evaluation of lewis and shr rat strains as a genetic model for the study of anxiety and pain
TRANSCRIPT
Behavioural Brain Research 129 (2002) 113–123
Research report
Evaluation of Lewis and SHR rat strains as a genetic model forthe study of anxiety and pain
Andre Ramos a,*, Anderson L. Kangerski a, Paula F. Basso a,Jose E. Da Silva Santos b, Jamil Assreuy b, Leandro F. Vendruscolo b,
Reinaldo N. Takahashi b
a Laboratorio de Genetica do Comportamento, Departamento de Biologia Celular, Embriologia e Genetica, Centro de Ciencias Biologicas,Uni�ersidade Federal de Santa Catarina, 88.040-900, Florianopolis, SC, Brazil
b Departamento de Farmacologia, Uni�ersidade Federal de Santa Catarina, 88.015-420, Florianopolis, SC, Brazil
Received 10 May 2001; received in revised form 16 July 2001; accepted 16 July 2001
Abstract
The study of inbred strains of rodents that differ for specific behaviours can help us to understand the biological mechanismsunderlying complex psychological traits. Lewis (LEW) and SHR inbred rat strains, for example, have been recently proposed asa genetic model for the study of anxiety. Our goal was to characterise two Brazilian substrains of LEW and SHR rats, that havenever been compared before, behaviourally and/or pharmacologically, in order to evaluate their potential contribution to studieson anxiety and pain. Male and female LEW and SHR rats were submitted after 8 weeks of age to five anxiety/emotionality tests:the open field (7 or 260 lux), the elevated plus-maze, the elevated T-maze and the black/white box. Rats of all groups were alsosubmitted to the formalin test of nociception and measurement of blood pressure. Significant strain differences (P�0.05) wereobserved in both sexes for all indices of anxiety and also for measures of pain and blood pressure. SHRs, compared with LEWs,explored more the aversive environments of all anxiety tests, showed less nociceptive responses and were hypertensive. Alldifferences in experimental anxiety parameters agree with previous differences reported between two French LEW and SHRsubstrains, suggesting that LEWs are more anxious than SHRs, thus consolidating these strains as a useful genetic model for thestudy of anxiety and pain. The possible involvement of tachykinergic mechanisms is discussed. © 2002 Elsevier Science B.V. Allrights reserved.
Keywords: Behaviour genetics; Anxiety; Nociception; Spontaneously hypertensive rats; Lewis; Open field; Elevated plus-maze
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1. Introduction
Different epidemiological studies indicate that anxi-ety-related disorders are the most frequent psychiatricdisorders in the modern human society. These studiespoint out to an early age-of-onset (7–12 years), alifetime rate of prevalence exceeding 10% of the popula-tion and a preponderance of females among differentcategories of anxious patients [17,18,43]. Nevertheless,
the development of more efficient and specific methodsof diagnosis and treatment of anxiety is limited by thelack of a comprehensive knowledge concerning thebiological mechanisms underlying anxiety-related traitsand disorders.
Classical studies on human personality indicate thatnormal personality traits which are related to anxietyare highly heritable (near 50%) [3]. Moreover, geneticeffects may explain as much as 47% of the variance insubclinical forms of phobic fears [34] and they alsohave significant influences on the development of vari-ous anxiety-related psychopathologies [23,32,40,43]. Ithas been thoroughly demonstrated that genetics modu-
* Corresponding author. Tel.: +55-48-331-5153; fax: +55-48-331-5148.
E-mail address: [email protected] (A. Ramos).
0166-4328/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 6 -4328 (01 )00337 -0
A. Ramos et al. / Beha�ioural Brain Research 129 (2002) 113–123114
lates many aspects of emotionality in laboratory ani-mals [25]. Therefore, genetic and biochemical analysesof animal models of anxiety (e.g. two inbred strainsthat differ for anxiety-related behaviours) represent apowerful tool to identify and dissect the etiologicalbases of anxiety disorders.
We have recently proposed [26,27] that the inbred ratstrains Lewis/NIco (LEW) and Spontaneously Hyper-tensive Rats/NIco (SHR) (commercialised by IFFACREDO, France) constitute a useful genetic model forthe study of anxiety. The use of inbred strains make iteasier to dissociate genetic and environmental compo-nents of a trait because the animals within each strainare theoretically identical in their genotype. The strainsLEW and SHR differed significantly from each otherwhen submitted to several behavioural tests of anxiety/emotionality. LEW rats of both sexes displayed higherlevels of avoidance of different types of anxiogenicstimuli (the open arms of the elevated plus-maze, thecentral area of the open field and the white compart-ment of the black/white box) than their SHR counter-parts. It is interesting to notice, however, thatdifferently from other genetic models of emotionalitythat tend to react either passively or actively to stress,the LEW and SHR strains did not differ in their levelsof activity in either novel or familiar environments[25–27]. In addition, pharmacological tests using anxi-olytic and anxiogenic drugs provided further support toour hypothesis that the behavioural differences ob-served between LEW and SHR rats were anxiety-re-lated [26].
Many strains of rodents are used in biomedical re-search all over the world. This fact leads to subpopula-tions of the same strain to be raised independently fromeach other by different laboratories and in differentcountries. Over time, accumulated mutations and ge-netic drift are expected to produce a certain degree ofvariability between these subpopulations, which maycause changes in traits of interest including behaviouralones. Significant anxiety-related differences have beenfound between substrains of rats obtained from differ-ent breeders [8,29]. More importantly, there are reportsof significant stress-related differences between sub-strains of Lewis rats and also between SHR and SHRstroke-prone rats [8,36]. Thus, concerning the afore-mentioned LEW/SHR model, in order to replicate itand to propose its use for the study of anxiety indifferent countries, we needed first to verify if otherpopulations of LEW and SHR rats would display thesame anxiety-related differences found between theFrench substrains LEW/NIco and SHR/NIco. Onemain objective of the present study was, therefore, tocharacterise two Brazilian colonies of LEW and SHRrats that have never been compared before in a batteryof behavioural tests of anxiety/emotionality in order tovalidate these substrains (and hence consolidate the
strains as such) as a genetic model of anxiety. The testsapplied, all widely-used, were, the open field (eitherdimly or strongly illuminated); the elevated plus-maze;the black/white box and the elevated T-maze.
An intercross between LEW/NIco and SHR/NIcorats has been recently used to identify and map, for thefirst time, quantitative trait loci (QTL) for emotional-ity-related behaviours in rats [28]. Among several sug-gestive loci, one major QTL was identified on ratchromosome 4 for central locomotion in the open fieldtest, a putative measure of anxiety. This genomic re-gion, which affected only females, was found to alsoharbour the gene Tac1r for the tachykinin receptorNK1 [28], which has been shown to affect a number ofanxiety-related traits in mice, rats and humans[6,16,39,44]. Thus, we have hypothesised that LEW andSHR strains may differ for the gene Tac1r. Becausesubstance P and its receptor NK1 are known for theirinvolvement in pain [31], we aimed to verify the hy-pothesis that LEW and SHR rats should show differ-ences in their behavioural measures of nociception inaddition to those related to anxiety. Therefore, wecompared these two strains in the widely-used formalintest of nociception.
Finally, because the original SHR strain (as stated byits name) has been created through a selective breedingprogram aiming at high spontaneous blood pressure(BP) [21], an additional objective of the present studywas to verify if the Brazilian substrains of LEW andSHR rats would also show, as expected, significantdifferences in their BP levels.
2. Materials and methods
2.1. Animals
Male and female Lewis (LEW) and SpontaneouslyHypertensive Rats (SHR) were obtained in 1999 fromUNICAMP (Campinas, SP, Brazil) and UFES (Vitoria,ES, Brazil), respectively. Since then, colonies of thesetwo inbred strains have been maintained in our labora-tory under a system of brother–sister mating. Rats ofboth sexes representing each of the two strains wereused in all experiments of this study. The animals wereweaned and separated by sex at 4 weeks of age and,thereafter, were kept in collective plastic cages (four orfive rats/cage) with food and water available ad libitumunder a 12-h light:12-h dark schedule (lights on at 07:00h) at 23�2 °C. At 8 and 9 weeks of age, a group of 51rats was submitted once to the following sequence ofbehavioural tests of anxiety/emotionality, with an inter-val of 48 h between tests, the open field (dim light), theelevated plus-maze and the black/white box. At thesame age, another group of 60 naıve rats was submittedonce to the open field (strong light) and, starting two
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days later, to the elevated T-maze. At 10–12 weeks ofage, after behavioural testing, rats of both groups weresubmitted either to the formalin test of nociception orto a test of BP. All tests were carried out between 13:00and 18:00 h.
2.2. Open field (dim light or strong light)
The apparatus, made of wood covered with imperme-able formica, had a white floor of 100×100 cm (di-vided by black lines into 25 squares of 20×20 cm) andwhite walls, 40 cm high. The test room had a controlledillumination, providing either 7 lux (dim-light condi-tion) or 260 lux (strong-light condition) inside theapparatus. Such a variation had the objective of pro-voking two different levels of stress in the animals, sincebright light is usually more aversive than low light [25].Each rat was placed in the centre of the open field andthe following variables were registered, through directvisual observation, for 5 min, number of peripheralsquares (adjacent to the walls) crossed (=outer loco-motion), number of central squares (away from thewalls) crossed (= inner locomotion), time in the centralarea (= inner time) and total number of faecal boli(=defecation). The percentage of inner locomotionwas calculated in relation to the total number ofsquares crossed in the whole arena. The floor wascleaned with a wet sponge and a dry paper towelbetween rats, the same procedure being adopted for allother behavioural tests.
2.3. Ele�ated plus-maze
The apparatus was made of wood covered with alayer of black formica and had four elevated arms (52cm from the floor) 50 cm long and 10 cm wide. Thearms were arranged in a cross-like disposition, with twoopposite arms being enclosed (by 40 cm high walls) andtwo being open, having at their intersection a centralplatform (10×13.5 cm) which gave access to any of thefour arms. The open arms were surrounded by a raisedledge (1 mm thick and 5 mm high) to avoid rats fallingoff the arms. The central platform was under 70 lux ofillumination. Each rat was placed in the central plat-form facing an open arm and the following behaviourswere registered, through direct visual observation, for 5min, the number of entries and the time spent (with allfour paws) inside each type of arm and the percentageof open-arm entries in relation to the total number ofarm entries.
2.4. Black and white box
The apparatus was made of wood covered withformica and presented two compartments. One larger(27×27×27 cm) and white, with the floor divided by
black lines in nine squares (9×9 cm), being stronglyilluminated by a 40 W white bulb. The other smaller(27×18×27 cm high) and black, with the floor di-vided by white lines in six squares (9×9 cm), beingilluminated only by a 40 W red bulb. Both white andred bulbs were located 30 cm above the apparatus’floor, thus providing a 1100 lux intensity inside thewhite compartment and a 25 lux intensity inside theblack compartment. The two compartments, separatedby a wall, were connected by a small square opening of7×7 cm. Each rat was placed in the centre of the whitecompartment facing the small opening and the follow-ing variables were registered, through direct visual ob-servation, for 5 min, number of squares crossed(locomotion) and time spent with all four paws in eachcompartment, number of transitions between compart-ments (one entry in the white plus one return to theblack) and total number of feacal boli produced (defe-cation). The time spent in the white compartment didnot include the initial latency to the first entry into theblack compartment.
2.5. Ele�ated T-maze
This test, derived from the elevated plus-maze, wasoriginally developed to separate conditioned from un-conditioned fear [45]. Differently from the classicalelevated plus-maze test that uses one single trial and isbased on innate fear of height and openness, the ele-vated T-maze involves several consecutive trials thusallowing the animal to acquire inhibitory avoidance ofthe open arms (conditioned fear). Depending on theprotocol, this test may also assess the escape responseof rats placed once at the end of an open arm, abehaviour thought to reflect unconditioned fear [10,45].Also in contrast with measures of anxiety from theelevated plus-maze, which respond variably to 5-HTacting drugs, the learned inhibitory avoidance assessedin the elevated T-maze has been shown to respondconsistently to benzodiazepines as well as to substancesthat are able to modify 5-HT activity [10].
The apparatus we have used was the same as the onedescribed above (see Section 2.3), differing only by thefact that one enclosed arm of the plus maze was totallyblocked by a black plastic barrier (as high as the arm’swall), giving to the maze a T shape formed by two openarms and one enclosed arm (the stem of the T). Theprocedure used herein, modified from the original pro-tocol, has been used by Carobrez and co-workers as amodel of emotional memory [30]. During the trainingsession (Day 1), each rat was placed at the end of theenclosed arm. Whenever, during a maximum period of5 min, the rat entered with all four paws into one of theopen arms, the rat was immediately removed from theapparatus and placed for 30 s in a plastic container (20cm in diameter and 25 cm high). Following this, the
A. Ramos et al. / Beha�ioural Brain Research 129 (2002) 113–123116
animal returned to the enclosed arm and the sessionwas resumed. This procedure was repeated as manytimes as necessary until reaching the criterion of 5 minwithout entering an open arm (inhibitory avoidance).The number of trials necessary for the rat to acquire theinhibitory avoidance was registered. During the testingsession (Day 2), each rat was placed at the end of theenclosed arm and the latency to enter an open arm(duration of inhibitory avoidance) was recorded up to amaximum of 5 min.
2.6. Formalin test
The formalin test of nociception was carried out inan open acrylic box measuring 28×28×28 cm, with amirror placed under the floor to allow unobstructedview of the rat’s paws. Each rat was injected with 50 �lof formalin 5% into the dorsal region of the righthindpaw and then observed for 60 min. The number offlinches of the injected paw was registered. The test hadtwo different phases possibly reflecting two differenttypes of pain [13], the early phase of nociceptive re-sponse, between 0 and 10 min after formalin injection,and the late phase, between 10 and 60 min after forma-lin injection.
2.7. Blood pressure
Under anesthesia with ketamine and xylazine (90 and15 mg/kg, respectively), heparinized PE 20 polyethylenecatheter was inserted into the right carotid artery forrecording of systolic and diastolic arterial pressure. Toprevent clotting, an intraperitoneal dose of heparin (300I.U.) was injected 10 min before the ketamine/xylazineinjection. Animals were allowed to breath sponta-neously via a tracheal cannula and body temperaturewas monitored by a rectal thermometer (maintained at36�1 °C). After the surgical procedure, a period of 30min was alowed for stabilization and the systolic anddiastolic arterial BP from male and female LEW andSHR rats were recorded during 60 min. BP data wererecorded (at a 10 s sampling rate) with a Digi-Med BPAnalyser system (Model 190) connected to a Digi-MedSystem Integrator (Model 200; Louisville, KY) soft-ware, running under Windows 98™ (Microsoft Corpo-ration, Redmond, WA). Results are expressed asmean�S.E.M. of systolic or diastolic arterial pressure(mmHg). At the end of the experiment, animals weresacrificed with pentobarbitone overdose.
2.8. Statistics
Previously to any comparison of means, all depen-dent variables were submitted to Levene’s test of homo-geneity of variances and to Shapiro–Wilks’ W-test ofnormality. Because most variables failed to satisfy ei-
ther one or both these criteria and because data trans-formation was mostly unsuccessful, all statisticalanalysis was based on nonparametric tests. Differencesbetween LEW and SHR strains were assessed for eachsex by the Mann–Whitney rank test. Similarly, malesand females were compared within each strain by themeans of the same test. All tests were performed usingthe Statistica® software package.
3. Results
3.1. Open field (dim light or strong light)
Under both dim- and strong-light conditions, LEWand SHR rats of both sexes differed significantly for allmeasures of approach/avoidance towards the centraland aversive area of the open field. Under dim light,SHR rats crossed more squares (P�0.0001 for malesand P�0.001 for females), spent more time (P�0.001for males and P�0.0001 for females) and showed ahigher percentage of locomotion (P�0.0001 for bothsexes) in the central area of the open field than theirLEW counterparts. Under strong light, similarly, theSHR strain crossed more squares, spent more time anddevoted a higher percentage of their locomotion (P�0.0001 for all variables and both sexes) in the centre ofthe open field than the LEW strain. On the other hand,LEW rats defecated more than SHR rats under bothdim-light (P�0.0001 for males and P�0.01 for fe-males) and strong-light (P�0.01 for males and P�0.05 for females) conditions. Interestingly, the twostrains did not differ, under either type of illumination,in their total locomotion in the open field (P�0.05 forboth sexes).
Significant sexual differences were also detected forsome variables. Under dim light, females displayedhigher levels of inner locomotion (P�0.05 for bothstrains), percentage of inner locomotion (P�0.05 forLEW only), inner time (P�0.05 for both strains) andtotal locomotion (P�0.05 for both strains) when com-pared with males. No differences were found for defe-cation scores. Under strong light, female rats (SHRonly) showed higher inner locomotion (P�0.01) andinner time (P�0.05) and lower defecation (P�0.05)than male rats. Yet, females of both strains showedhigher total locomotion (P�0.05 for LEW and P�0.001 for SHR) than males. Mean values grouped bystrain and sex are presented in Table 1 and Fig. 1.
3.2. Ele�ated plus-maze
Results grouped by strain and sex are presented inTable 2 and Fig. 2. The two strains differed for allindices of anxiety from the elevated plus-maze. SHRrats spent more time in the aversive open arms (P�
A. Ramos et al. / Beha�ioural Brain Research 129 (2002) 113–123 117
Table 1Open field (dim light and strong light) measures (X�S.E.M.) of 8-week old LEW and SHR rats grouped by sex
MalesVariable Females
SHR LEWLEW SHR
Dim light (n=14) (n=13) (n=10) (n=14)Inner locomotion **27.5�2.910.6�1.4 c17.7�2.4 ** c40.1�3.6
**32.9�3.5 c16.8�2.49.8�1.7 ** c46.8�4.1Inner time (s)**3.4�0.5Defecation 0.1�0.1 **1.8�0.8 0.0�0.0
(n=20)Strong light (n=10)(n=10) (n=20)**23.4�1.4 6.1�1.56.4�1.7 ** c c31.9�2.4Inner locomotion
4.0�1.6Inner time (s) **45.2�3.0 8.0�3.1 ** c55.8�4.3Defecation **3.1�0.7 c0.7�0.3 *1.4�0.6 0.1�0.1
For each sex and light condition, significant interstrain differences are represented by * and ** and, for each strain and light condition, significantsex differences are represented by c and c c (P�0.05 and P�0.01, Mann–Whitney U-test). Significance signs are always placed on the highergroup.
0.01 for both sexes), spent less time in the protectedclosed arms (P�0.05 for males and P�0.01 for fe-males) and made a higher percentage of open-armentries (P�0.05 for males only) than LEW rats. Inter-estingly, the two strains did not differ for the twomeasures of locomotion of the elevated plus-maze,
namely number of entries in the closed arms and totalnumber of arm entries (P�0.05 for both sexes).
Regarding sex effects, females showed lower scores ofanxiety and higher scores of locomotion than males.Females spent more time in the open arms (P�0.001for LEW and P�0.01 for SHR), spent less time in theclosed arms (P�0.05 for LEW and P�0.001 forSHR), made a higher percentage of open-arm entries(P�0.01 for LEW and P�0.05 for SHR) and mademore total-(P�0.001 for LEW and P�0.05 for SHR)and closed-arm entries (P�0.05 for LEW only) thanmales.
3.3. Black and white box
The results for this test are shown in Table 3 and Fig.3. Significant strain differences were found for all mea-sures of the black/white box, with the SHR strainshowing higher levels of approach towards the aversivewhite compartment. Thus, SHRs spent more time in thewhite compartment (P�0.001 for males and P�0.05for females), spent less time in the black compartment(P�0.0001 for males and P�0.05 for females), mademore transitions between compartments (P�0.01 formales and P�0.05 for females), displayed higher loco-motion in the white compartment (P�0.001 for malesand P�0.05 for females) and lower locomotion in theblack compartment (P�0.01 for females only) thanLEWs.
Females of both strains spent more time in the whitecompartment (P�0.05 for both strains), made moretransitions between compartments (P�0.01 for bothstrains), displayed higher locomotion in the white com-partment (P�0.01 for both strains) and higher loco-motion in the black compartment (P�0.001 for LEWand P�0.05 for SHR). Moreover, females LEW spentless time in the black compartment than their malecounterparts (P�0.05).
Fig. 1. Percentage of inner locomotion and total locomotion in theopen field for LEW and SHR rats tested under two light conditions,dim light (DL) and strong light (SL). Bars and vertical lines representthe means and SEM of animals grouped by sex, strain and lightcondition (n for all groups are shown in Table 1). For each sex andlight condition, significant interstrain differences are represented by *and ** and, for each strain and light condition, significant sexdifferences are represented by c and c c (P�0.05 and P�0.01,Mann–Whitney U-test). Significance signs are always placed on thehigher group.
A. Ramos et al. / Beha�ioural Brain Research 129 (2002) 113–123118
Table 2Elevated plus-maze measures (X�S.E.M.) of 8-week-old LEW andSHR rats grouped by sex
Open-armStrain Closed-arm Time closed armsnentries (%) entries (s)
Males20.1�2.9 9.2�0.6 * c176.9�12.9LEW 14
*29.0�3.4 9.2�0.5 cc138.4�13.313SHR
Females10LEW cc36.3�2.5 c11.6�0.8 **136.3�10.8
c40.3�2.9 10.6�0.814 85.9�4.8SHR
For each sex, significant interstrain differences are represented by *and ** and, for each strain, significant sex differences are representedby c and c c (P�0.05 and P�0.01, Mann–Whitney U-test).Significance signs are always placed on the higher group.
Fig. 3. Time in the white compartment (s) and total locomotion in theblack compartment in the black and white box for LEW and SHRrats. Bars and vertical lines represent the means and SEM of animalsgrouped by sex and strain (n for all groups are shown in Table 3). Foreach sex, significant interstrain differences are represented by * and** and, for each strain, significant sex differences are represented byc and c c (P�0.05 and P�0.01, Mann–Whitney U-test). Sig-nificance signs are always placed on the higher group.
3.4. Ele�ated T-maze
The results for this test, which has been proposed asa model of emotional learning, are also shown in Table3. No significant strain differences were found amongfemale rats. On the other hand, SHR males neededmore trials in the training session (P�0.01) to acquireinhibitory avoidance of the open arms and, during test(24 h later), showed a shorter latency (P�0.05) toenter an open arm (duration of inhibitory avoidance)
Fig. 2. Time in the open arms (s) and total number of arm entries inthe elevated plus-maze for LEW and SHR rats. Bars and vertical linesrepresent the means and SEM of animals grouped by sex and strain(n for all groups are shown in Table 2). For each sex, significantinterstrain differences are represented by * and ** and, for eachstrain, significant sex differences are represented by c and c c(P�0.05 and P�0.01, Mann–Whitney U-test). Significance signsare always placed on the higher group.
when compared with LEW males. Gender differenceswere found for duration of inhibitory avoidance onlyamong LEW rats, with females showing a shorter la-tency than males (P�0.05) during test.
3.5. Formalin test
As shown in Fig. 4, LEW and SHR rats of bothsexes displayed highly significant differences related tonociceptive responses in the formalin test. In the latephase (Phase 2), LEW rats seemed hyperalgesic whencompared with SHR rats, thus displaying a highernumber of paw flinchings after formalin injection (P�0.001 for males and P�0.01 for females). In the earlyphase (Phase 1), on the other hand, in spite of a trendin the same direction (LEW�SHR) being observed inboth sexes, strain differences were not significant andonly approached significance levels in males (P=0.057). No gender differences were observed for LEWrats in either phase of the test, but for the SHR strain,females showed even less flinchings (P�0.01) thanmales in Phase 2.
3.6. Blood pressure
As expected, SHR males and females were found tobe hypertensive in relation to their LEW counterparts.
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Table 3Measures of the black and white box and the elevated T-maze (X�S.E.M.) of 9-week-old LEW and SHR rats grouped by sex
Strain Elevated T-mazeBlack/white box
Transitions Locomotion Inhibitory avoidance latency (s)Time in blackn n Number of trialsin white (s)
Males0.5�0.2LEW 7.1�1.214 ** c c282.8�3.0 10 0.9�0.2 * c300.0�0.00
SHR 13 **2.2�0.4 **20.2�2.9 238.1�7.6 20 **1.9�0.2 237.9�22.5
Females10LEW cc2.5�0.7 cc 24.6�6.0 *259.7�8.9 10 1.1�0.2 191.4�39.3
* c c4.4�0.5 * c c36.9�4.0 226.0�9.1 20 1.5�0.3 176.0�29.2SHR 14
For each sex, significant interstrain differences are represented by * and ** and, for each strain, significant sex differences are represented by cand c c (P�0.05 and P�0.01, Mann–Whitney U-test). Significance signs are always placed on the higher group.
SHR rats had higher systolic (P�0.01 for both sexes)and higher diastolic (P�0.05 for both sexes) BP thanLEW rats. No significant sexual differences (P�0.05)were detected either for systolic or diastolic BP. Meanvalues for each group are presented in Table 4.
4. Discussion
The SHR strain, derived from Wistar rats, was devel-oped through selective breeding for high systolic BP in1963 [21]. Since then, SHR rats and their Wistar Kyoto(WKY) controls became one of the most widely usedmodels of hypertension. When compared with LEW/NIco, SHR/NIco rats have also shown to be clearlyhypertensive. However, factor analyses performed onan F2 population derived from these two strainsshowed that BP and anxiety-related behaviours werenot correlated [27]. In the present study, as expected,systolic and diastolic BP of SHR rats were significantlyhigher than those of LEW rats. The mean values ofsystolic BP found herein (between 90 and 135 mmHg),however, were overall lower than those found in ourprevious study with the /NIco substrains [27] (between157 and 230 mmHg). Such differences are likely theconsequence of changing methods of BP measurementbetween studies (indirectly on restrained conscious ratsvs. directly on anaesthetised rats). In order to verifywhether the strain differences in BP would change withage, we carried out a further experiment (data notshown) with male rats of 6–7 months of age. In theseolder rats, differences between LEW and SHR strains
Fig. 4. Number of times flinching the paw after injection of formalin5% in the formalin test of nociception for LEW and SHR rats. Phase1 (top) goes from 0 to 10 min and Phase 2 (bottom) from 10 to 60min after injection. Bars and vertical lines represent the means andS.E.M. of animals grouped by sex and strain (for LEW males, SHRmales and SHR females, n=9, for LEW females, n=6). For eachsex, significant interstrain differences are represented by * and ** and,for each strain, significant sex differences are represented by c andc c (P�0.05 and P�0.01, Mann–Whitney U-test). Significancesigns are always placed on the higher group.
Table 4Systolic and diastolic BP (X�S.E.M.) of 10 to12-week-old LEW andSHR rats grouped by sex
Diastolic BP (mmHg)Systolic BP (mmHg)Strain n
Males62.8�3.097.5�3.5LEW 6
*83.5�5.06 **124.5�6.6SHR
Females5 90.4�3.0LEW 60.4�7.1
*91.4�4.8**134.5�5.3SHR 8
For each sex, significant interstrain differences are represented by *and ** (P�0.05 and P�0.01, Mann–Whitney U-test). Significancesigns are always placed on the higher group.
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were slightly larger but mean BP values were overallsimilar.
One main goal of the present study was to character-ise the emotional profiles of two substrains of LEW andSHR rats in a battery of behavioural tests of anxiety/emotionality. Such a characterisation should serve toverify whether these substrains would display the sametype of anxiety-related differences as those previouslydescribed for another pair of LEW and SHR sub-strains, which have already been used as a model inquantitative and molecular studies on the genetics ofanxiety [27,28]. The open field, the elevated plus mazeand the black/white box are behavioural tests widelyrecognised as models of anxiety/emotionality [25]. Mea-sures of approach/avoidance which have been oftenused as indices of anxiety from these tests relate to theanimals’ reaction to specific anxiogenic stimuli that arepresent in each test apparatus. Thus, for the open field,the elevated plus maze and the black/white box, ahighly anxious/emotional rat is expected to highlyavoid the unprotected central part of the arena, theelevated open arms of the maze and the bright whitecompartment of the box, respectively [25].
SHR rats have been generally described as less fear-ful and more active than some other rat strains [7,9,35].When compared with LEW/NIco, SHR/NIco rats(from France), displayed higher levels of approach to-wards the aversive stimuli of the open field, elevatedplus-maze and black/white box, which suggested thatthey were less anxious than LEW/NIco [26,27]. Inter-estingly, the two strains did not differ for measures ofgeneral locomotion in the open field and the elevatedplus-maze [26,27]. Moreover, LEW/NIco rats wereshown to be more sensitive to social stress than SHR/NIco [2]. LEW/NIco and SHR/NIco rats also presentedstress-related physiological differences, with the formershowing higher increases in corticosterone after immo-bilisation, lower levels of tryptophan in midbrain [19]and higher extracellular serotonin levels in thehippocampus [24].
The results of the present study revealed that theBrazilian substrains of LEW and SHR rats possessemotional profiles which are very similar to those de-scribed for the corresponding/NIco substrains [26,27].In all behavioural tests applied herein, SHR rats ofboth sexes showed higher indices of approach (10 timesas much in some cases) towards the anxiogenic stimulithan their LEW counterparts. SHR rats explored morethe central area of the open field, the open arms of theplus maze and the white compartment of the black/white box. The approach/avoidance measures of theelevated plus-maze and the black/white box are wellaccepted as indices of anxiety [4,22,26]. Concerning theopen field measures, in spite of their being more contro-versial, a number of pharmacological studies show that
avoidance of the central area (where the animal cannotperform thigmotaxis) can be modulated by anxiolyticand anxiogenic coumpounds [1,7,20,33,41].
In the past few decades, several pairs of selectivelybred rat strains have been proposed as genetic modelsfor the study of emotionality [25]. Many of these mod-els (e.g. Maudsley lines, Roman lines and Tsukubastrains), however, besides contrasting for several wellaccepted fear-related behaviours, also show robust dif-ferences in their activity levels under different stressfulsituations, with the opposite strains reacting either pas-sively or actively to stress [25]. Such a contrast inlocomotion may represent a confounding factor sincethis behaviour does not respond consistently to anxi-olytic/anxiogenic drugs. It is useful, thus, the develop-ment of new tools (e.g. alternative genetic models) toallow the dissection of emotionality in its simpleranalysable components (e.g. locomotion levels in novelenvironments and approach/avoidance behaviours).Herein, in agreement with our previous studies [26,27],the LEW and SHR strains did not differ for measuresof locomotor activity such as total locomotion in theopen field [26,42] and total and closed-arm entries inthe elevated plus-maze [5,26]. Thus, the present charac-terisation of LEW and SHR rats consolidates these twostrains as a useful and unique genetic model for thestudy of anxiety, since they show a clear-cut contrastfor anxiety-related measures obtained from differentbehavioural tests and because such a contrast does notseem to be contaminated by differences in generallocomotor activity in stressful situations.
In its original protocol, the elevated T-maze (derivedfrom the elevated plus-maze) allowed the distinctionbetween learned fear and innate fear, which were hypo-thetically related to generalised anxiety disorder andpanic disorder, respectively, [10,45]. A modified proto-col (that ignores the escape behaviour as a measure ofinnate fear) has been proposed as a model of emotionallearning and memory [30]. In this version of the test,which is very similar to the one used in the presentstudy, all animals learn to completely avoid the openarms of the maze in the training session and such aninhibitory avoidance can be maintained for as long as 3months [30]. Thus, differently from the other tests ofemotionality used herein, the elevated T-maze is ex-pected to detect a learning-dependant form of anxiety.Because inhibitory avoidance in the elevated T-mazehas been shown to respond to low doses of benzodi-azepines as well as to 5-HT1A agonists, this test hasbeen proposed as an adequate model of generalisedanxiety disorder [10,45].
The present results revealed significant strain differ-ences only in males, with SHR rats taking more trials inthe training session to attain the inhibitory avoidancecriterion than LEW rats. Moreover, once acquired, theinhibitory avoidance of LEW males was totally re-
A. Ramos et al. / Beha�ioural Brain Research 129 (2002) 113–123 121
trieved by 100% of the subjects (i.e. all rats completelyavoided the open arms during test), whereas more than50% of the SHR males entered the open arms duringtest. Thus, a clear difference appears also in this testbetween LEW and SHR males. However, as thismodel does not allow the separation between emo-tional and memory factors, such a strain differencecould be attributed either to a difference in anxietylevels or to possible differences in learning. Furtherstudies should verify how these strains respond toother models of learning and memory which do notinvolve strong emotional components.
It has been repeatedly demonstrated that womenhave a higher prevalence of anxiety disorders than men[18,43], which suggests that some of the underlyingmechanisms of anxiety in humans are sex-dependant.In rodents, sex differences in emotionality have beenwidely described [11], but the biological bases for thisphenomenon are poorly understood. Taking the hu-man sex differences into consideration, one might ex-pect that an animal model of anxiety should showrodent females to be more anxious than males, but thisdoes not seem to be true. In the open field test, fe-males are generally considered to be less emotionalthan males [11]. In other tests of anxiety, the resultsconcerning sex differences are controversial [15]. In thepresent study, though, females could be considered asless anxious and more active than males in all be-havioural tests utilised, which does not seem to matchthe clinical situation in humans. Nevertheless, theLEW/SHR model has been shown to be useful for thestudy of sex-dependant anxiety, since our previousQTL study using these strains (see [28]) has revealed amajor locus that strongly affects an anxiety-relatedbehaviour exclusively in females.
The aforementioned QTL was mapped on rat chro-mosome 4 near the gene Tac1r which, due to its func-tion and localisation, was the best candidate geneidentified in that study [28]. If this gene is in factresponsible for the effects observed, we expect LEWand SHR rats to differ in relation to it and hence alsoin relation to its functional properties. Besides its re-cently reported role as a modulator of anxiety anddepression [6,16,39,44], the tachykinin substance P isknown since 1950s as a ‘pain neurotransmitter’. Thus,if LEW and SHR rats differ for the gene Tac1r and itsproduct (the substance P receptor NK1), one mayhypothesise that these two strains shall also differ inbehavioural models of nociception. Testing this hy-pothesis was one of the goals of the present study.
To the best of our knowledge, the results reportedherein corroborate for the first time such a hypothesis.SHR rats have been described in the literature ashaving an abnormal sensitivity to pain when comparedwith WKY rats, reacting as either hyperalgesic (e.g. inthe formalin test) or as hypoalgesic (e.g. in the hot-
plate test) [37,38]. In the present study, major differ-ences between the LEW and SHR strains wereobserved in both males and females in the secondphase of the formalin test of nociception. LEW ratsdisplayed more paw flinchings 10–60 min after forma-lin injection than their SHR counterparts, thus sug-gesting that the former are hyperalgesic in relation tothe latter. A non-significant trend in the same directionwas found for both sexes in the first phase (0–10 min)of the test. Whereas the early phase may involve directeffects on nociceptors, the late phase of the formalintest seems to be related to a more persistent type ofpain involving an inflammatory response [13]. Thus,the present data suggest that LEW and SHR ratsdiffer for longer-lasting pain, which is in agreementwith a hypothetical genetic difference between strainsconcerning tachykinin NK1 receptors [12]. Whetherthe LEW and SHR strains also differ in theirtachykinergic activity remains to be investigated. Nev-ertheless, the present results suggest that this pair ofstrains may be useful not only in the study of anxietybut also in the study of pain. From another perspec-tive, the higher nociceptive sensitivity of LEW ratsobserved herein also corroborates the accounts thatanxious state may increase pain [14]. Certainly, addi-tional studies are necessary to better understand thisproposed influence.
In summary, the present study reveals that twoBrazilian substrains of LEW and SHR rats presentoverall the same emotional profiles as those previouslydescribed for two French substrains, thus consolidat-ing the LEW and SHR strains as an excellent model tobe used (among others) in genetic, pharmacologicaland molecular studies aiming to understand the etiol-ogy of anxiety disorders. The present results on noci-ception are also in agreement with our hypothesis thatthese strains may differ in tachykinergic mechanismswhich influence anxiety and pain. Pharmacologicalstudies aiming to verify whether the LEW and SHRstrains differ in their sensitivity to centrally injectedsubstance P are already being carried out in our insti-tution.
Acknowledgements
This work was supported by Funpesquisa/UFSC(behavioural experiments), CNPq and PRONEX (BP).A. Ramos, J. Assreuy and R. N. Takahashi are recipi-ents of a fellowship from CNPq. A.L. Kangerski, L.F.Vendruscolo and P.F. Basso had scholarships fromCNPq. The authors wish to thank Dr Luiz CarlosSchenberg from UFES for providing the first breedingcouples of SHR rats. We also thank Edineia C. Cor-reia, Halana F. Andrezzo and Geison S. Izıdio fortheir technical assistance.
A. Ramos et al. / Beha�ioural Brain Research 129 (2002) 113–123122
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