects. Physiol. Rev. 62, 976–1059.

Díaz-Morán, S., Martínez-Membrives, E., López-Aumatell, R., Canete, T., Blázquez,G., Palencia, M., Mont-Cardona, M., Estanislau, C., Tobena, A., Fernández-Teruel,A., 2013. What can we learn on rodent fearfulness/anxiety from the geneticallyheterogeneous NIH-HS rat stock? Open J. Psychiatry 3, 238–250.

Díaz-Morán, S., Mont-Cardona, C., Canete, T., Blázquez, G., Martínez-Membrives, E.,López-Aumatell, R., Tobena, A., Fernández-Teruel, A., 2012. Coping style andstress hormone responses in genetically heterogeneous rats: comparison withthe Roman rat strains. Behav. Brain Res. 228, 203–210.

Drago, F., Canonico, P.L., Bitetti, R., Scapagnini, U., 1980. Systemic and intraventric-ular prolaction induces excessive grooming. Eur. J. Pharmacol. 65, 457–458.

Driscoll, P., Fernández-Teruel, A., Corda, M.G., Giorgi, O., Steimer, T., 2009. Someguidelines for defining personality differences in rats. In: Yong-Kyu, K. (Ed.),Handbook of Behavior Genetics. Springer, New York, pp. 281–300.

ence R

E

E

F

F

F

F

G

G

H

J

K

K

K

L

L

C. Estanislau et al. / Neurosci

scorihuela, R.M., Fernández-Teruel, A., Gil, L., Aguilar, R., Tobena, A., Driscoll,P., 1999. Inbred Roman high- and low-avoidance rats: differences in anx-iety, novelty-seeking, and shuttlebox behaviors. Physiol. Behav. 67, 19–26.

stanislau, C., 2012. Cues to the usefulness of grooming behavior in the evaluationof anxiety in the elevated plus-maze. Psychol. Neurosci. 5, 105–112.

ernández-Teruel, A., Driscoll, P., Gil, L., Aguilar, R., Tobena, A., Escorihuela,R.M., 2002. Enduring effects of environmental enrichment on novelty seek-ing, saccharin and ethanol intake in two rat lines (RHA/Verh and RLA/Verh)differing in incentive-seeking behavior. Pharmacol. Biochem. Behav. 73,225–231.

ernández-Teruel, A., Escorihuela, R.M., Núnez, J.F., Gomà, M., Driscoll, P., Tobena,A., 1992. Early stimulation effects on novelty-induced behavior in twopsychogenetically-selected rat lines with divergent emotionality profiles. Neu-rosci. Lett. 137, 185–188.

erré, P., Fernández-Teruel, A., Escorihuela, R.M., Driscoll, P., Corda, M.G., Giorgi, O.,Tobena, A., 1995. Behavior of the Roman/Verh high- and low-avoidance rat linesin anxiety tests: relationship with defecation and self-grooming. Physiol. Behav.58, 1209–1213.

ile, S., Wardill, A.G., 1975. Validity of head-dipping as a measure of exploration ina modified hole-board. Psychopharmacology 44, 53–59.

ispen, W.H., Wiegant, V.M., Greven, H.M., De Wied, D., 1975. The induction of exces-sive grooming in the rat by intraventricular application of peptides derived fromACTH: structure-activity studies. Life Sci 17, 645–652.

uitart-Masip, M., Giménez-Llort, L., Fernández-Teruel, A., Canete, T., Tobena, A.,Ogren, S.O., Terenius, L., Johansson, B., 2006. Reduced ethanol response in thealcohol-preferring RHA rats and neuropeptide mRNAs in relevant structures.Eur. J. Neurosci. 23, 531–540.

ansen, C., Spuhler, K., 1984. Development of the National Institutes of Health genet-ically heterogeneous stock. Alcohol Clin. Exp. Res. 8, 477–479.

olles, J., Rompa-Barendregt, J., Gispen, W.H., 1979. Novelty and grooming behaviorin the rat. Behav. Neural Biol. 25, 563–572.

alueff, A.V., Tuohimaa, P., 2005a. The grooming analysis algorithm discriminatesbetween different levels of anxiety in rats: potential utility for neurobehaviouralstress research. J. Neurosci. Methods 143, 169–177.

alueff, A.V., Tuohimaa, P., 2005b. Mouse grooming microstructure is a reliable anx-iety marker bidirectionally sensitive to GABAergic drugs. Eur. J. Pharmacol. 508,147–153.

omorowska, J., Pisula, W., 2003. Does changing levels of stress affect thecharacteristics of grooming behavior in rats? Int. J. Comp. Psychol. 16,237–246.

ópez-Aumatell, R., Blázquez, G., Gil, L., Aguilar, R., Canete, T., Giménez-Llort, L.,

Tobena, A., Fernández-Teruel, A., 2009a. The Roman high- and low-avoidancerat strains differ in fear-potentiated startle and classical aversive conditioning.Psicothema 21, 27–32.

ópez-Aumatell, R., Guitart-Masip, M., Vicens-Costa, E., Gimenez-Llort, L., Valdar, W.,Johannesson, M., Flint, J., Tobena, A., Fernández-Teruel, A., 2008. Fearfulness in a

esearch 77 (2013) 187–201 201

large N/Nih genetically heterogeneous rat stock: differential profiles of timidityand defensive flight in males and females. Behav. Brain Res. 188, 41–55.

López-Aumatell, R., Martínez-Membrives, E., Vicens-Costa, E., Canete, T., Blázquez,G., Mont-Cardona, C., Johannesson, M., Flint, J., Tobena, A., Fernández-Teruel, A.,2011. Effects of environmental and physiological covariates on sex differences inunconditioned and conditioned anxiety and fear in a large sample of geneticallyheterogeneous (N/Nih-HS) rats. Behav. Brain Funct. 7, 48.

López-Aumatell, R., Vicens-Costa, E., Guitart-Masip, M., Martínez-Membrives, M.,Valdar, W., Johannesson, M., Canete, T., Blázquez, G., Driscoll, P., Flint, J., Tobena,A., Fernández-Teruel, A., 2009b. Unlearned anxiety predicts learned fear: a com-parison among heterogeneous rats and the Roman rat strains. Behav. Brain Res.202, 92–101.

Mairesse, J., Viltart, O., Salome, N., Giuliani, A., Catalani, A., Casolini, P., Morley-Fletcher, S., Nicoletti, F., Maccari, S., 2007. Prenatal stress alters the negativecorrelation between neuronal activation in limbic regions and behavioralresponses in rats exposed to high and low anxiogenic environments. Psychoneu-roendocrinology 32, 765–776.

Meisenberg, G., 1988. Vasopression-induced grooming and scratching behavior inmice. Ann. N.Y. Acad. Sci. 525, 257–269.

Rosas, J.M., Callejas-Aguilera, J.E., Escarabajal, M., Gómez, M.J., de la Torre, L., Agüero,A., Tobena, A., Fernández-Teruel, A., Torres, C., 2007. Successive negative contrasteffect in instrumental runway behaviour: a study with Roman high- (RHA) andRoman low- (RLA) avoidance rats. Behav. Brain Res. 185, 1–8.

Spruijt, B., De Graan, P.N.E., Eberle, N., Gispen, W.H., 1985. Comparison of structuralrequirements of �-MSH and ACTH for inducing excessive grooming and pigmentdispersion. Peptides 6, 1185–1189.

Spruijt, B.M., Van Hoof, J.A.R.A.M., Gispen, W.H., 1992. Ethology and neurobiology ofgrooming behavior. Physiol. Rev. 72, 825–852.

Steimer, T., Driscoll, P., 2003. Divergent stress responses and coping styles inpsychogenetically selected Roman high-(RHA) and low-(RLA) avoidance rats:behavioral, neuroendocrine and developmental aspects. Stress 6, 87–100.

Steimer, T., Escorihuela, R.M., Fernández-Teruel, A., Driscoll, P., 1998. Long-termbehavioural and neuroendocrine changes in Roman high-(RHA/Verh) and low-(RLA-Verh) avoidance rats following neonatal handling. Int. J. Dev. Neurosci. 16,165–174.

Steimer, T., la Fleur, S., Schulz, P.E., 1997. Neuroendocrine correlates of emotionalreactivity and coping in male rats from the Roman high (RHA/Verh)- and low(RLA/Verh)-avoidance lines. Behav. Genet. 27, 503–512.

van Erp, A.M.M., Kruk, M.R., Meelis, W., Willekens-Bramer, D.C., 1994. Effect of envi-ronmental stressors on time course, variability and form of self-grooming in therat: handling, social contact, defeat, novelty, restraint and fur moistening. Behav.Brain Res. 65, 47–55.

Vicens-Costa, E., Martínez-Membrives, E., López-Aumatell, R., Guitart-Masip, M.,Canete, T., Blázquez, G., Tobena, A., Fernández-Teruel, A., 2011. Two-way avoid-ance acquisition is negatively related to conditioned freezing and positivelyassociated with startle reactions: a dissection of anxiety and fear in geneticallyheterogeneous rats. Physiol. Behav. 103, 148–156.