- Email: [email protected]
Unconditioned anxiety and social behaviour in two rat lines selectively bred for high and low anxiety-related behaviour
Behavioural Brain Research 111 (2000) 153 – 163 www.elsevier.com/locate/bbr
Research report
Unconditioned anxiety and social behaviour in two rat lines selectively bred for high and low anxiety-related behaviour Markus S.H. Henniger * , Frauke Ohl, Sabine M. Ho¨lter, Petra Weißenbacher, Nicola Toschi, Patrick Lo¨rscher, Alexandra Wigger, Rainer Spanagel, Rainer Landgraf Max Planck Institute of Psychiatry, Kraepelinstr. 2 -10, 80804 Munich, Germany Received 25 October 1999; received in revised form 13 January 2000; accepted 13 January 2000
Abstract Individuals of high anxiety-related behaviour (HAB) and low anxiety-related behaviour (LAB) rat lines were selectively bred for differences in anxiety-related behaviour on the elevated plus-maze. The goal of this study was to investigate whether this behavioural difference is restricted to the test used as the selection criterion or whether it is a stable and robust trait also in other conflict or non-conflict situations. Therefore, behaviour of male and female HAB and LAB rats was examined in two further tests of unconditioned anxiety: the black–white box and the social interaction test. Furthermore, behaviour of group-housed male HAB and LAB rats was studied in their home cages. In addition to standard statistics, discriminant analyses were performed. The difference in anxiety-related behaviour between the two lines was highly consistent in all tests of unconditioned anxiety. There were also differences in home cage behaviour, LAB rats being more active than HAB rats; this is likely to be a consequence of the LAB rats displaying a higher aggressiveness in social behaviour, compared to HAB rats. In all tests used HAB and LAB rats were clearly distinguished by discriminant analysis. However, while in the elevated plus-maze and the black – white box test the most important parameters for discrimination between the two lines were mainly those generally seen as closely related to anxiety, the discrimination in the social interaction paradigm was primarily due to differences in locomotor activity. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Emotionality; Elevated plus-maze; Black–white box; Social interaction test; Home cage behaviour
1. Introduction We recently reported on two Wistar rat lines which were selectively bred over several years for extremes in anxiety-related behaviour on the elevated plus-maze [12 – 14]: the high anxiety-related behaviour (HAB) and the low anxiety-related behaviour (LAB) lines. These lines represent a useful animal model for studying the neurobiology of inborn anxiety and the mechanism(s) of actions of anxiolytic compounds [13]. Anxiety has been shown to be a highly complex construct and it has been suggested that different tests of anxiety-related behaviour measure various aspects of * Corresponding author. Tel.: +49-89-30622641; fax: + 49-8930622569. E-mail address: [email protected] (M.S.H. Henniger)
anxiety [2,24]. When using animal models of inborn anxiety it is, therefore, of great interest to evaluate whether differences in anxiety-related behaviour occur globally or are restricted to variables measured by the test used as selection criterion. Therefore, we tested HAB/LAB rats in different paradigms, which have been shown to measure distinct dimensions of emotionality. First, the elevated plus-maze was used to confirm the selected line differences. Then we tested HAB and LAB rats in two other tests of unconditioned anxiety, the black–white box and the social interaction test. The elevated plus-maze test is a widely used test paradigm to investigate anxiety-related behaviour (for reviews see Refs. [6,10,25]) and has extensively been validated for use in both rats [19] and mice [15]. It is based on the test-induced conflict in rodents between
0166-4328/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 0 ) 0 0 1 5 1 - 0
154
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
the aversion of being exposed to an open and elevated platform on the one hand and the motivation to explore the new environment on the other. As a consequence, the less anxious the individuals are, the more they explore the open arms. The black–white box test was developed [5] and validated [4,17] as a test of anxiety in mice. In this test an animal experiences a conflict situation between the motivation to either explore or to avoid an exposed, brightly lit, area. An individual, which explores the exposed area more and for longer is defined as being less anxious. In some studies the black – white box paradigm was also used for rats, with differences in experimental design and procedure [3,26,27]. For example, some authors have found it to be advantageous, as we have in the present study, to initially place the animal in the darkened side of the box, rather than in the brightly lit side [28]. In the social interaction test, developed and validated for rats by File et al. [8,9], a pair of rats which are unknown to each other, are placed into a neutral arena and the time the individuals spend in social interaction is measured. It has been shown that the more time a pair of rats spends in active social interaction, the less anxious they are [9]. In addition to the described tests of unconditioned anxiety, we observed behaviour of group-housed HAB and LAB rats in their home cages during the first hour of the activity phase. This home cage analysis was conducted to get a better idea about possible behavioural differences of HAB/LAB rats in a nonconflict situation. Taken together, the aim of this study was to investigate behaviour of the HAB and LAB rat lines in different conflict and non-conflict situations. In addition to standard statistics, discriminant analysis was used to examine under inclusion of all parameters of the respective tests: (a) the accuracy of discrimination of HAB and LAB lines for the different paradigms; and (b) those parameters of each test which determine the discrimination between the two lines.
2. Materials and methods
2.1. Animals Subjects were individuals of two Wistar rat lines selected and bred in our institute as previously described [13,14]. Briefly, adult Wistar rats, obtained from a commercial supplier (Charles River, Sulzfeld, Germany) were tested in an elevated plus-maze and animals with HAB and animals with LAB, respectively, were then mated for years to establish the HAB and LAB lines. At the age of 10 weeks, the offspring from both breeding lines was tested on the elevated plus-maze and
for each new generation, HAB and LAB breeding pairs were selected. HAB animals showing low levels and LAB rats displaying high levels of anxiety-related behaviour, as well as individuals showing no clear tendency towards either of these two extremes, were excluded from further breeding as well as from further behavioural testing. In the present study, female rats (HAB n= 10, LAB n = 13; generation F7) were tested (as mentioned above) on the elevated plus-maze at an age of 10 weeks. At an age of 6–10 months the same individuals were tested in the black–white box. Three weeks thereafter they were tested in the social interaction paradigm. All three tests of unconditioned anxiety were also conducted with male rats (elevated plus-maze: HAB n=9, LAB n = 10, age 10 weeks; black–white box: HAB n = 10, LAB n= 8, age 14 weeks; social interaction: 10 individuals per line, age 13 weeks; all tests: generation F10). Home cage observation was conducted with male, 16–20-week old rats (17 individuals per line, generations F9 and 10). Animals were housed in groups of up to five samesexed individuals of the same line (cage size: 58×38× 20 cm height), except before the social interaction test, when rats were housed individually for 2 weeks (cage size: 42× 26× 20 cm height). All animals were maintained on a 12-h light/dark cycle (lights on: 6:00 h– 18:00 h) with food and water available ad libitum. At least 16 h before testing or familiarising with the test arena (social interaction, see below) the animals were brought to the testing room. All tests of unconditioned anxiety were conducted between 8:00 h and 12:30 h. Home cage behaviour was observed during the first hour after the lights in the housing room were switched off (18:00 h–19:00 h). The experiments were approved by the Committee on Animal Care and Use of the relevant local governmental body.
2.2. Ele6ated plus-maze The elevated plus-maze we used was described previously [13,14]. Briefly, it was made of dark grey PVC and consisted of two open arms (50× 10 cm) and two enclosed arms (50 ×10×38 cm high walls) extending from a central platform (10×10 cm). The apparatus was elevated 73 cm above the floor. The maze, surrounded by an opaque curtain, was lit by two white bulbs above the open arms and two red bulbs above the closed arms (mean light intensity over the whole maze: 100 lux). At the beginning of a test, each rat was placed on the central platform, facing a closed arm. During the 5-min test period, behaviour was monitored by means of a video camera mounted above the maze and the following measurements were taken by a trained observer: (1) number of entries into open and closed
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
arms (an entry was counted when both forepaws were placed on the respective arm); (2) time spent on each type of arm; (3) latency until the first entry into an open arm; (4) number of full open arm entries (with all four paws) into the open arms. The number of entries into and time spent on the open arms were expressed as percentage of total number of arm entries and total time on all arms, respectively. Before each trial the maze was cleaned with water containing a detergent and dried with a towel.
2.3. Black–white box The open-topped test box was made of white PVC and divided into two compartments, connected by a small opening (7.5×7.5 cm). One compartment (54× 36 × 27 cm high) remained white and was illuminated by cold light with an intensity of 350 lux, the other one (27× 36× 27 cm high) was painted black and illuminated with an intensity of 70 lux. The white and black floors were divided into 24 and 12 squares (9 ×9 cm), respectively. At the beginning of a 5-min test period each rat was placed in the centre of the black compartment, facing the opening (and not in the white compartment as is usually the case — see Section 1). The rats were observed via a mirror mounted above the test box, and the following parameters were scored directly: (1) number of entries into the white compartment (defined as placing both forepaws into the white compartment); (2) time spent in the white compartment; (3) latency until the first entry into the white compartment; (4) number of exploratory rearings in the black and white compartment; (5) number of line crossings in the black and white compartment. From (4) and (5) the number of rearings and of line crossings in the black and the white compartment per min spent in the respective compartment was calculated. The box was cleaned before each trial.
2.4. Social interaction The test arena was made of white PVC (54× 36 × 27 cm). It was illuminated by cold light with an intensity of 40 lux and the floor was divided into six squares (18× 18 cm). At the beginning of a test a pair of rats was placed in the centre of the arena, the rats facing each other. The rats had been familiarised individually with the apparatus on two days prior to testing for 10 min each day. Both members of a pair belonged to the same line of rats but were unknown to each other. The weight of two rats of a pair differed up to 50 g (female) and 40 g (male), respectively. The behaviour was observed for 10 min via a mirror as well as monitored by a camera mounted vertically over the apparatus. The following parameters were scored: (1) time spent in active social interaction (sniffing, following, grooming, kicking, mounting, jumping on, wrestling and boxing with, crawling under or over the partner); (2) time spent in passive social interaction
155
(the rats sitting or lying with their bodies in contact, but without interacting with each other); (3) the number of line crossings of both rats. The arena was cleaned before each trial.
2.5. Home cage obser6ation Behaviour of HAB/LAB rats was observed in their home cages (five cages with two to five individuals per line) during the first hour after the lights in the housing room were switched off. The room was illuminated with dim red light during the observation phase. The animals were observed twice during the observation phase with 20 min between the two observation points on four consecutive days. At each timepoint of observation the behaviour of each animal was scored directly. The following parameters were taken: fighting, threatening, social grooming, rearing, sniffing, burying, self-grooming, sleeping/resting, feeding/drinking. As the scoring was carried out groupwise (i.e. there was no individual recognition of any single animal) the percentage of each behaviour compared with total behaviour at this observation point was calculated for each cage. The mean of the eight observation points was then calculated for each cage and mean data of the five HAB cages were compared to those of the five LAB cages (n= 5 cages per line).
2.6. Statistics Data were analysed using two different techniques, which afford complementary information. First, the group means of every particular variable (i.e. every parameter measured in any test paradigm) were compared by Mann–Whitney U-tests. This allowed the estimation of the statistical significance levels of measured differences in means across breeding lines (note: in the case of zero entries into the white box, time averaged quantities, such as line crossings or rearings in the white box per min spent in the white box, are not meaningful; such cases were excluded from all calculations). PB0.05 was considered statistically significant. Further, since multivariate analysis of variance (MANOVA) rejected the hypothesis of all equal means as expected, discriminant analyses [11] were performed on the parameter set measured in each test for each gender. The different qualitative properties of the data we examined are: (1) classification power of each paradigm with respect to group participation. This gives an idea of the usefulness of each test for our animal model; the information is globally reflected in the percentage of correct group assignments achieved when using the computed discriminant functions to predict group participation. (2) Discriminating power of each variable. This is a measure of the unique contribution of a variable to the discriminating power of the statistical model (and/or to its power to predict
156
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
group participation), and in the case of two groups it can be directly related to the size of the (standardized) regression coefficient for that particular variable in the discriminant function. This information is determined by computing the F-value for any particular variable together with its statistical significance. (3) Tolerance of each variable within a particular paradigm. This is an effort towards identifying the true number of independent properties (degrees of freedom) measured by each paradigm. The tolerance value of each variable is computed as 1 − R 2 of the respective variable with all previous variables included in the model. In multiple regression techniques, R 2 is a direct measure of the difference between the true values and the values estimated by the statistical model. Thus the tolerance is the proportion of the variance, which is unique to a particular variable, and represents a measure of that variable’s redundancy within the model. The more its contribution to the model, the higher the tolerance score for a specific variable (for example, a tolerance value of 20% means that the variable is 80% redundant with the other variables in the model).
3. Results
3.1. Ele6ated plus-maze Fig. 1 illustrates that HAB females displayed a lower percentage of entries into and time spent on the open arms compared to LABs. Also, the number of full open arm entries of female HAB rats, taken as an additional indicator of anxiety, was significantly lower than in the LAB group. However, the latency to the first open arm entry was not different between the lines. There was also no difference in general locomotor activity, measured as the number of entries into the closed arms. For male rats, similar results are shown in Fig. 2, HAB males displaying a lower percentage of entries into and time spent on the open arms than LAB males. The number of full open arm entries was significantly lower in male HAB than in LAB rats. The latency to the first entry into an open arm was significantly higher in male HAB rats than in their LAB counterparts. The number of closed arm entries did not differ significantly between the two lines.
Fig. 1. Baseline scores (means +S.E.M.) of female HAB/LAB rats in an elevated plus-maze paradigm. HAB, high anxiety-related behaviour (n= 10), LAB, low anxiety-related behaviour (n = 13). ** PB 0.01; *** PB 0.001 compared to HAB rats (Mann – Whitney U-test).
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
157
Fig. 2. Baseline scores (means + S.E.M.) of male HAB/LAB rats in an elevated plus-maze paradigm. HAB, high anxiety-related behaviour (n= 9), LAB, low anxiety-related behaviour (n= 10). **PB 0.01; ***PB0.001 compared to HAB rats (Mann – Whitney U-test).
3.2. Black–white box As can be seen from Fig. 3, HAB females entered the white compartment less often and spent less time in it than female LAB rats. The difference between the two lines in latency to the first entry into the white compartment did not reach significance. The number of line crossings per min in the black compartment was significantly lower in HAB than in LAB females, while there was no significant difference between the two lines in the number of line crossings per min in the white compartment. Female LAB but not HAB rats displayed significantly more line crossings per min in the white than in the black compartment. Compared to their LAB counterparts, HAB females displayed significantly less rearings per min in the black compartment as well as in the white compartment. Female LAB but not HAB rats displayed significantly more rearings per min in the white compartment than in the black compartment. Fig. 4 displays similar results for male rats. HAB males also entered the white compartment less often and spent less time in it than LAB males did. The latency to the first entry into the white compartment
was significantly higher in male HAB rats compared to their LAB counterparts. The number of line crossings per min in the black compartment as well as the number of line crossings per min in the white compartment was significantly lower in HAB than in LAB males. Male HAB rats displayed significantly less line crossings per min in the white than in the black compartment, whereas LAB males tended to display more line crossings per min in the white than in the black compartment. Compared to their LAB counterparts, male HAB rats displayed significantly less rearings per min in the black compartment as well as in the white compartment. Male HAB rats displayed significantly less rearings per min in the white compartment than in the black compartment, whereas there was no difference respecting this parameter in LAB males.
3.3. Social interaction Fig. 5 shows that female HAB rats spent significantly less time in active social interaction than LAB females, while there was no significant difference in passive social interaction. The number of line crossings in HAB females was significantly lower compared to LAB females.
158
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
HAB males also spent significantly less time in active social interaction than LAB males (Fig. 6). The time male HAB rats spent in passive social interaction was significantly higher compared to their LAB counterparts. In comparison to LAB males, the number of line crossings of HAB males was significantly lower.
3.4. Home cage beha6iour Behaviour of male HAB and LAB rats in their home cages is illustrated in Fig. 7. HAB rats displayed significantly less rearings but more sleeping/resting than their LAB. There were no significant differences in fighting, threatening, social grooming, sniffing, burying, selfgrooming and feeding/drinking.
3.5. Discriminant analyses HAB and LAB rats were discriminated with a high accuracy in all tests of unconditioned anxiety as well as in social behaviour in their home cages. In the elevated plus-maze paradigm the most important variable for
discrimination between the two lines was the percent time spent on the open arms. The most important parameters in the black–white box test were the number of rearings per min in the black compartment and the number of entries into the white compartment, respectively. In the social interaction test, the number of line crossings, and in home cage observation, the number of rearings, were most important for discrimination between the lines. For the exact percentage of correct discrimination between the two lines as well as the discriminating factors (with their corresponding Fand P-values and values of tolerance) refer to Table 1.
4. Discussion According to the breeding selection, female and male individuals of the HAB/LAB lines differed significantly in anxiety-related behaviour on the elevated plus-maze, with HAB rats entering the open arms less often and spending less time on the open arms than their LAB counterparts. Individuals of both lines did not differ in
Fig. 3. Baseline scores (means+S.E.M.) of female HAB/LAB rats in a black – white box paradigm. HAB, high anxiety-related behaviour (n= 10), LAB, low anxiety-related behaviour (n = 13). *PB 0.05, ***PB 0.001 compared to HAB rats in the same compartment, °PB 0.05, °°°PB0.001 compared to LAB rats in the white compartment (Mann–Whitney U-test).
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
159
Fig. 4. Baseline scores (means+S.E.M.) of male HAB/LAB rats in a black – white box paradigm. HAB, high anxiety-related behaviour (n= 10), LAB, low anxiety-related behaviour (n =8). *P B 0.05, **PB 0.01, ***PB 0.001 compared to HAB rats in the same compartment, °PB 0.05, °°°PB 0.001 compared to HAB rats in the white compartment (Mann – Whitney U-test).
Fig. 5. Baseline scores (means+ S.E.M.) of female HAB/LAB rats in a social interaction paradigm. HAB, high anxiety-related behaviour (n=5 pairs), LAB, low anxiety-related behaviour (n =6 pairs). *P B 0.05; **PB0.01 compared to HAB rats (Mann – Whitney U-test).
the number of closed arm entries, implying that there were no differences in locomotion (Figs. 1 and 2). These results are in good agreement with data previously shown [14].
The difference in anxiety-related behaviour between the two lines was highly consistent in both the black– white box and the social interaction test. In the black– white box paradigm, male and female HAB rats entered
160
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
the white compartment less often and spent less time in it than their LAB counterparts did (Figs. 3 and 4). Likewise, in the social interaction test, male as well as female HAB rats spent less time in active social interaction than LAB rats (Figs. 5 and 6). These results underline former observations from the elevated plus-
maze and the open field test that the difference in anxiety-related behaviour between HAB and LAB rats is independent of gender [14]. However, in the black–white box as well as in the social interaction test, there was not only a distinct difference in anxiety-related behaviour but also a sig-
Fig. 6. Baseline scores of male HAB/LAB rats in a social interaction paradigm. HAB, high anxiety-related behaviour (n =5 pairs), LAB, low anxiety-related behaviour (n=5 pairs). **PB 0.01 compared to HAB rats (Mann – Whitney U-test).
Fig. 7. Behaviour of male HAB/LAB rats in their home cages. Bars represent the percentage of the particular behaviour in respect to total behaviour in the cages during the first hour of the activity phase (mean+S.E.M.). HAB, high anxiety-related behaviour (n = 5 cages), LAB, low anxiety-related behaviour (n=5 cages). *PB 0.05 compared to HAB rats (Mann – Whitney U-test).
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
161
Table 1 Results of discriminant analyses of HAB/LAB rats in different paradigmsa Paradigm
% Correct
Discriminating factors
F
P
Tolerance
92.3
%Time open arms Latency %Entries open arms Full entries open arms Closed entries
13.43 6.29 3.51 1.43 0.11
0.002 0.02 0.08 0.25 0.74
0.70 0.57 0.76 0.63 0.55
78
90
%Time open arms Latency Closed entries Full entries open arms %Entries open arms
9.24 2.96 0.04 0.03 0.00
0.008 0.10 0.85 0.87 0.99
0.94 0.94 0.96 0.26 0.30
90
100
Rearings black/min black Time white Entries white Latency Crossings black/min black
7.22 2.52 2.35 1.01 0.36
0.01 0.13 0.14 0.33 0.56
0.73 0.69 0.58 0.67 0.39
90
100
Entries white Rearings black/min black Latency Crossings black/min black Time white
20.87 1.69 0.48 0.44 0.21
0.0004 0.21 0.50 0.52 0.66
1.00 1.00 0.50 0.62 0.52
Line crossings Passive social interaction Active social interaction
18.71 0.78 0.10
0.002 0.40 0.76
1.00 0.99 0.63
Line crossings Passive social interaction Active social interaction
51.99 4.47 2.25
0.0004 0.08 0.18
0.62 0.65 0.92
0.02 0.23 0.36 0.42 0.43 0.57 0.78 0.79 0.99
0.94 0.94 0.97 0.59 0.76 0.55 0.70 0.93 0.46
HAB Elevated plus-maze Females
100
LAB
HAB: n = 10 LAB: n = 13 Elevated plus-maze Males HAB: n = 9 LAB: n = 10 Black–white box Females HAB: n = 10 LAB: n = 13 Black–white box Males HAB: n = 10 LAB: n = 8 Social interaction Females HAB: n = 5 pairs LAB: n = 6 pairs
100
Social interaction Males HAB: n = 5 pairs LAB: n = 5 pairs
100
100
Home cage behaviour Males
100
80
HAB: n = 5 cages LAB: n = 5 cages
83.3
Rearing Threatening Social grooming Burying Feeding/drinking Sniffing Fighting Self-grooming Sleeping/resting
8.73 1.54 0.97 0.74 0.71 0.35 0.09 0.08 0.00
a For each paradigm the percentage of accurate discrimination of each line is given. Additionally the variables of each test are listed in order of discriminating power with their respective F- and corresponding P-value. The value of tolerance indicates the amount of redundancy of the respective variable for discrimination of the two lines (a lower tolerance indicates higher redundancy). HAB, high anxiety-related behaviour; LAB, low anxiety-related behaviour.
nificant difference in locomotor activity, with female and male HAB rats being less active than their LAB counterparts. Although this difference might be interpreted as anxiety-related behaviour being, at least in part, secondary to the difference in locomotor activity, it has to be considered that movement inhibition may be one characteristic of anxious animals [7,25]. Furthermore, more comprehensive statistical analysis using discriminant analysis revealed that in the black – white box test, the parameters that are most important for the discrimination between the two lines are anxiety-related and
exploratory behaviours. These parameters are the number of entries into the white compartment and the number of rearings per min in the black compartment, respectively. Analogously, in the elevated plus-maze test, the most important parameter for the discrimination between the two lines is the percent time spent on the open arms, the parameter that is generally seen as the one that is most closely related to innate anxiety. In both paradigms, the locomotor activity (as measured in the black–white box test by the number of line crossings in the black compartment per min spent in it and in the
162
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
elevated plus-maze paradigm by the number of closed arm entries) has no significant importance for discrimination between the two lines. In the social interaction paradigm the data indicate the opposite. The most important parameter that discriminates between the two lines is the locomotor activity, whereas the active social interaction, classically seen as the most relevant parameter for anxiety-related behaviour, is the least important parameter for discrimination. These results confirm recent factor analytical studies that found locomotion being the parameter that loaded highest on the factor interpreted as anxiety in the social interaction paradigm [21,22]. A possible explanation for those results may be that the inhibition of active social interaction seems to be related to competing defensive reactions such as freezing, thigmotaxis and displacement behaviours [24]: at least two of those three factors imply a reduction of locomotion. Interestingly, in the factor analytical studies locomotion in the black – white box but not in the elevated plus-maze test also loaded highly on the factor ‘anxiety’ [21,22]. The difference in locomotor activity between HAB and LAB rats has also been observed in an open-field experiment [14]. However, when singly-housed male HAB and LAB rats were tested under baseline conditions in their home cage with a radiotelemetric system, no difference in locomotion was found [13]. These findings suggest that individuals of the HAB and LAB lines do not differ in their basal locomotor activity, but in their reactions to novel and stressful situations. This suggestion is supported by the fact that HAB and LAB rats differed in their behaviour in a forced swim test, a behavioural test developed for screening anti-depressant drugs [20]: HAB rats started floating earlier, spent significantly more time in this immobile posture and struggled less than LAB rats [14]. The behavioural difference in this inescapable situation reflects a difference in coping behaviour [1,16]. In this respect, LAB rats display an active coping behaviour whereas HAB rats behave in a rather passive, ‘depressive-like’ manner. Thus, the results of the tests used here may not only reflect a difference between the two lines in their innate state of anxiety but also a difference in coping with a novel and stressful environment. This is in line with the notion that in experimental rats, emotionality and coping behaviour are linked to each other [18,29]. Likewise, there is also a linkage between human conditions, hypothetically considered as parallels of emotionality and coping in animals, namely anxiety and depression, respectively [23,30]. Individuals of the HAB and LAB rat lines did not only differ in their behaviour in conflict situations, as in the tests of anxiety described above, but also in a non-conflict situation: when group-housed HAB and LAB rats were observed under basal conditions in their home cages within the first hour of the activity phase, HAB rats
displayed significantly less rearings and more sleeping/ resting than their LAB counterparts Fig. 7. The latter is in contrast to the finding that individuals of the HAB and LAB lines did not differ in locomotor activity when measured under baseline conditions in their home cage with a radiotelemetric system [13]. This difference is likely due to the fact that the radiotelemetric measurements were conducted with singly housed rats whereas the home cage observation was done with group-housed animals: The differences between group-housed HAB and LAB rats in rearings and sleeping/resting behaviour were probably not due to a difference in activity between the two lines per se but may be a consequence of an increased level of aggressive interactions in the home cages of the LAB rats compared to the HAB rats, although the differences in the behavioural parameters fighting and threatening taken alone did not reach significance. However, when the data of fighting and threatening were taken together to the parameter ‘aggressive behaviour’, the difference in this parameter was significant (PB 0.05, Mann–Whitney U-test), with LAB rats being more aggressive than HAB rats. In conclusion, this study has shown consistent behavioural differences between HAB and LAB rats, regardless of the gender, in three tests of unconditioned anxiety, indicating that the emotional difference between the two lines is a robust trait independent of the test used. Moreover, the selective breeding of HAB and LAB rats resulted also in differences in social behaviour, with LAB rats tending to display more aggressive behaviour than HAB rats. Acknowledgements The authors wish to thank Albin Varga for reliable and professional animal care. This study was supported by the Deutsche Forschungsgemeinschaft. References [1] Armario A, Galvada` A, Martı´ O. Forced swimming test in rats: effect of desipramine administration and the period of exposure to the test on struggling behaviour, swimming, immobility and defecation rate. Eur J Pharmacol 1988;158:207 – 12. [2] Belzung C, Le Pape G. Comparison of different behavioral test situations used in psychopharmacology for measurement of anxiety. Physiol Behav 1994;56:623 – 8. [3] Chaouloff F, Durand M, Morme`de P. Anxiety- and activity-related effects of diazepam and chlordiazepoxide in rat light/dark and dark/light test. Behav Brain Res 1997;85:27 – 35. [4] Costall B, Jones BJ, Kelly ME, Naylor RJ, Tomkins DM. Exploration of mice in a black and white test box: validation as a model of anxiety. Pharmacol Biochem Behav 1989;32:777–85. [5] Crawley J, Goodwin FK. Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacol Biochem Behav 1980;13:167 – 70. [6] Dawson GR, Tricklebank MD. Use of the elevated plus maze in the search for novel anxiolytic agents. Trends Pharmacol Sci 1995;16:33 – 6.
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163 [7] Escorihuela RM, Ferna´ndez-Teruel A, Gil L, Aguilar R, Toben˜a A, Driscoll P. Inbred Roman high- and low avoidance rats: differences in anxiety, novelty-seeking, and shuttlebox behaviors. Physiol Behav 1999;67:19–26. [8] File SE. The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs. J Neurosci Methods 1980;2:219 –38. [9] File SE, Hyde JRG. Can social interaction be used to measure anxiety? Br J Pharmacol 1978;62:19–24. [10] Hogg S. A review of the validity and variability of the elevated plus-maze as an animal model of anxiety. Pharmacol Biochem Behav 1996;54:21 – 30. [11] Huberty CJ. Applied Discriminant Analysis. New York: Wiley, 1994. [12] Landgraf R, Wigger A, Holsboer F, Neumann ID. Hyper-reactive hypothalamo-pituitary adrenocortical axis in rats bred for high anxiety-related behaviour. J Neuroendocrinol 1999;11:405 – 7. [13] Liebsch G, Linthorst CE, Neumann ID, Reul JMHM, Holsboer F, Landgraf R. Behavioral, physiological, and neuroendocrine stress responses and differential sensitivity to diazepam in two Wistar rat lines selectively bred for high- and low-anxiety-related behavior. Neuropsychopharmacology 1998;19:381–96. [14] Liebsch G, Montkowski A, Holsboer F, Landgraf R. Behavioral profiles of two Wistar rat lines selectively bred for high or low anxiety-related behavior. Behav Brain Res 1998;94:301–10. [15] Lister RG. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology 1987;92:180–5. [16] Martı´ J, Armario A. Effects of diazepam and desipramine in the forced swimming test: influence of previous experience with situation. Eur J Pharmacol 1993;236:295–9. [17] Onaivi ES, Martin BR. Neuropharmacological and physiological validation of a computer-controlled two-compartment black and white box for the assessment of anxiety. Prog Neuropsychopharmacol Biol Psychiatr 1989;13:963–76. [18] Overstreet DH, Rezvani AH, Janowsky DS. Maudsley reactive and nonreactive rats differ only in some tasks reflecting emotionality. Physiol Behav 1992;52:149–52.
.
163
[19] Pellow S, Chopin P, File SE, Briley M. Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 1985;14:149 – 67. [20] Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal model sensitive to antidepressant treatment. Nature 1977;266:730 – 2. [21] Ramos A, Berton O, Morme`de P, Chaouloff F. A multiple-test study of anxiety-related behaviours in six inbred rat strains. Behav Brain Res 1997;85:57 – 69. [22] Ramos A, Morme`de P. Stress and emotionality: a multidimensional and genetic approach. Neurosci Biobehav Rev 1998;22:33 – 57. [23] Regier DA, Rae DS, Narrow WE, Kaelber CT, Schatzberg AF. Prevalence of anxiety disorders and their comorbidity with mood and addictive disorders. Br J Psychiatr 1998;173(34):24–8. [24] Rodgers RJ. Animal models of ‘anxiety’: where next? Behav Pharmacol 1997;8:477 – 96. [25] Rodgers RJ, Cao BJ, Dalvi A, Holmes A. Animal models of anxiety: an ethological perspective. Braz J Med Biol Res 1997;30:289 – 304. [26] Sa´nchez C. 5-HT receptors play an important role in modulation of behavior of rats in a two-compartment black and white box. Behav Pharmacol 1996;7:788 – 97. [27] Smythe JW, Murphy D, Bhatnagar S, Timothy C, Costall B. Muscarinic antagonists are anxiogenic in rats tested in the black – white box. Pharmacol Biochem Behav 1996;54:57–63. [28] Steimer T, Driscoll P, Schulz PE. Brain metabolism of progesterone, coping behaviour and emotional reactivity in male rats from two psychogenetically selected lines. J Neuroendocrinol 1997;169 – 175. [29] Steimer T, la Fleur S, Schulz PE. Neuroendocrine correlates of emotional reactivity and coping in male rats from the Roman High (RHA/Verh)- and low (RLA/Verh)-Avoidance lines. Behavior Genetics. 1997;503 – 512. [30] Wittchen H-U, Essau CA, Krieg J-C. Anxiety disorders: similarities and differences of comorbidity in treated and untreated groups. Br J Psychiatr 1991;159(12):23– 33.
Research report
Unconditioned anxiety and social behaviour in two rat lines selectively bred for high and low anxiety-related behaviour Markus S.H. Henniger * , Frauke Ohl, Sabine M. Ho¨lter, Petra Weißenbacher, Nicola Toschi, Patrick Lo¨rscher, Alexandra Wigger, Rainer Spanagel, Rainer Landgraf Max Planck Institute of Psychiatry, Kraepelinstr. 2 -10, 80804 Munich, Germany Received 25 October 1999; received in revised form 13 January 2000; accepted 13 January 2000
Abstract Individuals of high anxiety-related behaviour (HAB) and low anxiety-related behaviour (LAB) rat lines were selectively bred for differences in anxiety-related behaviour on the elevated plus-maze. The goal of this study was to investigate whether this behavioural difference is restricted to the test used as the selection criterion or whether it is a stable and robust trait also in other conflict or non-conflict situations. Therefore, behaviour of male and female HAB and LAB rats was examined in two further tests of unconditioned anxiety: the black–white box and the social interaction test. Furthermore, behaviour of group-housed male HAB and LAB rats was studied in their home cages. In addition to standard statistics, discriminant analyses were performed. The difference in anxiety-related behaviour between the two lines was highly consistent in all tests of unconditioned anxiety. There were also differences in home cage behaviour, LAB rats being more active than HAB rats; this is likely to be a consequence of the LAB rats displaying a higher aggressiveness in social behaviour, compared to HAB rats. In all tests used HAB and LAB rats were clearly distinguished by discriminant analysis. However, while in the elevated plus-maze and the black – white box test the most important parameters for discrimination between the two lines were mainly those generally seen as closely related to anxiety, the discrimination in the social interaction paradigm was primarily due to differences in locomotor activity. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Emotionality; Elevated plus-maze; Black–white box; Social interaction test; Home cage behaviour
1. Introduction We recently reported on two Wistar rat lines which were selectively bred over several years for extremes in anxiety-related behaviour on the elevated plus-maze [12 – 14]: the high anxiety-related behaviour (HAB) and the low anxiety-related behaviour (LAB) lines. These lines represent a useful animal model for studying the neurobiology of inborn anxiety and the mechanism(s) of actions of anxiolytic compounds [13]. Anxiety has been shown to be a highly complex construct and it has been suggested that different tests of anxiety-related behaviour measure various aspects of * Corresponding author. Tel.: +49-89-30622641; fax: + 49-8930622569. E-mail address: [email protected] (M.S.H. Henniger)
anxiety [2,24]. When using animal models of inborn anxiety it is, therefore, of great interest to evaluate whether differences in anxiety-related behaviour occur globally or are restricted to variables measured by the test used as selection criterion. Therefore, we tested HAB/LAB rats in different paradigms, which have been shown to measure distinct dimensions of emotionality. First, the elevated plus-maze was used to confirm the selected line differences. Then we tested HAB and LAB rats in two other tests of unconditioned anxiety, the black–white box and the social interaction test. The elevated plus-maze test is a widely used test paradigm to investigate anxiety-related behaviour (for reviews see Refs. [6,10,25]) and has extensively been validated for use in both rats [19] and mice [15]. It is based on the test-induced conflict in rodents between
0166-4328/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 0 ) 0 0 1 5 1 - 0
154
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
the aversion of being exposed to an open and elevated platform on the one hand and the motivation to explore the new environment on the other. As a consequence, the less anxious the individuals are, the more they explore the open arms. The black–white box test was developed [5] and validated [4,17] as a test of anxiety in mice. In this test an animal experiences a conflict situation between the motivation to either explore or to avoid an exposed, brightly lit, area. An individual, which explores the exposed area more and for longer is defined as being less anxious. In some studies the black – white box paradigm was also used for rats, with differences in experimental design and procedure [3,26,27]. For example, some authors have found it to be advantageous, as we have in the present study, to initially place the animal in the darkened side of the box, rather than in the brightly lit side [28]. In the social interaction test, developed and validated for rats by File et al. [8,9], a pair of rats which are unknown to each other, are placed into a neutral arena and the time the individuals spend in social interaction is measured. It has been shown that the more time a pair of rats spends in active social interaction, the less anxious they are [9]. In addition to the described tests of unconditioned anxiety, we observed behaviour of group-housed HAB and LAB rats in their home cages during the first hour of the activity phase. This home cage analysis was conducted to get a better idea about possible behavioural differences of HAB/LAB rats in a nonconflict situation. Taken together, the aim of this study was to investigate behaviour of the HAB and LAB rat lines in different conflict and non-conflict situations. In addition to standard statistics, discriminant analysis was used to examine under inclusion of all parameters of the respective tests: (a) the accuracy of discrimination of HAB and LAB lines for the different paradigms; and (b) those parameters of each test which determine the discrimination between the two lines.
2. Materials and methods
2.1. Animals Subjects were individuals of two Wistar rat lines selected and bred in our institute as previously described [13,14]. Briefly, adult Wistar rats, obtained from a commercial supplier (Charles River, Sulzfeld, Germany) were tested in an elevated plus-maze and animals with HAB and animals with LAB, respectively, were then mated for years to establish the HAB and LAB lines. At the age of 10 weeks, the offspring from both breeding lines was tested on the elevated plus-maze and
for each new generation, HAB and LAB breeding pairs were selected. HAB animals showing low levels and LAB rats displaying high levels of anxiety-related behaviour, as well as individuals showing no clear tendency towards either of these two extremes, were excluded from further breeding as well as from further behavioural testing. In the present study, female rats (HAB n= 10, LAB n = 13; generation F7) were tested (as mentioned above) on the elevated plus-maze at an age of 10 weeks. At an age of 6–10 months the same individuals were tested in the black–white box. Three weeks thereafter they were tested in the social interaction paradigm. All three tests of unconditioned anxiety were also conducted with male rats (elevated plus-maze: HAB n=9, LAB n = 10, age 10 weeks; black–white box: HAB n = 10, LAB n= 8, age 14 weeks; social interaction: 10 individuals per line, age 13 weeks; all tests: generation F10). Home cage observation was conducted with male, 16–20-week old rats (17 individuals per line, generations F9 and 10). Animals were housed in groups of up to five samesexed individuals of the same line (cage size: 58×38× 20 cm height), except before the social interaction test, when rats were housed individually for 2 weeks (cage size: 42× 26× 20 cm height). All animals were maintained on a 12-h light/dark cycle (lights on: 6:00 h– 18:00 h) with food and water available ad libitum. At least 16 h before testing or familiarising with the test arena (social interaction, see below) the animals were brought to the testing room. All tests of unconditioned anxiety were conducted between 8:00 h and 12:30 h. Home cage behaviour was observed during the first hour after the lights in the housing room were switched off (18:00 h–19:00 h). The experiments were approved by the Committee on Animal Care and Use of the relevant local governmental body.
2.2. Ele6ated plus-maze The elevated plus-maze we used was described previously [13,14]. Briefly, it was made of dark grey PVC and consisted of two open arms (50× 10 cm) and two enclosed arms (50 ×10×38 cm high walls) extending from a central platform (10×10 cm). The apparatus was elevated 73 cm above the floor. The maze, surrounded by an opaque curtain, was lit by two white bulbs above the open arms and two red bulbs above the closed arms (mean light intensity over the whole maze: 100 lux). At the beginning of a test, each rat was placed on the central platform, facing a closed arm. During the 5-min test period, behaviour was monitored by means of a video camera mounted above the maze and the following measurements were taken by a trained observer: (1) number of entries into open and closed
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
arms (an entry was counted when both forepaws were placed on the respective arm); (2) time spent on each type of arm; (3) latency until the first entry into an open arm; (4) number of full open arm entries (with all four paws) into the open arms. The number of entries into and time spent on the open arms were expressed as percentage of total number of arm entries and total time on all arms, respectively. Before each trial the maze was cleaned with water containing a detergent and dried with a towel.
2.3. Black–white box The open-topped test box was made of white PVC and divided into two compartments, connected by a small opening (7.5×7.5 cm). One compartment (54× 36 × 27 cm high) remained white and was illuminated by cold light with an intensity of 350 lux, the other one (27× 36× 27 cm high) was painted black and illuminated with an intensity of 70 lux. The white and black floors were divided into 24 and 12 squares (9 ×9 cm), respectively. At the beginning of a 5-min test period each rat was placed in the centre of the black compartment, facing the opening (and not in the white compartment as is usually the case — see Section 1). The rats were observed via a mirror mounted above the test box, and the following parameters were scored directly: (1) number of entries into the white compartment (defined as placing both forepaws into the white compartment); (2) time spent in the white compartment; (3) latency until the first entry into the white compartment; (4) number of exploratory rearings in the black and white compartment; (5) number of line crossings in the black and white compartment. From (4) and (5) the number of rearings and of line crossings in the black and the white compartment per min spent in the respective compartment was calculated. The box was cleaned before each trial.
2.4. Social interaction The test arena was made of white PVC (54× 36 × 27 cm). It was illuminated by cold light with an intensity of 40 lux and the floor was divided into six squares (18× 18 cm). At the beginning of a test a pair of rats was placed in the centre of the arena, the rats facing each other. The rats had been familiarised individually with the apparatus on two days prior to testing for 10 min each day. Both members of a pair belonged to the same line of rats but were unknown to each other. The weight of two rats of a pair differed up to 50 g (female) and 40 g (male), respectively. The behaviour was observed for 10 min via a mirror as well as monitored by a camera mounted vertically over the apparatus. The following parameters were scored: (1) time spent in active social interaction (sniffing, following, grooming, kicking, mounting, jumping on, wrestling and boxing with, crawling under or over the partner); (2) time spent in passive social interaction
155
(the rats sitting or lying with their bodies in contact, but without interacting with each other); (3) the number of line crossings of both rats. The arena was cleaned before each trial.
2.5. Home cage obser6ation Behaviour of HAB/LAB rats was observed in their home cages (five cages with two to five individuals per line) during the first hour after the lights in the housing room were switched off. The room was illuminated with dim red light during the observation phase. The animals were observed twice during the observation phase with 20 min between the two observation points on four consecutive days. At each timepoint of observation the behaviour of each animal was scored directly. The following parameters were taken: fighting, threatening, social grooming, rearing, sniffing, burying, self-grooming, sleeping/resting, feeding/drinking. As the scoring was carried out groupwise (i.e. there was no individual recognition of any single animal) the percentage of each behaviour compared with total behaviour at this observation point was calculated for each cage. The mean of the eight observation points was then calculated for each cage and mean data of the five HAB cages were compared to those of the five LAB cages (n= 5 cages per line).
2.6. Statistics Data were analysed using two different techniques, which afford complementary information. First, the group means of every particular variable (i.e. every parameter measured in any test paradigm) were compared by Mann–Whitney U-tests. This allowed the estimation of the statistical significance levels of measured differences in means across breeding lines (note: in the case of zero entries into the white box, time averaged quantities, such as line crossings or rearings in the white box per min spent in the white box, are not meaningful; such cases were excluded from all calculations). PB0.05 was considered statistically significant. Further, since multivariate analysis of variance (MANOVA) rejected the hypothesis of all equal means as expected, discriminant analyses [11] were performed on the parameter set measured in each test for each gender. The different qualitative properties of the data we examined are: (1) classification power of each paradigm with respect to group participation. This gives an idea of the usefulness of each test for our animal model; the information is globally reflected in the percentage of correct group assignments achieved when using the computed discriminant functions to predict group participation. (2) Discriminating power of each variable. This is a measure of the unique contribution of a variable to the discriminating power of the statistical model (and/or to its power to predict
156
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
group participation), and in the case of two groups it can be directly related to the size of the (standardized) regression coefficient for that particular variable in the discriminant function. This information is determined by computing the F-value for any particular variable together with its statistical significance. (3) Tolerance of each variable within a particular paradigm. This is an effort towards identifying the true number of independent properties (degrees of freedom) measured by each paradigm. The tolerance value of each variable is computed as 1 − R 2 of the respective variable with all previous variables included in the model. In multiple regression techniques, R 2 is a direct measure of the difference between the true values and the values estimated by the statistical model. Thus the tolerance is the proportion of the variance, which is unique to a particular variable, and represents a measure of that variable’s redundancy within the model. The more its contribution to the model, the higher the tolerance score for a specific variable (for example, a tolerance value of 20% means that the variable is 80% redundant with the other variables in the model).
3. Results
3.1. Ele6ated plus-maze Fig. 1 illustrates that HAB females displayed a lower percentage of entries into and time spent on the open arms compared to LABs. Also, the number of full open arm entries of female HAB rats, taken as an additional indicator of anxiety, was significantly lower than in the LAB group. However, the latency to the first open arm entry was not different between the lines. There was also no difference in general locomotor activity, measured as the number of entries into the closed arms. For male rats, similar results are shown in Fig. 2, HAB males displaying a lower percentage of entries into and time spent on the open arms than LAB males. The number of full open arm entries was significantly lower in male HAB than in LAB rats. The latency to the first entry into an open arm was significantly higher in male HAB rats than in their LAB counterparts. The number of closed arm entries did not differ significantly between the two lines.
Fig. 1. Baseline scores (means +S.E.M.) of female HAB/LAB rats in an elevated plus-maze paradigm. HAB, high anxiety-related behaviour (n= 10), LAB, low anxiety-related behaviour (n = 13). ** PB 0.01; *** PB 0.001 compared to HAB rats (Mann – Whitney U-test).
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
157
Fig. 2. Baseline scores (means + S.E.M.) of male HAB/LAB rats in an elevated plus-maze paradigm. HAB, high anxiety-related behaviour (n= 9), LAB, low anxiety-related behaviour (n= 10). **PB 0.01; ***PB0.001 compared to HAB rats (Mann – Whitney U-test).
3.2. Black–white box As can be seen from Fig. 3, HAB females entered the white compartment less often and spent less time in it than female LAB rats. The difference between the two lines in latency to the first entry into the white compartment did not reach significance. The number of line crossings per min in the black compartment was significantly lower in HAB than in LAB females, while there was no significant difference between the two lines in the number of line crossings per min in the white compartment. Female LAB but not HAB rats displayed significantly more line crossings per min in the white than in the black compartment. Compared to their LAB counterparts, HAB females displayed significantly less rearings per min in the black compartment as well as in the white compartment. Female LAB but not HAB rats displayed significantly more rearings per min in the white compartment than in the black compartment. Fig. 4 displays similar results for male rats. HAB males also entered the white compartment less often and spent less time in it than LAB males did. The latency to the first entry into the white compartment
was significantly higher in male HAB rats compared to their LAB counterparts. The number of line crossings per min in the black compartment as well as the number of line crossings per min in the white compartment was significantly lower in HAB than in LAB males. Male HAB rats displayed significantly less line crossings per min in the white than in the black compartment, whereas LAB males tended to display more line crossings per min in the white than in the black compartment. Compared to their LAB counterparts, male HAB rats displayed significantly less rearings per min in the black compartment as well as in the white compartment. Male HAB rats displayed significantly less rearings per min in the white compartment than in the black compartment, whereas there was no difference respecting this parameter in LAB males.
3.3. Social interaction Fig. 5 shows that female HAB rats spent significantly less time in active social interaction than LAB females, while there was no significant difference in passive social interaction. The number of line crossings in HAB females was significantly lower compared to LAB females.
158
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
HAB males also spent significantly less time in active social interaction than LAB males (Fig. 6). The time male HAB rats spent in passive social interaction was significantly higher compared to their LAB counterparts. In comparison to LAB males, the number of line crossings of HAB males was significantly lower.
3.4. Home cage beha6iour Behaviour of male HAB and LAB rats in their home cages is illustrated in Fig. 7. HAB rats displayed significantly less rearings but more sleeping/resting than their LAB. There were no significant differences in fighting, threatening, social grooming, sniffing, burying, selfgrooming and feeding/drinking.
3.5. Discriminant analyses HAB and LAB rats were discriminated with a high accuracy in all tests of unconditioned anxiety as well as in social behaviour in their home cages. In the elevated plus-maze paradigm the most important variable for
discrimination between the two lines was the percent time spent on the open arms. The most important parameters in the black–white box test were the number of rearings per min in the black compartment and the number of entries into the white compartment, respectively. In the social interaction test, the number of line crossings, and in home cage observation, the number of rearings, were most important for discrimination between the lines. For the exact percentage of correct discrimination between the two lines as well as the discriminating factors (with their corresponding Fand P-values and values of tolerance) refer to Table 1.
4. Discussion According to the breeding selection, female and male individuals of the HAB/LAB lines differed significantly in anxiety-related behaviour on the elevated plus-maze, with HAB rats entering the open arms less often and spending less time on the open arms than their LAB counterparts. Individuals of both lines did not differ in
Fig. 3. Baseline scores (means+S.E.M.) of female HAB/LAB rats in a black – white box paradigm. HAB, high anxiety-related behaviour (n= 10), LAB, low anxiety-related behaviour (n = 13). *PB 0.05, ***PB 0.001 compared to HAB rats in the same compartment, °PB 0.05, °°°PB0.001 compared to LAB rats in the white compartment (Mann–Whitney U-test).
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
159
Fig. 4. Baseline scores (means+S.E.M.) of male HAB/LAB rats in a black – white box paradigm. HAB, high anxiety-related behaviour (n= 10), LAB, low anxiety-related behaviour (n =8). *P B 0.05, **PB 0.01, ***PB 0.001 compared to HAB rats in the same compartment, °PB 0.05, °°°PB 0.001 compared to HAB rats in the white compartment (Mann – Whitney U-test).
Fig. 5. Baseline scores (means+ S.E.M.) of female HAB/LAB rats in a social interaction paradigm. HAB, high anxiety-related behaviour (n=5 pairs), LAB, low anxiety-related behaviour (n =6 pairs). *P B 0.05; **PB0.01 compared to HAB rats (Mann – Whitney U-test).
the number of closed arm entries, implying that there were no differences in locomotion (Figs. 1 and 2). These results are in good agreement with data previously shown [14].
The difference in anxiety-related behaviour between the two lines was highly consistent in both the black– white box and the social interaction test. In the black– white box paradigm, male and female HAB rats entered
160
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
the white compartment less often and spent less time in it than their LAB counterparts did (Figs. 3 and 4). Likewise, in the social interaction test, male as well as female HAB rats spent less time in active social interaction than LAB rats (Figs. 5 and 6). These results underline former observations from the elevated plus-
maze and the open field test that the difference in anxiety-related behaviour between HAB and LAB rats is independent of gender [14]. However, in the black–white box as well as in the social interaction test, there was not only a distinct difference in anxiety-related behaviour but also a sig-
Fig. 6. Baseline scores of male HAB/LAB rats in a social interaction paradigm. HAB, high anxiety-related behaviour (n =5 pairs), LAB, low anxiety-related behaviour (n=5 pairs). **PB 0.01 compared to HAB rats (Mann – Whitney U-test).
Fig. 7. Behaviour of male HAB/LAB rats in their home cages. Bars represent the percentage of the particular behaviour in respect to total behaviour in the cages during the first hour of the activity phase (mean+S.E.M.). HAB, high anxiety-related behaviour (n = 5 cages), LAB, low anxiety-related behaviour (n=5 cages). *PB 0.05 compared to HAB rats (Mann – Whitney U-test).
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
161
Table 1 Results of discriminant analyses of HAB/LAB rats in different paradigmsa Paradigm
% Correct
Discriminating factors
F
P
Tolerance
92.3
%Time open arms Latency %Entries open arms Full entries open arms Closed entries
13.43 6.29 3.51 1.43 0.11
0.002 0.02 0.08 0.25 0.74
0.70 0.57 0.76 0.63 0.55
78
90
%Time open arms Latency Closed entries Full entries open arms %Entries open arms
9.24 2.96 0.04 0.03 0.00
0.008 0.10 0.85 0.87 0.99
0.94 0.94 0.96 0.26 0.30
90
100
Rearings black/min black Time white Entries white Latency Crossings black/min black
7.22 2.52 2.35 1.01 0.36
0.01 0.13 0.14 0.33 0.56
0.73 0.69 0.58 0.67 0.39
90
100
Entries white Rearings black/min black Latency Crossings black/min black Time white
20.87 1.69 0.48 0.44 0.21
0.0004 0.21 0.50 0.52 0.66
1.00 1.00 0.50 0.62 0.52
Line crossings Passive social interaction Active social interaction
18.71 0.78 0.10
0.002 0.40 0.76
1.00 0.99 0.63
Line crossings Passive social interaction Active social interaction
51.99 4.47 2.25
0.0004 0.08 0.18
0.62 0.65 0.92
0.02 0.23 0.36 0.42 0.43 0.57 0.78 0.79 0.99
0.94 0.94 0.97 0.59 0.76 0.55 0.70 0.93 0.46
HAB Elevated plus-maze Females
100
LAB
HAB: n = 10 LAB: n = 13 Elevated plus-maze Males HAB: n = 9 LAB: n = 10 Black–white box Females HAB: n = 10 LAB: n = 13 Black–white box Males HAB: n = 10 LAB: n = 8 Social interaction Females HAB: n = 5 pairs LAB: n = 6 pairs
100
Social interaction Males HAB: n = 5 pairs LAB: n = 5 pairs
100
100
Home cage behaviour Males
100
80
HAB: n = 5 cages LAB: n = 5 cages
83.3
Rearing Threatening Social grooming Burying Feeding/drinking Sniffing Fighting Self-grooming Sleeping/resting
8.73 1.54 0.97 0.74 0.71 0.35 0.09 0.08 0.00
a For each paradigm the percentage of accurate discrimination of each line is given. Additionally the variables of each test are listed in order of discriminating power with their respective F- and corresponding P-value. The value of tolerance indicates the amount of redundancy of the respective variable for discrimination of the two lines (a lower tolerance indicates higher redundancy). HAB, high anxiety-related behaviour; LAB, low anxiety-related behaviour.
nificant difference in locomotor activity, with female and male HAB rats being less active than their LAB counterparts. Although this difference might be interpreted as anxiety-related behaviour being, at least in part, secondary to the difference in locomotor activity, it has to be considered that movement inhibition may be one characteristic of anxious animals [7,25]. Furthermore, more comprehensive statistical analysis using discriminant analysis revealed that in the black – white box test, the parameters that are most important for the discrimination between the two lines are anxiety-related and
exploratory behaviours. These parameters are the number of entries into the white compartment and the number of rearings per min in the black compartment, respectively. Analogously, in the elevated plus-maze test, the most important parameter for the discrimination between the two lines is the percent time spent on the open arms, the parameter that is generally seen as the one that is most closely related to innate anxiety. In both paradigms, the locomotor activity (as measured in the black–white box test by the number of line crossings in the black compartment per min spent in it and in the
162
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163
elevated plus-maze paradigm by the number of closed arm entries) has no significant importance for discrimination between the two lines. In the social interaction paradigm the data indicate the opposite. The most important parameter that discriminates between the two lines is the locomotor activity, whereas the active social interaction, classically seen as the most relevant parameter for anxiety-related behaviour, is the least important parameter for discrimination. These results confirm recent factor analytical studies that found locomotion being the parameter that loaded highest on the factor interpreted as anxiety in the social interaction paradigm [21,22]. A possible explanation for those results may be that the inhibition of active social interaction seems to be related to competing defensive reactions such as freezing, thigmotaxis and displacement behaviours [24]: at least two of those three factors imply a reduction of locomotion. Interestingly, in the factor analytical studies locomotion in the black – white box but not in the elevated plus-maze test also loaded highly on the factor ‘anxiety’ [21,22]. The difference in locomotor activity between HAB and LAB rats has also been observed in an open-field experiment [14]. However, when singly-housed male HAB and LAB rats were tested under baseline conditions in their home cage with a radiotelemetric system, no difference in locomotion was found [13]. These findings suggest that individuals of the HAB and LAB lines do not differ in their basal locomotor activity, but in their reactions to novel and stressful situations. This suggestion is supported by the fact that HAB and LAB rats differed in their behaviour in a forced swim test, a behavioural test developed for screening anti-depressant drugs [20]: HAB rats started floating earlier, spent significantly more time in this immobile posture and struggled less than LAB rats [14]. The behavioural difference in this inescapable situation reflects a difference in coping behaviour [1,16]. In this respect, LAB rats display an active coping behaviour whereas HAB rats behave in a rather passive, ‘depressive-like’ manner. Thus, the results of the tests used here may not only reflect a difference between the two lines in their innate state of anxiety but also a difference in coping with a novel and stressful environment. This is in line with the notion that in experimental rats, emotionality and coping behaviour are linked to each other [18,29]. Likewise, there is also a linkage between human conditions, hypothetically considered as parallels of emotionality and coping in animals, namely anxiety and depression, respectively [23,30]. Individuals of the HAB and LAB rat lines did not only differ in their behaviour in conflict situations, as in the tests of anxiety described above, but also in a non-conflict situation: when group-housed HAB and LAB rats were observed under basal conditions in their home cages within the first hour of the activity phase, HAB rats
displayed significantly less rearings and more sleeping/ resting than their LAB counterparts Fig. 7. The latter is in contrast to the finding that individuals of the HAB and LAB lines did not differ in locomotor activity when measured under baseline conditions in their home cage with a radiotelemetric system [13]. This difference is likely due to the fact that the radiotelemetric measurements were conducted with singly housed rats whereas the home cage observation was done with group-housed animals: The differences between group-housed HAB and LAB rats in rearings and sleeping/resting behaviour were probably not due to a difference in activity between the two lines per se but may be a consequence of an increased level of aggressive interactions in the home cages of the LAB rats compared to the HAB rats, although the differences in the behavioural parameters fighting and threatening taken alone did not reach significance. However, when the data of fighting and threatening were taken together to the parameter ‘aggressive behaviour’, the difference in this parameter was significant (PB 0.05, Mann–Whitney U-test), with LAB rats being more aggressive than HAB rats. In conclusion, this study has shown consistent behavioural differences between HAB and LAB rats, regardless of the gender, in three tests of unconditioned anxiety, indicating that the emotional difference between the two lines is a robust trait independent of the test used. Moreover, the selective breeding of HAB and LAB rats resulted also in differences in social behaviour, with LAB rats tending to display more aggressive behaviour than HAB rats. Acknowledgements The authors wish to thank Albin Varga for reliable and professional animal care. This study was supported by the Deutsche Forschungsgemeinschaft. References [1] Armario A, Galvada` A, Martı´ O. Forced swimming test in rats: effect of desipramine administration and the period of exposure to the test on struggling behaviour, swimming, immobility and defecation rate. Eur J Pharmacol 1988;158:207 – 12. [2] Belzung C, Le Pape G. Comparison of different behavioral test situations used in psychopharmacology for measurement of anxiety. Physiol Behav 1994;56:623 – 8. [3] Chaouloff F, Durand M, Morme`de P. Anxiety- and activity-related effects of diazepam and chlordiazepoxide in rat light/dark and dark/light test. Behav Brain Res 1997;85:27 – 35. [4] Costall B, Jones BJ, Kelly ME, Naylor RJ, Tomkins DM. Exploration of mice in a black and white test box: validation as a model of anxiety. Pharmacol Biochem Behav 1989;32:777–85. [5] Crawley J, Goodwin FK. Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacol Biochem Behav 1980;13:167 – 70. [6] Dawson GR, Tricklebank MD. Use of the elevated plus maze in the search for novel anxiolytic agents. Trends Pharmacol Sci 1995;16:33 – 6.
M.S.H. Henniger et al. / Beha6ioural Brain Research 111 (2000) 153–163 [7] Escorihuela RM, Ferna´ndez-Teruel A, Gil L, Aguilar R, Toben˜a A, Driscoll P. Inbred Roman high- and low avoidance rats: differences in anxiety, novelty-seeking, and shuttlebox behaviors. Physiol Behav 1999;67:19–26. [8] File SE. The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs. J Neurosci Methods 1980;2:219 –38. [9] File SE, Hyde JRG. Can social interaction be used to measure anxiety? Br J Pharmacol 1978;62:19–24. [10] Hogg S. A review of the validity and variability of the elevated plus-maze as an animal model of anxiety. Pharmacol Biochem Behav 1996;54:21 – 30. [11] Huberty CJ. Applied Discriminant Analysis. New York: Wiley, 1994. [12] Landgraf R, Wigger A, Holsboer F, Neumann ID. Hyper-reactive hypothalamo-pituitary adrenocortical axis in rats bred for high anxiety-related behaviour. J Neuroendocrinol 1999;11:405 – 7. [13] Liebsch G, Linthorst CE, Neumann ID, Reul JMHM, Holsboer F, Landgraf R. Behavioral, physiological, and neuroendocrine stress responses and differential sensitivity to diazepam in two Wistar rat lines selectively bred for high- and low-anxiety-related behavior. Neuropsychopharmacology 1998;19:381–96. [14] Liebsch G, Montkowski A, Holsboer F, Landgraf R. Behavioral profiles of two Wistar rat lines selectively bred for high or low anxiety-related behavior. Behav Brain Res 1998;94:301–10. [15] Lister RG. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology 1987;92:180–5. [16] Martı´ J, Armario A. Effects of diazepam and desipramine in the forced swimming test: influence of previous experience with situation. Eur J Pharmacol 1993;236:295–9. [17] Onaivi ES, Martin BR. Neuropharmacological and physiological validation of a computer-controlled two-compartment black and white box for the assessment of anxiety. Prog Neuropsychopharmacol Biol Psychiatr 1989;13:963–76. [18] Overstreet DH, Rezvani AH, Janowsky DS. Maudsley reactive and nonreactive rats differ only in some tasks reflecting emotionality. Physiol Behav 1992;52:149–52.
.
163
[19] Pellow S, Chopin P, File SE, Briley M. Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 1985;14:149 – 67. [20] Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal model sensitive to antidepressant treatment. Nature 1977;266:730 – 2. [21] Ramos A, Berton O, Morme`de P, Chaouloff F. A multiple-test study of anxiety-related behaviours in six inbred rat strains. Behav Brain Res 1997;85:57 – 69. [22] Ramos A, Morme`de P. Stress and emotionality: a multidimensional and genetic approach. Neurosci Biobehav Rev 1998;22:33 – 57. [23] Regier DA, Rae DS, Narrow WE, Kaelber CT, Schatzberg AF. Prevalence of anxiety disorders and their comorbidity with mood and addictive disorders. Br J Psychiatr 1998;173(34):24–8. [24] Rodgers RJ. Animal models of ‘anxiety’: where next? Behav Pharmacol 1997;8:477 – 96. [25] Rodgers RJ, Cao BJ, Dalvi A, Holmes A. Animal models of anxiety: an ethological perspective. Braz J Med Biol Res 1997;30:289 – 304. [26] Sa´nchez C. 5-HT receptors play an important role in modulation of behavior of rats in a two-compartment black and white box. Behav Pharmacol 1996;7:788 – 97. [27] Smythe JW, Murphy D, Bhatnagar S, Timothy C, Costall B. Muscarinic antagonists are anxiogenic in rats tested in the black – white box. Pharmacol Biochem Behav 1996;54:57–63. [28] Steimer T, Driscoll P, Schulz PE. Brain metabolism of progesterone, coping behaviour and emotional reactivity in male rats from two psychogenetically selected lines. J Neuroendocrinol 1997;169 – 175. [29] Steimer T, la Fleur S, Schulz PE. Neuroendocrine correlates of emotional reactivity and coping in male rats from the Roman High (RHA/Verh)- and low (RLA/Verh)-Avoidance lines. Behavior Genetics. 1997;503 – 512. [30] Wittchen H-U, Essau CA, Krieg J-C. Anxiety disorders: similarities and differences of comorbidity in treated and untreated groups. Br J Psychiatr 1991;159(12):23– 33.