A genetic and multifactorial analysis of anxiety-related behaviours in Lewis and SHR intercrosses

Behavioural Brain Research 96 (1998) 195 – 205

Research report

A genetic and multifactorial analysis of anxiety-related behaviours in Lewis and SHR intercrosses Andre´ Ramos a,b,*, Yannick Mellerin a, Pierre Morme`de a, Francis Chaouloff

a

a b

Neuroge´ne´tique et Stress, INSERM U471, IRNA, Institut Franc¸ois Magendie, rue Camille Saint-Sae¨ns, 33077 Bordeaux Ce´dex, France Departamento de Biologia Celular, Embriologia e Gene´tica, Uni6ersidade Federal de Santa Catarina, 88.040 -900 Floriano´polis, SC, Brazil Received 14 August 1997; received in revised form 13 November 1997; accepted 4 February 1998

Abstract Lewis (LEW) and spontaneously hypertensive rats (SHR) have been shown to differ in a series of fear-related behaviours measured in different anxiety/emotionality tests. In the present study, we have investigated some of the genetic mechanisms underlying these differences. To this end, male and female rats from the two inbred strains were crossed to produce two parental (LEW and SHR), two F1 (LEW or SHR mother), and two F2 (LEW or SHR grandmother) groups. All rats were tested in the elevated plus-maze and in the open field, besides being characterised for systolic blood pressure (BP). LEW rats approached the open arms of the plus-maze and the central area of the open field less than SHRs. The two strains also differed in their BP (SHR\ LEW). LEW/SHR differences were found to be due to direct effects of the genes, rather than to indirect maternal and grand-maternal effects. Central locomotion in the open field was shown to be the most heritable of all the traits considered herein. A factor analysis on the segregating F2 population produced three independent factors. The first one was associated to measures of anxiety from the elevated plus-maze, and the second to measures of locomotion in novel environments. Factor scores revealed that the parental strains differ in relation to the first but not to the second factor. This study demonstrates the usefulness of coupling genetic and multifactorial methods to investigate behavioural traits and it confirms LEW and SHR strains as an interesting genetic tool for the study of anxiety. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Behavior genetics; Anxiety; Emotionality; Factor analysis; Spontaneously hypertensive rats; Lewis; Elevated plus-maze; Open field

1. Introduction The genetic study of animal strains that differ in their responses to stressful environments has proven to be a valuable tool in the study of the neurobiological and behavioural mechanisms of adaptation [12,18,19,37]. Genetic animal models can be used both to increase and to dissect the interindividual variability of stress-related responses found in many animal species [59]. Such an approach has thus been proposed as an experimental method for the investigation of the bases underlying different human psychological disorders [10,29,47]. * Corresponding author. Fax: [email protected]

+ 33 557573752; e-mail: an-

Two inbred rat strains, namely Lewis (LEW) and spontaneously hypertensive rats (SHR) have been recently proposed as a genetic model for the study of anxiety [59,60]. In a thorough behavioural study conducted in our laboratory involving male and female rats from six inbred strains, LEW and SHR rats displayed contrasting responses to several types of stressful environments, including two recognised models of anxiety (the elevated plus-maze, and the black and white box) [60]. In all cases, SHR rats displayed a higher approach towards the aversive stimuli (thought to be anxiogenic) than their LEW counterparts. This pair of strains, contrary to other proposed genetic models of anxiety [18,30,63], did not differ in their general locomotion and defecation scores in either novel or familiar envi-

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ronments. These two traits appear, in fact, to vary independently from some classical measures of anxiety [59]. Since it is very unlikely that one single pair of strains will ever represent all aspects of anxiety-related traits, the investigation of new models (e.g. LEW/ SHR) should improve our current understanding of such complex phenotypes. The goal of the present study was to investigate some inheritance mechanisms of a number of behaviours thought to reflect anxiety in the LEW/SHR model. Through a crossbreeding approach we aimed, first, to dissociate some of the components explaining the interstrain differences observed in this model which should be useful in the planning of future molecular experiments. To this end, six different genetic groups, involving pure and crossbred animals derived from LEW and SHR strains, were simultaneously produced and studied in our laboratory. These animals were tested in the elevated plus-maze, which is a recognised model of anxiety [57], and in the open-field test, which allows the measurement of different emotionality/locomotor activity indices [1,35]. Moreover, since the SHR strain has been intentionally selected in the past for high spontaneous blood pressure (BP) [54], rats from all groups were also characterised for this phenotypic trait. A series of quantitative genetic parameters were then estimated for each individual variable. A growing body of experimental evidence suggests that fear-related responses do not vary along a single axis. Instead, factor analyses involving a variety of emotionality/anxiety measures have repeatedly revealed a number of independent dimensions, most of them being potentially related to emotionality [1,29,36,59]. The inconsistency of findings on the behavioural effects of certain anxiety-modulating substances also suggests that different putative measures of anxiety may reflect distinct emotional states in the animal [2,41]. Moreover, even behavioural and/or physiological traits that are associated within single strains (and that appear, hence, to be biologically related) may segregate independently from each other across generations [12,19,44]. The use of segregating F2 populations derived from inbred strains may shed some light on this matter. Since unlinked genes that are eventually associated within parental strains will be reorganised by meiosis, traits not sharing the same biological substrate will tend to dissociate in segregating generations (and vice versa). It should be noticed, however, that this sole approach, although suggestive of, does not provide a clear-cut answer on the presence or absence of genetic cosegregation of traits, since environmental and genetic components may be confounded within the overall F2 variability. Nevertheless, the coupling of intercross experiments (e.g. F2) and multivariate

statistical methods (e.g. factor analysis) has proven to be a simple and efficient way of dissecting the different aspects of emotional responses [12,19,44]. Therefore, another goal of the present study was to determine, through a factor analysis carried out on a segregating F2 population, how the different behavioural traits were associated with each other. Moreover, we aimed at identifying which behavioural dimension characterises best the LEW/SHR pair of strains. Lastly, correlational analyses should bring some information about the possible relationship between BP and anxiety-related behaviours within this model.

2. Materials and methods

2.1. Animals A total of 267 rats of both sexes were used in this study. All animals were derived from two inbred strains, namely LEW and SHR. Male and female rats from these two strains were purchased from IFFA Credo (Les Oncins, France) and then mated in our laboratory to produce (at the same time and under the same environmental conditions) the following genetic types: LEW, SHR, F1 from LEW mother (F1L), and F1 from SHR mother (F1S). Simultaneously to this, F1L and F1S rats previously produced in our laboratory were mated (brother–sister) to produce two additional genetic types: F2 from LEW grandmother (F2L), and F2 from SHR grandmother (F2S). F1L/F1S-type crosses were not carried out. Within each type of F2, all animals derived from one single couple of grandparents, but they were produced through two consecutive series of matings using the same F1s as parents. Rats from the six genetic types (Table 1) were all born between February 16 and March 3, 1996 (except for the second half of F2 pups which were born between April 8 and 15). As seen in Table 1, the poor reproductive ability of LEW rats (when mated intrastrain) resulted in a lower number of LEW pups available for the tests. The animals were weaned and separated by sex at 4 weeks of age and, thereafter, kept in collective plastic cages (three or four rats/cage) with food and water available ad libitum under a 12-h light/dark schedule (lights on at 07:00 h). At 8 and 9 weeks of age, respectively, each rat was tested once in the elevated plus-maze and in the open field, with an interval of 1 week between the two testing sessions. Between the 10th and the 11th week of age, BP was determined for all animals. The behavioural tests and the measures of BP, detailed below, were carried out between 12:30 and 18:00 h. In order to minimise the effects of

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Table 1 Number of matings and total number of male and female descendants classified by genetic type Genetic type

LEW

SHR

F1L

F1S

F2L

F2S

Parents Males Females

4 LEW 4 LEW

4 SHR 4 SHR

4 SHR 4 LEW

4 LEW 4 SHR

3 F1L 6 F1L

3 F1S 6 F1S

Offspring Males Females

5 6

12 12

10 10

10 10

48 48

48 48

time, animals were tested in successive blocks, each block containing one rat from each genetic group considered. Males and females were tested on alternate days. Following this schedule, small-sized groups (LEW, SHR, F1L and F1S) were tested in the first half of the experimental week, with the large-sized groups (F2L and F2S) being tested afterwards. Prior to the first test (plus maze), the animals were naive to all manipulation, except for regular cage-cleaning. Each rat left its social partners and home cage only during the testing period, when it was transported by the experimenter inside a small plastic cage to a separate testing room where the apparatus was set up. This study was conducted in conformity with the French publication on animal experimentation (no. 87-848).

2.2. Ele6ated plus-maze As already described [13], the apparatus was made of perspex, with four elevated arms (66 cm from the floor) 45-cm long and 10-cm wide. The arms were arranged in a cross-like disposition, with two opposite arms being enclosed (by 50-cm high walls) and two being open, having at their intersection a central square platform (10 × 10 cm) which gave access to any of the four arms. All floor surfaces were black, walls were opaque grey and the central platform was under an illumination of 70 lux. Each rat was placed in the central platform facing an open arm and its behaviour was video-recorded for 5 min. The number of entries and the time spent (with all four paws) inside each arm [57] were recorded and the percentage of open-arm entries was calculated in relation to the total number of entries in both types of arms. The floor of the maze was cleaned with a wet sponge and a dry paper towel between rats.

of decreasing the aversiveness of the test, thus dissociating novelty from other types of aversive stimuli. Each rat was placed in the centre of the open field and the following variables were recorded, by the use of a video camera, for 5 min: number of outer squares (those adjacent to the walls) crossed (outer locomotion), number of inner squares crossed (inner locomotion), and total number of faecal boli (defecation). The whole area was cleaned between rats as described in Section 2.2.

2.4. Blood pressure BP was determined by a non-invasive, indirect method adapted to a sphingomanometric system produced by Letica (Barcelona, Spain). The system comprised a central digital unit (LE 5000) which was connected to a pulse signal transducer (to be placed around the rat’s tail) and to a tail cuff (manually inflated by a rubber hand pump). A modified protocol was developed with the aim of providing adequate pulse signal levels with minimal stress to the rats. Prior to BP determination, the animals were transferred in their home cages to a quiet, dimly illuminated room with an ambient temperature of 38–40°C. Animals were then left undisturbed for a minimum of 30 min. Following that, each rat was transferred to an adjacent testing room and placed in a cylindrical restrainer which was introduced through an opening into a dark warming box (38°C). The rat’s tail was kept outside the box to allow for the placement of the pulse signal transducer and tail cuff. Once a constant pulse signal was detected (5.99 3.8 min after the introduction in the box), four to six recordings of BP were obtained and the average of these readings was calculated.

2.5. Statistics 2.3. Open field (no6el en6ironment/dim light) The apparatus made of wood, as already reported [14], had a white floor of 100×100 cm, divided into 25 squares of 20 ×20 cm. The walls, 40-cm high, were also painted white. The test room had a dim illumination, with 7 lux being measured inside the apparatus. Such a procedure, as previously described [60], had the objective

Differences between the two parental strains (LEW and SHR) were assessed for each sex by the Mann– Whitney rank test. The same test was used to compare males and females within each genetic type. As one main goal of this study was to investigate how the different phenotypic traits related to each other, data from the segregating F2 generation were submitted to correla-

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tional and multivariate analyses. In this case, all sex-dependent variables were normalised for such an effect by adding the difference between male and female means to all male individuals. Correlations between systolic BP and behavioural variables were determined by the Spearman rank correlation test. All phenotypic variables were submitted to a principal component analysis with a varimax rotation. Only factors with eigenvalues greater than one were kept. From the factor loadings (which represent the correlations between a factor and the different variables) obtained with the F2 data, factor scores were calculated [20] for individuals of all genetic groups. Data used in the calculation of these scores were not corrected for sex.

2.6. Genetic parameters Data from the six genetic types allowed the estimation of genetic parameters which partitioned the differences found between the parental strains into more meaningful components. These parameters were only determined for those variables that differed between the two strains, since in all the other cases (LEW= SHR), Kruskal–Wallis analyses revealed no differences between the six genetic types. Four crossbreeding parameters adapted from Dickerson’s model [8,24] were estimated. The ‘maternal effect’ (g m) represents how much of the interstrain difference found for a given trait is determined by differential influences of LEW and SHR mothers on their pups during gestation and lactation. Such influences, whereas environmental for the pup, may be a consequence of the mother’s genotype (e.g. milk production genes). Similarly, the ‘grand-maternal’ effect (g n) relates to the possible influences of the grand-mother (i.e. maternal effect upon maternal effect) on a trait. In contrast to these two indirect effects (g m and g n), the ‘direct genetic effect’ (g o) represents the amount of the difference between strains that is determined directly by the genes of the animals being tested. Finally, ‘heterosis’ (h o) is the deviation of the F1 generation from the mean value of the two parental strains, which, when significant, may suggest the presence of dominance. The models for the estimation of g m, g n, g o and h o are presented below. Mean values and variances from the different genetic groups were used to estimate the parameters as well as to verify, through a t-test, whether they were significantly different from zero. g m =(F1S− F1L)− (F2S −F2L) g n = F2S− F2L g o = (SHR− LEW)− (F1S − F1L) h o = [(F1S+F1L)/2] −[(SHR +LEW)/2] Whenever h o was shown to be significant, the pres-

ence of dominance was tested through the ‘deviation from the dominance model’ (d), which comprises both ‘maternal heterosis’ and ‘epistatic recombination loss’ [24] (the present design did not allow the dissociation of these two effects). The parameter ‘d’ was estimated according to the formula: d = 0.5 (F2S+ F2L)− 0.25(F1S+ F1L+ SHR+LEW) In addition to these, another genetic parameter estimated was ‘heritability in the broad sense’ (h 2), which represents the relative importance of heredity in the determination of a phenotypic trait [27]. Based on the variances of the different generations, h 2 is defined as the proportion of the phenotypic variability observed in a population that is explained by its total genetic variability. As the variability within inbred strains and within their F1 generations is essentially environmental (i.e. theoretically all animals have the same genotype), whereas the variability within the segregating F2 generation results from both environmental and genetic factors, h 2 = {VF2 − [(VLEW + VSHR + VF1S + VF1L)/4]}/VF2) As h 2, according to this estimation method, represents a proportion and not a mean value, it is not tested for significance. Males and females produced similar overall results when analysed either separately or as a single population. Therefore, for the estimations of all genetic parameters, both sexes were combined within each genetic group.

3. Results

3.1. Interstrain and gender differences As shown in Table 2, significant differences between the two parental strains were found for all variables related to the approach/avoidance towards the open arms of the plus maze (time spent and percentage of entries in the open arms and time spent in the closed arms) and towards the centre of the open field (inner locomotion). In all cases (non-significant only for percentage of open-arm entries in males), SHR rats approached the aversive stimuli more (open arms, and centre of the open field) than LEW rats. Moreover, SHR rats of both sexes displayed higher BP than their LEW counterparts. No strain differences were found (in both males and females) for total and closed arm entries in the plus maze and for outer locomotion and defecation in the open field. Significant sex differences were found, for SHR rats only, in outer (females \males, PB0.05) and inner (females\males, PB 0.01) open-field locomotion as well as BP (males\ females, PB 0.001).

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3.2. Crossings and genetic parameters The distribution of the six genetic types along the variables for which LEW and SHR rats were different (Section 3.1) is shown in Fig. 1 and Fig. 2. For all the remaining variables (not shown), no differences were found between any of the genetic types and the parental strains. As illustrated in Figs. 1 and 2, the two parental strains were placed in the two extreme positions whereas their F1 and F2 crosses occupied the intermediate ranges. Besides the sex effects already mentioned for the parental strains (see above), differences between males and females were significant for: time spent in the open arms of the plus maze (F2S only, P B 0.05); inner locomotion in the open field (F2L (P B 0.001) and F2S (P B0.05) rats); and BP (F1L (P B 0.01), F1S (P B 0.01), F2L (P B0.05) and F2S (PB 0.05) rats). Of all the crossbreeding parameters submitted to significance tests (g m, g n, g o and h o), only those significantly different from zero are shown in Table 3. Direct genetic effects (g o) were significant for all five variables. Maternal and grand-maternal effects (g m and g n), conversely, were not significant for any of them, thus revealing that the differences observed between LEW and SHR rats were essentially due to the direct effects of genes. Table 2 Elevated plus-maze, open field and systolic BP measures (X 9 S.E.M.) of LEW and SHR rats grouped by sex Variable

Males LEW

Plus maze Time in open arms (s) Time in closed arms (s) Open arm entries (%) Closed arm entries Total arm entries Open field Outer locomotion Inner locomotion Defecation BP (mmHg)

Females SHR

5.8 9 3.7 30.39 7.8*

LEW

5.292.4

SHR

49.7 9 12.1*

236 9 10 1689 13*

216 911

1459 13**

16.3 9 7.6 28.89 5.0

10.1 96.8

35.39 5.0*

3.8 9 0.7 4.69 0.5

7.09 1.5

5.29 0.4

4.8 9 1.1 6.89 0.8

7.891.6

8.5 9 0.9

47.8 9 7.5 58.29 4.7

74.3 912.3 79.39 4.4 c

1.0 90.0 4.79 0.7** 1.39 0.2

13.19 2.1** c c

0.6 90.6 0.19 0.1 16194 2299 2**

0.1 90.1 1949 2** c c

0.0 90.0 1579 2

For each sex, significant interstrain differences are represented by * and ** and for each strain, significant sex differences are represented by c and c c (PB0.05 and PB0.01).

Fig. 1. Means and S.E.M.s of time spent in the open arms (top), time spent in the closed arms (middle), and percentage of entries in the open arms (bottom) of the elevated plus-maze for male and female rats of six genetic types: LEW (Lewis), F1L (F1 from LEW mothers), F1S (F1 from SHR mothers), F2L (F2 from Lewis grandmother), F2S (F2 from SHR grandmother), and SHR (spontaneously hypertensive rats).

Significant heterosis effects (h o) were found for the time spent in the closed arms of the plus maze and for BP. In order to test for the presence of dominance within these two phenotypic traits, the deviations from the dominance model (d) were estimated. Such a parameter was not significant for any of the two variables, thus suggesting the presence of dominance in the genetic bases of both these traits. Of all phenotypic traits, inner locomotion in the open field produced the highest estimate of heritability whereas the percentage of entries in the open arms of the elevated plus-maze produced the lowest one.

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Table 4 Factor analysis (PCA) of nine phenotypic traits measured in the F2 generation derived from LEW and SHR rats. Factor loadings higher than 0.4, produced by a varimax rotation, are shown for each factor Variable

Factor 1

Plus maze Time in open arms(s) Time in closed arms(s) Open arm entries (%) Closed arm entries Total arm entries

Factor 2

0.89 −0.77 0.88 0.41

Open field Outer locomotion Inner locomotion Defecation

0.93 0.85 0.60

BP (mmHg)

3.3. Multi6ariate analyses Spearman rank correlation analyses revealed no significant correlation between BP and all the behavioural variables measured in this study (in all cases r B 9 0.12 and P \0.1 for n =192). A principal component analysis on the data from the segregating F2 generation produced three factors with eigenvalues higher than one, representing 64.6% of the total variability. The factor loadings for each

0.49 0.50 0.56 −0.54

% Of the total variance

Fig. 2. Means and S.E.M.s of inner locomotion in the open field (top) and systolic blood pressure (bottom) for male and female rats of six genetic types: LEW (Lewis), F1L (F1 from LEW mothers), F1S (F1 from SHR mothers), F2L (F2 from Lewis grandmother), F2S (F2 from SHR grandmother), and SHR (spontaneously hypertensive rats).

Factor 3

27

25

13

of the nine variables are shown in Table 4 (only loadings higher than 0.4 are presented to facilitate interpretation). Factor 1 was associated to all measures of approach/avoidance towards the open arms of the elevated plus-maze. Factor 2, on the other hand, correlated with measures of locomotion in both the elevated plus-maze and the open field. All variables from the open-field test as well as BP loaded together on factor 3. Based on the factor scores calculated for individuals of all groups, the mean values of males and females of the parental strains in relation to the first two factors were calculated. Significant differences (PB 0.05) between strains were found for the scores of factor 1 in both male ( −0.659 0.28 versus 0.549 0.30; LEWB SHR) and female (−0.989 0.25 versus 1.0490.36; LEWB SHR) groups, whereas no strain differences were found in relation to factor 2 (− 1.299 0.22, −0.729 0.15, 0.109 0.49 and −0.2290.16 for LEW males, SHR males, LEW females and SHR females, respectively).

Table 3 Estimate9S.E.M. of genetic parameters for five phenotypic traits that differ between LEW and SHR rats

gm gn go ho h2

Time open

Time closed

% Open entries

Inner locomotion

BP

— — 30.6 910.9** — 0.22

— — −66.19 16.4** −20.898.2* 0.46

— — 16.5 97.9* — 0.10

— — 9.1 9 1.7** — 0.59

— — 52.4 95.3** −9.292.6** 0.57

g m, g n and g o, Maternal, grand-maternal and direct genetic effects; h o, heterosis; h 2, heritability; ‘time open’ and ‘time closed’, time spent in the open arms and in the closed arms of the elevated plus-maze, respectively; ‘% open entries’, percentage of entries in the open arms of the elevated plus-maze in relation to the total number of arm entries; ‘inner locomotion’, number of inner squares crossed in the open field; and ‘BP’, systolic BP. Values not shown ( —) are not different from zero (P\0.05). Significance levels are represented by * and ** (PB0.05 and PB0.01). Note that according to their present calculation formula (Section 2.6) h 2 values are not provided here with S.E.M.s.

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4. Discussion

4.1. Interstrain and gender differences Besides being a widely used model of hypertension, SHR rats have also often been characterised as less fearful than rats from other strains. When compared with Wistar Kyoto rats, SHRs showed lower startle response to auditory stimulus [42], higher preference for the aversive open arms of the elevated plus-maze [32,62], and more visits to the aversive central area of the open field [11,32]. A similar profile regarding the elevated plus-maze and the open field tests was obtained when SHRs were compared with outbred Wistar rats [34]. Behavioural studies comparing LEW and Fisher 344 rats have reported a lower central locomotion in the open field for the former, with both strains displaying an equally high avoidance of the white compartment in the black and white box [15,33]. The profile of LEW rats in both these tests (avoidance of aversive stimuli) seems to be a sign of higher fearfulness. The results reported in the present study are thus consistent with previous data on the behaviour of LEW and SHR rats. As shown in Table 2, SHRs of both sexes spent more time in the open arms and less time in the closed arms of the plus maze than their LEW counterparts. The percentage of entries in the open arms (another classical index of anxiety) was also higher for SHR rats (significant only for females). No strain differences were found for total and closed-arm entries in the plus maze, which are usually considered as measures of locomotion [21,57]. In the open field, male and female SHRs displayed a higher locomotion in the central area than LEW rats, with no differences being found in their outer locomotion and defecation scores. These results, obtained with animals born in our laboratory, are in complete agreement with our recent study on six inbred strains (including LEW and SHR) which were purchased and transported to our laboratory at 5 weeks of age [60]. Consistent responses of LEW and SHR rats to single doses of diazepam (anxiolytic) and pentylenetetrazole (anxiogenic) during a plus-maze test [60] suggest that the behavioural differences observed between these two strains may be associated with different baseline levels of anxiety (LEW\SHR). SHR rats, genetically selected for high BP [54], also have a sympathetic nervous system (SNS) highly responsive to a variety of stressors [59]. Considering the potential relationship between stress, SNS activity and BP [26], one might ask if hypertension in SHRs is somehow associated with an ‘abnormal’ sensitivity to emotional situations. Since high emotionality is most often thought to accompany high BP [40,61], contrary to the apparent profile of SHRs, we wished to clarify

201

this issue and investigate the possible relationship between BP and anxiety-related behaviours. In the present study, SHRs showed higher systolic BP than LEW rats, which confirms a previous hypertension study on these two strains [46]. Among the segregating F2 generation that presented an independent recombination of LEW and SHR alleles, no correlation was found between systolic BP and any of the behavioural measures studied herein. This suggests that BP and anxiety-related behaviours in the plus maze do not present a cause/consequence relationship and do not share the same genetic basis within this specific genetic model. Significant sex effects were found in only one of the parental strains (SHR). The higher open-field outer locomotion observed in SHR females is in agreement with previous studies on sex-dependent activity scores [35,45,60]. For this same variable, however, LEW males were not significantly different from females. Considering the mean values for this strain (Table 2, females\ males), it is likely that the lack of a significant sex effect was due to the low number of animals available in this group. SHR females also displayed higher open-field inner locomotion than males and, conversely, SHR males had higher BP than females. Neither of these variables was affected by sex within the LEW strain, showing that these sex differences are strain specific (sex/genotype interaction). Hypertension had already been shown to be higher and to develop earlier in male than in female SHRs [54]. In addition, interactions between sex and genotype have been reported in recent genetic studies on hypertension [17,26,51] as well as on other physiological and behavioural traits [26,52]. Female ovarian hormones are known to influence some anxiety-related behaviours [53], therefore, the consideration of sex differences and sex-interacting effects is certainly an important issue in the identification of the mechanisms underlying emotional responses [3].

4.2. Genetic parameters The role played by genetic factors in different human psychological disorders and personality traits has been demonstrated and analysed by an increasing number of studies [9,25,65]. Regarding anxiety, a recent twin/adoption study has shown that over 30% of the total variability for some anxiety-related measures could be explained by genetics [38]. Moreover, Lesch et al. [48] found that a polymorphism in the regulatory region of the serotonin (5-HT) transporter gene was responsible for a small but significant proportion of the variability in human neuroticism scores (an anxiety-related trait). As far as animals are concerned, there are different strains of rodents proposed

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as genetic models to be used in the investigation of anxiety disorders [18,23,30,63]. Through the use of sophisticated crossing programs, classical studies on behaviour genetics conducted in the 1960s have analysed the genetic components underlying some emotionality-related behaviours in rodents [5,7,43,58]. In particular, the study of segregating populations (F2, F3 and backcrosses) and/ or diallel crosses along with their inbred parental strains has allowed detailed analyses of the genetic architecture of open-field behaviours in rats (e.g. Maudsley reactive and non-reactive strains) and mice (e.g. BALB/cJ and C57BL/6J). To our knowledge, however, no information is available about the inheritance mechanisms of animal behavioural traits measured in well-recognised tests of anxiety (such as the elevated plus-maze, for example). One of the aims of the present study was thus to shed some light on this subject. The results reported herein confirm that the behavioural contrasts previously observed between LEW and SHR rats [60] were genetic in origin. The ‘overall genetic effect’ is represented here by the differences found between the two parental strains (Table 2), which were born, raised and tested simultaneously and under the same environmental conditions. Such an effect was then analysed by the estimation of some genetic parameters (Table 3) based on the mean values and variances of all six genetic types. Since rats (like all mammals) receive from their mothers not only half of their genetic material, but also a number of physical, chemical and psychological influences during and after gestation [8], one might expect F1 rats to differ depending on their mothers’ genotype. Significant maternal effects have already been found in mice for grooming behaviour in the open field [18]. In the present study, however, that was shown not to be the case. The absence of maternal (g m) and grand-maternal (g n) effects for all traits indicates that the differences between the two strains are not due to differential influences of LEW and SHR mothers on their litters. Moreover, these results indicate that there is no major effect from genes located on the X chromosome, transmitted from mothers to all male offspring. The lack of indirect effects (g m, g n) combined with the significant direct genetic effects (g o) for all variables reveal that the LEW/SHR contrasts are essentially explained by the direct effects of the genes carried by the animals being tested. When F1 animals deviate from the mean between the two parental strains, this deviation is called heterosis [27]. Significant heterosis effects (h o) for the time spent in the closed arms of the plus maze and for BP suggest the presence of dominance in the genetic control of these two variables. To further verify this possibility, we estimated the deviation (d) from a

theoretical model of dominance. As ‘d’ was not significant for any of these two variables, the hypothesis of dominance is corroborated. In both cases, thus, negative values of h o indicate that the LEW phenotype was dominant over the SHR phenotype. Finally, heritability values (h 2) give an idea about the relative importance of genetic factors for each trait [27]. In a study on mice, the heritability estimated for total open-field activity and defecation was 26 and 11%, respectively [30]. In previous studies on rats, however, much higher values have been obtained for these two variables, with defecation presenting higher heritability (\90%) than activity in the open field [5]. If one considers, as a rule of thumb, that h 2 values over 0.5 are fairly high for a quantitative trait, we can say, according to the present study, that the measures of anxiety from the elevated plus-maze are not highly heritable, whereas the open-field inner locomotion and BP are highly affected by the animals’ genotypes. Therefore, one could predict that openfield inner locomotion and BP should give the most significant and rapid results in future experiments of selection and/or molecular research of genes using the LEW/SHR model. It is important to notice, however, that heritability values should be interpreted with caution, since they depend on the environmental conditions and on the genetic characteristics of the population used in each experiment [27].

4.3. Multi6ariate analyses An increasing number of studies has factor analysed stress-related responses in animals and found that sets of putative measures of emotionality vary along two or more independent axes [1,16,29,59,60]. Nevertheless, a unidimensional view of emotionality has underlied the interpretations of many studies on this subject [28,30,35]. Three orthogonal (and hence independent) factors were revealed in the present study. The factor loadings (Table 4) represent the correlations between a given variable and each of the factors. Thus, factor 1 correlated strongly with all putative measures of anxiety from the elevated plus-maze. Total number of arm entries in the plus maze loaded moderately on this factor and strongly on factor 2, a factor also associated with the number of entries in the closed arms and outer locomotion in the open field. The variables with high loadings on factor 1 have been widely validated as indices of anxiety, whereas total- and closedarm entries are considered as measures of locomotion in the plus maze [4,21,50,57]. We can thus suggest that factor 1 represents an anxiety-related dimension and factor 2 a dimension of locomotion in novel environments (both the plus maze and the open field were novel to the animals).

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Ambulation and defecation in the open field have been considered for a long time as measures of emotionality in rodents [35,39,50]. Nevertheless, a number of factorial studies involving measures from different tests of emotionality/anxiety indicate that locomotion in novel environments and/or defecation in stressful situations vary independently from other measures of anxiety [19,49,60,64]. In the present study, defecation in the open field provided the highest loading on factor 3, which also received contributions from BP (negatively related) and from inner and outer open-field locomotion (positively related). Considering that there are no strong loadings on factor 3 and that it accounts for only 13% of the total phenotypic variability, some care should be taken in the interpretation of this factor, as it clearly represents only marginal correlations between variables. Factor 3 does reveal, however, that neither open-field defecation nor BP are correlated with the classical measures of anxiety from the elevated plus-maze. Central locomotion in the open field has been shown to increase following injection of the anxiolytic chlordiazepoxide [32] and after habituation with the test apparatus [55]. This variable has thus been considered by some authors as being anxiety-related [18]. In a previous study [60], we found central locomotion in the open field to be associated with anxiety measures from the elevated plus-maze and black/white box. In the present study, however, central open-field locomotion, even when expressed as a percentage of total locomotion (data not shown), was not associated with any of the plus-maze variables. As mentioned before, factor analysis carried out on a F2 generation (as in the present study) is likely to be more realistic regarding trait associations than data from single strains (as in our previous study). The present findings suggest, therefore, that the central locomotion in the open field and the approach of the open arms in the plus maze, in spite of being seemingly associated in the parental strains, are independently inherited, indicating that these two measures may reflect two different types of emotional state. Based on extensive reports of a variety of interstrain differences, some pairs of rodent strains are thought to contrast in relation to a general trait of emotionality [6,22,31], which is often considered as being equivalent to fearfulness and/or anxiety [30,35]. Other studies, however, either through multivariate comparisons of inbred strains [56,60,64] or through intercrossing approaches [12,19], suggest that emotionality is multidimensional. In a recent molecular study on mice [30], three genomic regions were found to cosegregate simultaneously with open-field activity, open-field defecation, activity in a Y maze, and openarm activity in the plus maze. These findings might suggest that these different measures, being affected

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by common genes, are all part of the same psychological dimension. Firstly, however, one should remember that genes may act not only at the central level of emotional experience, but also at the perception level (input) and/or at the peripheral level of behavioural expression (output). Therefore, a gene affecting perception (e.g. olfaction) might have pleiotropic effects on a wide range of behaviours not presenting an obvious biological/psychological interrelationship. Secondly, it should be noticed that the plus-maze variable found to be affected by the aforementioned genomic regions [30] was not one of the classical plusmaze measures of anxiety (percentage of open-arm entries and time in open arms) and that those were found, in that study, not to be correlated with the emotionality measures from the open field. In the present study, individual rat scores calculated for the different factors showed significant differences between the LEW and SHR strains in relation to factor 1 with no differences being found in relation to factor 2. These results suggest again that the LEW/ SHR model varies phenotypically along an anxiety-related axis and not along an axis of locomotion in novel environments [60]. It is clear, therefore, that the recognition of the different dimensions assessed by a set of emotionality measures is particularly important before one makes assumptions about the psychological significance of a specific pair of strains. In summary, the present report confirmed that LEW and SHR rats differ in a number of behavioural traits as well as in their BP. Some genetic components of these differences have been estimated, showing that it is the direct effect of the genes, rather than the indirect maternal and grand-maternal influences, that are responsible for the contrasts between the two strains. Among all the variables analysed, inner locomotion in the open field and BP are those presenting the highest estimates of heritability. The multidimensional character of the emotional response has been further demonstrated through this genetic and multivariate approach, which allowed us to recognise the main behavioural dimension along which LEW and SHR rats differ from each other. Previous pharmacological evidence suggests that such a dimension is anxiety-related. Molecular experiments aiming at the identification of the chromosomic regions involved in the control of the traits studied herein are already being carried out in our laboratory.

Acknowledgements The authors would like to thank Dr Jean-Michel Brun for his valuable help in the estimation of the genetic parameters. A. Ramos had a scholarship from

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Coordenac¸ao de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES)/Brazil.

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