- Email: [email protected]
The Roman high- and low-avoidance rats respond differently to novelty in a familiarized environment
Behavioural Processes 63 (2003) 63–72
The Roman high- and low-avoidance rats respond differently to novelty in a familiarized environment Wojciech Pisula The Warsaw School of Social Psychology and The Polish Academy of Sciences, Chodakowska 19/31, PL 03-815 Warsaw, Poland Received 7 October 2002; received in revised form 27 January 2003; accepted 5 February 2003
Abstract The purpose of the present study was to investigate the response to novelty in Roman high- and low-avoidance rats under non-stressful conditions. To reduce fear, a procedure of repetitive placing in the experimental chamber consisting of start, screen, and tunnel zones was applied. Each animal was placed in the experimental chamber daily for a 6 min period. The first 11 sessions were the habituation sessions. In the 12th session, the novelty was introduced into the screen and tunnel zones. The subsequent two sessions were conducted under novelty conditions. Behavioral activities such as walking, object contacts, time spent in given zones, and entering the tunnels were measured. All the comparisons were made for two 3-min intervals, using a three-factor MANOVA, involving 2sex × 2subline × 83-mininterval . All subjects increased time spent in the tunnel zone, but RHA/Verh rats responded to a greater extent, especially the males. All subjects spent shorter times in the screen zone, but the RHA/Verh rats responded to a much greater extent. RHA/Verh rats, especially males responded with a substantial increase of time spent inside the tunnels. All subjects responded with an increased amount of object contacts. In general the RHA/Verh subjects showed a more pronounced response to novelty, as evidenced by a significant shift toward the tunnel zone. They spent more time in this zone than their RLA/Verh counterparts. Among the RLA/Verh rats, males tended to behave similarly to RHA/Verh rats, especially during the second 3 min interval of session “12.” The differences between the rat lines obtained in this study may be attributed to mechanisms specific to exploration, making them promising subjects to study the relationships between reactivity, novelty detection, adaptation, and environmental information processing. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Exploratory behavior; Investigatory responses; Novelty; Response to novelty; Roman low-avoidance rats; Roman high-avoidance rats
1. Introduction One of the main goals of studying exploratory behavior is to fully understand the process of adaptation of an organism to environmental change. That change may be coped within different ways. High novel intensities, or biologically significant stimuli (predator-like-patterns) usually evoke stereotypical responses, called species specific defense responses E-mail address: [email protected] (W. Pisula).
(SSDR) such as freezing behavior, tail separation (lizards), burying, spreading a putrid odor or fleeing (for an extensive review see Fanselow and De Oca, 1998). The reactions to stimulation of low intensity, or of such an intensity that is not immediately relevant for survival, are less apparent. However, they are not less important since, in many species, they form the main part of the behavioral repertoire. Most manipulations in studies of animal responses to novelty have involved spatial rearrangement of
0376-6357/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0376-6357(03)00032-9
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W. Pisula / Behavioural Processes 63 (2003) 63–72
existing physical objects or the introduction of novel objects (e.g. Calhoun, 1963; Gouteux et al., 1999; Picq and Dhenain, 1998; Renner and Seltzer, 1994). Little attention has been paid to visual patterns, even though there is strong evidence that rats may discriminate visual stimuli very efficiently (Gaffan and Woolmore, 1996). The purpose of the present study was to determine the behavioral response of genetically selected rats to mild—low stress—novelty, in order to observe the relative importance of visual stimulation and rearrangement of physical objects. The Swiss sublines of Roman high- (RHA/Verh) and low- (RLA/Verh) avoidance rats have been used as genetic models in various types of studies, mainly based on differences in their emotional reactivity (Gentsch et al., 1982; Fernández-Teruel et al., 1992; Steimer et al., 1997), and/or in their “sensationseeking” characteristics (Siegel, 1997). Emotional processes are considered to be important in the regulation of exploratory behavior (Pisula and Matysiak, 1998). It has been found (Pisula and Osi´nski, 2000) that RLA/Verh and RHA/Verh rats differ in both quantitative and qualitative aspects of exploratory behavior. Also, under our testing conditions, males of both sublines were found to be more exploratory than their respective females, and this finding contradicted some traditional views (Hughes, 1968; Gray, 1971; Fernández-Teruel et al., 1991, 1994; Escorihuela et al., 1997). RHA/Verh rats showed a less diverse but more exploratory repertoire than did RLA/Verh, and males of both sublines showed more behavioral sequences than females. These notable differences were found under novel low-stress conditions, leading to the conclusion that emotional reactivity may have profound effects on behavioral regulation which are not limited to high intensity stimulation. This contradicts the classical approach (see Strelau, 1983, 1996), in which emotional reactivity is hypothesized to regulate the manifestation of individual differences in highly stressful situations mainly. If the emotional profile of an individual does influence the regulation of behavior in situations that are not emotogenic, the theory of exploratory behavior motivation may need to be reformulated. These ongoing studies with the Roman high- and low-avoidance rat sublines may provide important data for this debate. In the present study, emotional response was purposefully reduced by a procedure of repetitive placing
in the experimental chamber. This procedure has proven to be effective with AUGUST rats in a previous study (Pisula, in press). Moreover, all rats were systematically handled following arrival at our lab, as handling has been found to reduce emotional response (Thompson and Lippman, 1975; Pisula et al., 1992), and consequently, to reduce the differences between the Roman rat sublines (Fernández-Teruel et al., 1997).
2. Materials and methods 2.1. Subjects The RHA/Verh and RLA/Verh rats were obtained from Dr. Thierry Steimer, Clinical Psychopharmacology Unit, University Hospital of Geneva. The animals were from the 105th generation of an outbred, closed colony. They arrived to Warsaw, at the age of 7 weeks. Thirty two subjects (eight females and males of each subline), about 90 days of age at the onset of the experiment were used. The rats were gently handled for 5–8 min a day. This entailed being petted and carried by hand. The handling procedure started within a week after arrival to our laboratory, and was conducted by the author and a technician (same person throughout the experiment). Rats were housed in transparent plastic (polycarbonate) cages (610 mm length; 415 mm width; 215 mm height; floor area 2065 mm), four subjects per cage. The housing conditions fulfilled the criteria set by the local and the European Ethic Commission. 2.2. Apparatus The box used for exploratory behavior measured 83.5 cm length × 57.5 cm width × 80 cm height, and is represented in Fig. 1. It was illuminated with a red light of low intensity (50 lx). 2.3. Procedure In order to control the level of novelty, a procedure of habituation to the experimental chamber was utilized. Every day, from the beginning of the experiment onward, each animal was placed in the experimental chamber for a 6-min period. The
W. Pisula / Behavioural Processes 63 (2003) 63–72
65
Fig. 1. Experimental cage used in this study. Section “A”—start zone (the box seen in this zone was used as a rat transporter); section “B”—tunnel zone; section “C”—screen zone (note the initial position of the displayed symbols).
experimenter would leave the experimental room immediately after placing the transporter with the rat in the zone “A” of the experimental chamber (see Fig. 1). The first 11 sessions were the habituation sessions, with the chamber in a standard mode (see Table 1). At the 12th session the experimental manipulation, introduction of the novelty, took place. The subsequent two sessions were conducted with the chamber being turned into the experimental mode. Table 1 also shows the details of the experimental situation. Sessions 10 through 13 were video recorded. The experimental room was dark. The video camera did not generate sounds within the human or ultrasound frequencies that could affect the animal. The camera
was placed approximately 2 m from the front cover of the experimental chamber. The front was made of transparent plexiglass. For the purpose of analysis, the following behavioral activities were measured: walking, floor sniffing, object contacts, time spent in the given zones, and entering the tunnels. All 6-min sessions were divided into two 3-min intervals for the analysis. A three-factor MANOVA, involving 2sex × 2subline × 83-mininterval was performed.
3. Results Table 2 shows the detailed effects obtained in this analysis.
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W. Pisula / Behavioural Processes 63 (2003) 63–72
Table 1 Experimental conditions I Session number
II Tunnel zone (tunnel’s arrangement)
III Start zone
IV Screen zone
1–11
See Fig. 1.
Every 0.3 s a rectangle was displayed for 0.3 s in one of the four horizontally oriented positions 䊐 䊐 䊏 䊐
12–13
Same
orientation changed to vertical
Columns I, II, and III are self-explanatory. In column IV, symbol “䊐” represents one of four possible positions taken by symbol “䊏”. Sessions 10 through 13 were video tapped.
Table 2 The results of the MANOVA analysis Variable
Effect
F
d.f.
P
Duration of time spent in the tunnel zone
Line Trials Sex by trials Line by trials
22.30 15.76 2.45 3.77
1, 7, 7, 7,
28 196 196 196
0.001 0.001 0.05 0.01
Duration of time spent in the start zone
Line Trials Line by trials
9.48 9.90 3.37
1, 28 7, 196 7, 196
0.01 0.001 0.01
Duration of time spent in the screen zone
Line Trials Line by trials
14.67 5.49 2.54
1, 28 7, 196 7, 196
0.001 0.001 0.05
Duration of time spent inside the tunnels
Line Trials Line by trials
63.68 34.38 12.29
1, 28 7, 196 7, 196
0.001 0.001 0.001
Duration of object contact
Sex Sex by line Trials Sex by line by trials
11.98 4.23 24.61 2.44
1, 1, 7, 7,
28 28 196 196
0.001 0.05 0.001 0.05
Duration of floor sniffing
Sex by line Trials Line by trials Sex by line by trials
16.91 12.84 2.58 2.61
1, 7, 7, 7,
28 196 196 196
0.001 0.001 0.05 0.05
Walking (onsets)
Sex Line Trials
7.69 19.25 23.36
1, 28 1, 28 7, 196
0.01 0.001 0.001
The experimental design applied in this study was based on a repeated measures, and therefore our main focus is on the interactions among line, sex, and trial effects. These results may be summarized as follows: The experimental manipulation (introducing the novelty) resulted in an increase in time spent in the tunnel zone in all subjects, but RHA/Verh rats responded to a greater extent, especially the males (Fig. 2). All subjects responded to the experimental manipulation with a decrease in time spent in the start zone, with RHA/Verh rats showing greater response (Fig. 3). Within the RLA/Verh subline, however, males showed a tendency to shorten their stay in this zone, similar to their RHA/Verh counterparts of both sexes. All subjects spent less time in the screen zone; this was more so for the RHA/Verh rats (Fig. 4). The RHA/Verh rats, and especially the RHA males showed a spectacular increase in time spent inside the tunnels. These interaction effects overshadow the main effect of trials, which is a general increase in both sublines (Fig. 5). All subjects responded with increased object (tunnels and the walls dividing the cage into three zones) contact. The interaction effect (Table 2) indicates a greater response in males than in females (Fig. 6). All rats showed decreased floor sniffing but RLA/Verh subjects, and especially females, responded to a greater extent (Fig. 7).
W. Pisula / Behavioural Processes 63 (2003) 63–72
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Fig. 2. Duration of time that rats spent in the tunnel zone over the recorded sessions.
Fig. 3. Duration of time that rats spent in the start zone over the recorded sessions.
All rats walked less after experimental manipulations than in the previous trials (Fig. 8).
4. Discussion Novelty is often associated with stressful, emotogenic situations. This experiment was designed to
eliminate emotional response to novelty, while at the same time investigating the response to novelty of RHA/Verh and RLA/Verh rats. Since individual differences in temperamental traits such as emotional reactivity have been proposed to be more likely to occur when facing high- or extreme intensity stimulation (Strelau, 1983, 1996), it could have been expected that the usual differences between the Roman
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W. Pisula / Behavioural Processes 63 (2003) 63–72
Fig. 4. Duration of time that rats spent in the screen zone over the recorded sessions.
Fig. 5. Duration of time that rats spent inside the tunnels over the recorded sessions.
rat sublines might not be evident. Rats were systematically handled and were provided with an opportunity to adapt to the experimental cage through several days of exposure. The subjects showed no behavioral changes that could be interpreted in terms of emotional response, such as freezing, grooming, or burying. This result is in accordance with an earlier study with AUGUST rats, and proves that the procedure
involving repetitive placing in the experimental cage under non-stressful conditions is an effective method of reducing novelty-related anxiety in rats. Therefore, the differences revealed in the present study may be attributed to the characteristics of the individuals, which are specific to novelty related behavior, and not merely to activational effects related to emotional arousal.
W. Pisula / Behavioural Processes 63 (2003) 63–72
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Fig. 6. Duration of time that rats spent contacting objects over the recorded sessions.
Fig. 7. Duration of time that rats spent on sniffing.
All subjects responded to novelty with decreased walking and an increased duration of object contact, though the strength of these effects was modified by the interaction of sex and line characteristics. This finding contradicts the predictions formulated on the basis of stimulus-seeking behavior theory. Within this theoretical framework, exploratory behavior is defined, as “. . . activity, which is diffuse and com-
pletely random in a novel environment” (Pisula and Matysiak, 1998, p. 199). Thus, the increase of general motor activity, such as walking, is easily predictable. The results of this study clearly demonstrate that when adapting to a low emotogenic novelty an animal changes its activity budget allocation by redirecting its activity toward the source of environmental change. Thus, the level of novelty of the environment
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W. Pisula / Behavioural Processes 63 (2003) 63–72
Fig. 8. Number of walking onsets over the recorded sessions.
is controlled by the level and the direction of the activity. In general RHA/Verh subjects showed a more pronounced response to novelty than did the RLA/Verh rats. They significantly shifted their activity toward the tunnel zone and, spent more time in that zone than did their RLA/Verh counterparts. On the other hand, male RLA/Verh rats, tended to behave similarly to RHA/Verh rats, especially during the second 3-min interval of session 12. The increase of time spent in the tunnel zone was associated with a decrease of time spent in the two other zones, especially the screen zone. Whereas rats can discriminate complex visual stimuli (Gaffan and Woolmore, 1996) and detect the changes in light brightness (Hughes, 1999), these findings show that when they are attracted by two distinct sorts of novelty (visual versus space rearrangement), they ignore the visual change and rather engage in investigation of the spatial properties of their surroundings. One may easily imagine that the potentially adaptive significance of spatial change is much greater than the change of location of a picture on the wall. The functional aspect of this behavioral characteristic of rats may be related to Barnett’s hypothesis that “The principle means of avoiding predators are the use of pathways under cover, and flight to a burrow or other place of concealment. These actions depend on previous experience of the topography of
their living space. Given such experience, they can run from any one point to another, by the shortest route and in the least possible time” (Barnett, 1975, p. 49). This hypothesis has been further supported by experiments by Renner (1988). The main difference between RHA/Verh and RLA/Verh subjects was found in time spent inside the tunnels. The RHA/Verh rats responded to a change in the tunnel’s arrangement with very intensive and prolonged exploration of the tunnels. Within this subline, males scored significantly higher than females on this measurement. There was no difference between the sexes within RLA/Verh subline although they also increased, to a much lesser extent, time spent in the tunnels. It is also noteworthy that the increase of exploration by RLA/Verh rats was delayed and occurred mainly during the last recorded session. Some authors tend to view the exploratory responses in terms of adaptation to novelty. They believe that the more intensive and profound exploratory acts are, the longer is the time required to adapt to novelty (see Hughes, 1999). Such an interpretation would lead to the conclusion that a lack of response would be the best sign of adaptation to novelty. There is no doubt that a prolonged and thorough investigation of an altered environment permits a more adequate representation of this environment. In this study, active exploration of RHA/Verh rats in response to novelty clearly corresponds with the
W. Pisula / Behavioural Processes 63 (2003) 63–72
behavior pattern described in an earlier study (Pisula and Osi´nski, 2000), and with an active style of coping in these rats (Steimer et al., 1997). Furthermore, the results of this and previous studies, (Pisula, 1997; Pisula and Osi´nski, 2000) do not agree with another widely-held view regarding sex differences in emotional reactivity. A traditional viewpoint held that female rats tend to explore a new environment more—to ambulate and rear more, to groom and to defecate less, and to acquire the active avoidance response faster—than males do (Gray, 1971; Hughes, 1968). A similar pattern of sex differences has been found in outbred and inbred Roman rats as observed by Fernández-Teruel et al. (1991, 1994) and Escorihuela et al. (1997). On the other hand, the manifestation of sex differences in the exploration box is dependent on the test conditions, and tends to increase over measurement duration (Pisula, 1997). The response to novelty seems to be one of such situations in which sex differences are very clearly delineated. In this study, it was the males that approached the source of novelty more than females. In conclusion, genetic and gender differences in the processing of novel environments possessing varied degrees of stimulation may provide insight into the cognitive traits of a given species. The psychogenetically selected Roman rat sublines appear to be attractive and promising subjects for further studies along these lines.
Acknowledgements I am especially grateful to Dr. Thierry Steimer, Dr. Peter Driscoll, and Prof. Jerome Siegel for their help in preparation of this study and this manuscript. This study was funded by Polish Committee for Scientific Research Grant # 1H01F03117.
References Barnett, S.A., 1975. The Rat: A Study of Behaviour. University of Chicago Press, Chicago. Calhoun, J.B., 1963. The Ecology and Sociology of the Norway Rat. US Department of Health, Education, and Welfare, Bethesda, MD. Escorihuela, R.M., Fernández-Teruel, A., Tobena, A., Langhans, W., Bättig, K., Driscoll, P., 1997. Labyrinth exploration,
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emotional reactivity, and conditioned fear in young Roman/Verh inbred rats. Behav. Genet. 27, 573–578. Fanselow, M.S., De Oca, B.M., 1998. Defensive behaviors. In: Greenberg, G., Haraway, M.M. (Eds.), Comparative Psychology: A Handbook. Garland, New York, pp. 653– 665. Fernández-Teruel, A., Escorihuela, R.M., Driscoll, P., Tobena, A., Bättig, K., 1991. Infantile (handling) stimulation and behavior in young Roman high- and low-avoidance rats. Physiol. Behav. 50, 563–565. Fernández-Teruel, A., Escorihuela, R.M., Nunez, J.F., Goma, M., Driscoll, P., Tobena, A., 1992. Early stimulation effects on novelty-induced behavior in two psychogenetically-selected rat lines with divergent emotionality profiles. Neurosci. Lett. 137, 185–188. Fernández-Teruel, A., Escorihuela, R.M., Driscoll, P., Tobena, A., Bättig, K., 1994. Evaluating activity and emotional reactivity in a hexagonal tunnel maze: correlational and factorial analysis from a study with the Roman/Verh rat lines. Behav. Genet. 24, 419–425. Fernández-Teruel, A., Escorihuela, R.M., Castellano, B., Gonzalez, B., Tobena, A., 1997. Neonatal handling and environmental enrichment effects on emotionality, novelty/reward seeking, age related cognitive and hippocampal impairments: focus on Roman rats lines. Behav. Genet. 27, 513–526. Gaffan, E.A., Woolmore, A.L., 1996. Complex visual learning by rats. Learn. Motiv. 27, 375–399. Gentsch, C., Lichsteiner, M., Driscoll, P., Feer, H., 1982. Differential hormonal and physiological responses to stress an Roman high and low avoidance rats. Physiol. Behav. 28, 259– 263. Gouteux, S., Vauclair, J., Thinus-Blanc, C., 1999. Reaction to spatial novelty and exploratory strategies in baboons. Anim. Learn. Behav. 27, 323–332. Gray, J.A., 1971. Sex differences in emotional behavior in mammals including man: endocrine bases. Acta Psychol. 35, 29–46. Hughes, R., 1968. Behaviour of male and female rats with free choice of two environments differing in novelty. Anim. Behav. 16, 92–96. Hughes, R.N., 1999. Sex differences in novelty-related location preferences of hooded rats. Q. J. Exp. Psychol. 52B, 235– 252. Picq, J., Dhenain, M., 1998. Reaction to new objects and spatial changes in young and aged mouse lemurs (Microcebus murinus). Q. J. Exp. Psychol. 51B, 337–348. Pisula, W., in press. Response to novelty in low-stress conditions in rats. Pol. Psychol. Bull. 35. Pisula, W., 1997. Rat behavior in the exploration box—cluster analysis. Int. J. Comp. Psychol. 10, 74–89. Pisula, W., Matysiak, J., 1998. Stimulus-seeking behavior. In: Greenberg, G., Haraway, M.M. (Eds.), Comparative Psychology: A Handbook. Garland, New York, pp. 198–202. Pisula, W., Osi´nski, J.T., 2000. A comparative study of the behavioral patterns of RLA/Verh and RHA/Verh rats in the exploration box. Behav. Genet. 30, 375–384. Pisula, W., Ostaszewski, P., Matysiak, J., 1992. Effects of physical environment and social experiences on stimulus seeking
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behavior and emotionality in rats (Rattus norvegicus). Int. J. Comp. Psychol. 5, 124–137. Renner, M., 1988. Learning during exploration: the role of behavioral topography during exploration in determining subsequent adaptive behavior. Int. J. Comp. Psychol. 2, 43–56. Renner, M.J., Seltzer, C.P., 1994. Sequential structure in behavioral components of object investigation by Long-Evans rats (Rattus norvegicus). J. Comp. Psychol. 108, 335–343. Siegel, J., 1997. Augmenting and reducing of visual evoked potentials in high and low sensation-seeking humans, cats, and rats. Behav. Genet. 27, 557–563.
Steimer, T., Le Fleur, S., Schultz, P., 1997. Neuroendocrine correlates of emotional reactivity and coping in male rats from Roman high- (RHA.Verh) and low- (RLA/Verh) avoidance lines. Behav. Genet. 27, 503–512. Strelau, J., 1983. Temperament, Personality, Activity. Academic Press, London. Strelau, J., 1996. Temperament: A Psychological Perspective. Plenum Press, New York. Thompson, R.W., Lippman, L.G., 1975. The effects of age and postweaning and adult handling habituation on activity and exploration in the rats. Bull. Psychon. Soc. 5, 285–288.
The Roman high- and low-avoidance rats respond differently to novelty in a familiarized environment Wojciech Pisula The Warsaw School of Social Psychology and The Polish Academy of Sciences, Chodakowska 19/31, PL 03-815 Warsaw, Poland Received 7 October 2002; received in revised form 27 January 2003; accepted 5 February 2003
Abstract The purpose of the present study was to investigate the response to novelty in Roman high- and low-avoidance rats under non-stressful conditions. To reduce fear, a procedure of repetitive placing in the experimental chamber consisting of start, screen, and tunnel zones was applied. Each animal was placed in the experimental chamber daily for a 6 min period. The first 11 sessions were the habituation sessions. In the 12th session, the novelty was introduced into the screen and tunnel zones. The subsequent two sessions were conducted under novelty conditions. Behavioral activities such as walking, object contacts, time spent in given zones, and entering the tunnels were measured. All the comparisons were made for two 3-min intervals, using a three-factor MANOVA, involving 2sex × 2subline × 83-mininterval . All subjects increased time spent in the tunnel zone, but RHA/Verh rats responded to a greater extent, especially the males. All subjects spent shorter times in the screen zone, but the RHA/Verh rats responded to a much greater extent. RHA/Verh rats, especially males responded with a substantial increase of time spent inside the tunnels. All subjects responded with an increased amount of object contacts. In general the RHA/Verh subjects showed a more pronounced response to novelty, as evidenced by a significant shift toward the tunnel zone. They spent more time in this zone than their RLA/Verh counterparts. Among the RLA/Verh rats, males tended to behave similarly to RHA/Verh rats, especially during the second 3 min interval of session “12.” The differences between the rat lines obtained in this study may be attributed to mechanisms specific to exploration, making them promising subjects to study the relationships between reactivity, novelty detection, adaptation, and environmental information processing. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Exploratory behavior; Investigatory responses; Novelty; Response to novelty; Roman low-avoidance rats; Roman high-avoidance rats
1. Introduction One of the main goals of studying exploratory behavior is to fully understand the process of adaptation of an organism to environmental change. That change may be coped within different ways. High novel intensities, or biologically significant stimuli (predator-like-patterns) usually evoke stereotypical responses, called species specific defense responses E-mail address: [email protected] (W. Pisula).
(SSDR) such as freezing behavior, tail separation (lizards), burying, spreading a putrid odor or fleeing (for an extensive review see Fanselow and De Oca, 1998). The reactions to stimulation of low intensity, or of such an intensity that is not immediately relevant for survival, are less apparent. However, they are not less important since, in many species, they form the main part of the behavioral repertoire. Most manipulations in studies of animal responses to novelty have involved spatial rearrangement of
0376-6357/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0376-6357(03)00032-9
64
W. Pisula / Behavioural Processes 63 (2003) 63–72
existing physical objects or the introduction of novel objects (e.g. Calhoun, 1963; Gouteux et al., 1999; Picq and Dhenain, 1998; Renner and Seltzer, 1994). Little attention has been paid to visual patterns, even though there is strong evidence that rats may discriminate visual stimuli very efficiently (Gaffan and Woolmore, 1996). The purpose of the present study was to determine the behavioral response of genetically selected rats to mild—low stress—novelty, in order to observe the relative importance of visual stimulation and rearrangement of physical objects. The Swiss sublines of Roman high- (RHA/Verh) and low- (RLA/Verh) avoidance rats have been used as genetic models in various types of studies, mainly based on differences in their emotional reactivity (Gentsch et al., 1982; Fernández-Teruel et al., 1992; Steimer et al., 1997), and/or in their “sensationseeking” characteristics (Siegel, 1997). Emotional processes are considered to be important in the regulation of exploratory behavior (Pisula and Matysiak, 1998). It has been found (Pisula and Osi´nski, 2000) that RLA/Verh and RHA/Verh rats differ in both quantitative and qualitative aspects of exploratory behavior. Also, under our testing conditions, males of both sublines were found to be more exploratory than their respective females, and this finding contradicted some traditional views (Hughes, 1968; Gray, 1971; Fernández-Teruel et al., 1991, 1994; Escorihuela et al., 1997). RHA/Verh rats showed a less diverse but more exploratory repertoire than did RLA/Verh, and males of both sublines showed more behavioral sequences than females. These notable differences were found under novel low-stress conditions, leading to the conclusion that emotional reactivity may have profound effects on behavioral regulation which are not limited to high intensity stimulation. This contradicts the classical approach (see Strelau, 1983, 1996), in which emotional reactivity is hypothesized to regulate the manifestation of individual differences in highly stressful situations mainly. If the emotional profile of an individual does influence the regulation of behavior in situations that are not emotogenic, the theory of exploratory behavior motivation may need to be reformulated. These ongoing studies with the Roman high- and low-avoidance rat sublines may provide important data for this debate. In the present study, emotional response was purposefully reduced by a procedure of repetitive placing
in the experimental chamber. This procedure has proven to be effective with AUGUST rats in a previous study (Pisula, in press). Moreover, all rats were systematically handled following arrival at our lab, as handling has been found to reduce emotional response (Thompson and Lippman, 1975; Pisula et al., 1992), and consequently, to reduce the differences between the Roman rat sublines (Fernández-Teruel et al., 1997).
2. Materials and methods 2.1. Subjects The RHA/Verh and RLA/Verh rats were obtained from Dr. Thierry Steimer, Clinical Psychopharmacology Unit, University Hospital of Geneva. The animals were from the 105th generation of an outbred, closed colony. They arrived to Warsaw, at the age of 7 weeks. Thirty two subjects (eight females and males of each subline), about 90 days of age at the onset of the experiment were used. The rats were gently handled for 5–8 min a day. This entailed being petted and carried by hand. The handling procedure started within a week after arrival to our laboratory, and was conducted by the author and a technician (same person throughout the experiment). Rats were housed in transparent plastic (polycarbonate) cages (610 mm length; 415 mm width; 215 mm height; floor area 2065 mm), four subjects per cage. The housing conditions fulfilled the criteria set by the local and the European Ethic Commission. 2.2. Apparatus The box used for exploratory behavior measured 83.5 cm length × 57.5 cm width × 80 cm height, and is represented in Fig. 1. It was illuminated with a red light of low intensity (50 lx). 2.3. Procedure In order to control the level of novelty, a procedure of habituation to the experimental chamber was utilized. Every day, from the beginning of the experiment onward, each animal was placed in the experimental chamber for a 6-min period. The
W. Pisula / Behavioural Processes 63 (2003) 63–72
65
Fig. 1. Experimental cage used in this study. Section “A”—start zone (the box seen in this zone was used as a rat transporter); section “B”—tunnel zone; section “C”—screen zone (note the initial position of the displayed symbols).
experimenter would leave the experimental room immediately after placing the transporter with the rat in the zone “A” of the experimental chamber (see Fig. 1). The first 11 sessions were the habituation sessions, with the chamber in a standard mode (see Table 1). At the 12th session the experimental manipulation, introduction of the novelty, took place. The subsequent two sessions were conducted with the chamber being turned into the experimental mode. Table 1 also shows the details of the experimental situation. Sessions 10 through 13 were video recorded. The experimental room was dark. The video camera did not generate sounds within the human or ultrasound frequencies that could affect the animal. The camera
was placed approximately 2 m from the front cover of the experimental chamber. The front was made of transparent plexiglass. For the purpose of analysis, the following behavioral activities were measured: walking, floor sniffing, object contacts, time spent in the given zones, and entering the tunnels. All 6-min sessions were divided into two 3-min intervals for the analysis. A three-factor MANOVA, involving 2sex × 2subline × 83-mininterval was performed.
3. Results Table 2 shows the detailed effects obtained in this analysis.
66
W. Pisula / Behavioural Processes 63 (2003) 63–72
Table 1 Experimental conditions I Session number
II Tunnel zone (tunnel’s arrangement)
III Start zone
IV Screen zone
1–11
See Fig. 1.
Every 0.3 s a rectangle was displayed for 0.3 s in one of the four horizontally oriented positions 䊐 䊐 䊏 䊐
12–13
Same
orientation changed to vertical
Columns I, II, and III are self-explanatory. In column IV, symbol “䊐” represents one of four possible positions taken by symbol “䊏”. Sessions 10 through 13 were video tapped.
Table 2 The results of the MANOVA analysis Variable
Effect
F
d.f.
P
Duration of time spent in the tunnel zone
Line Trials Sex by trials Line by trials
22.30 15.76 2.45 3.77
1, 7, 7, 7,
28 196 196 196
0.001 0.001 0.05 0.01
Duration of time spent in the start zone
Line Trials Line by trials
9.48 9.90 3.37
1, 28 7, 196 7, 196
0.01 0.001 0.01
Duration of time spent in the screen zone
Line Trials Line by trials
14.67 5.49 2.54
1, 28 7, 196 7, 196
0.001 0.001 0.05
Duration of time spent inside the tunnels
Line Trials Line by trials
63.68 34.38 12.29
1, 28 7, 196 7, 196
0.001 0.001 0.001
Duration of object contact
Sex Sex by line Trials Sex by line by trials
11.98 4.23 24.61 2.44
1, 1, 7, 7,
28 28 196 196
0.001 0.05 0.001 0.05
Duration of floor sniffing
Sex by line Trials Line by trials Sex by line by trials
16.91 12.84 2.58 2.61
1, 7, 7, 7,
28 196 196 196
0.001 0.001 0.05 0.05
Walking (onsets)
Sex Line Trials
7.69 19.25 23.36
1, 28 1, 28 7, 196
0.01 0.001 0.001
The experimental design applied in this study was based on a repeated measures, and therefore our main focus is on the interactions among line, sex, and trial effects. These results may be summarized as follows: The experimental manipulation (introducing the novelty) resulted in an increase in time spent in the tunnel zone in all subjects, but RHA/Verh rats responded to a greater extent, especially the males (Fig. 2). All subjects responded to the experimental manipulation with a decrease in time spent in the start zone, with RHA/Verh rats showing greater response (Fig. 3). Within the RLA/Verh subline, however, males showed a tendency to shorten their stay in this zone, similar to their RHA/Verh counterparts of both sexes. All subjects spent less time in the screen zone; this was more so for the RHA/Verh rats (Fig. 4). The RHA/Verh rats, and especially the RHA males showed a spectacular increase in time spent inside the tunnels. These interaction effects overshadow the main effect of trials, which is a general increase in both sublines (Fig. 5). All subjects responded with increased object (tunnels and the walls dividing the cage into three zones) contact. The interaction effect (Table 2) indicates a greater response in males than in females (Fig. 6). All rats showed decreased floor sniffing but RLA/Verh subjects, and especially females, responded to a greater extent (Fig. 7).
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Fig. 2. Duration of time that rats spent in the tunnel zone over the recorded sessions.
Fig. 3. Duration of time that rats spent in the start zone over the recorded sessions.
All rats walked less after experimental manipulations than in the previous trials (Fig. 8).
4. Discussion Novelty is often associated with stressful, emotogenic situations. This experiment was designed to
eliminate emotional response to novelty, while at the same time investigating the response to novelty of RHA/Verh and RLA/Verh rats. Since individual differences in temperamental traits such as emotional reactivity have been proposed to be more likely to occur when facing high- or extreme intensity stimulation (Strelau, 1983, 1996), it could have been expected that the usual differences between the Roman
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Fig. 4. Duration of time that rats spent in the screen zone over the recorded sessions.
Fig. 5. Duration of time that rats spent inside the tunnels over the recorded sessions.
rat sublines might not be evident. Rats were systematically handled and were provided with an opportunity to adapt to the experimental cage through several days of exposure. The subjects showed no behavioral changes that could be interpreted in terms of emotional response, such as freezing, grooming, or burying. This result is in accordance with an earlier study with AUGUST rats, and proves that the procedure
involving repetitive placing in the experimental cage under non-stressful conditions is an effective method of reducing novelty-related anxiety in rats. Therefore, the differences revealed in the present study may be attributed to the characteristics of the individuals, which are specific to novelty related behavior, and not merely to activational effects related to emotional arousal.
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Fig. 6. Duration of time that rats spent contacting objects over the recorded sessions.
Fig. 7. Duration of time that rats spent on sniffing.
All subjects responded to novelty with decreased walking and an increased duration of object contact, though the strength of these effects was modified by the interaction of sex and line characteristics. This finding contradicts the predictions formulated on the basis of stimulus-seeking behavior theory. Within this theoretical framework, exploratory behavior is defined, as “. . . activity, which is diffuse and com-
pletely random in a novel environment” (Pisula and Matysiak, 1998, p. 199). Thus, the increase of general motor activity, such as walking, is easily predictable. The results of this study clearly demonstrate that when adapting to a low emotogenic novelty an animal changes its activity budget allocation by redirecting its activity toward the source of environmental change. Thus, the level of novelty of the environment
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Fig. 8. Number of walking onsets over the recorded sessions.
is controlled by the level and the direction of the activity. In general RHA/Verh subjects showed a more pronounced response to novelty than did the RLA/Verh rats. They significantly shifted their activity toward the tunnel zone and, spent more time in that zone than did their RLA/Verh counterparts. On the other hand, male RLA/Verh rats, tended to behave similarly to RHA/Verh rats, especially during the second 3-min interval of session 12. The increase of time spent in the tunnel zone was associated with a decrease of time spent in the two other zones, especially the screen zone. Whereas rats can discriminate complex visual stimuli (Gaffan and Woolmore, 1996) and detect the changes in light brightness (Hughes, 1999), these findings show that when they are attracted by two distinct sorts of novelty (visual versus space rearrangement), they ignore the visual change and rather engage in investigation of the spatial properties of their surroundings. One may easily imagine that the potentially adaptive significance of spatial change is much greater than the change of location of a picture on the wall. The functional aspect of this behavioral characteristic of rats may be related to Barnett’s hypothesis that “The principle means of avoiding predators are the use of pathways under cover, and flight to a burrow or other place of concealment. These actions depend on previous experience of the topography of
their living space. Given such experience, they can run from any one point to another, by the shortest route and in the least possible time” (Barnett, 1975, p. 49). This hypothesis has been further supported by experiments by Renner (1988). The main difference between RHA/Verh and RLA/Verh subjects was found in time spent inside the tunnels. The RHA/Verh rats responded to a change in the tunnel’s arrangement with very intensive and prolonged exploration of the tunnels. Within this subline, males scored significantly higher than females on this measurement. There was no difference between the sexes within RLA/Verh subline although they also increased, to a much lesser extent, time spent in the tunnels. It is also noteworthy that the increase of exploration by RLA/Verh rats was delayed and occurred mainly during the last recorded session. Some authors tend to view the exploratory responses in terms of adaptation to novelty. They believe that the more intensive and profound exploratory acts are, the longer is the time required to adapt to novelty (see Hughes, 1999). Such an interpretation would lead to the conclusion that a lack of response would be the best sign of adaptation to novelty. There is no doubt that a prolonged and thorough investigation of an altered environment permits a more adequate representation of this environment. In this study, active exploration of RHA/Verh rats in response to novelty clearly corresponds with the
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behavior pattern described in an earlier study (Pisula and Osi´nski, 2000), and with an active style of coping in these rats (Steimer et al., 1997). Furthermore, the results of this and previous studies, (Pisula, 1997; Pisula and Osi´nski, 2000) do not agree with another widely-held view regarding sex differences in emotional reactivity. A traditional viewpoint held that female rats tend to explore a new environment more—to ambulate and rear more, to groom and to defecate less, and to acquire the active avoidance response faster—than males do (Gray, 1971; Hughes, 1968). A similar pattern of sex differences has been found in outbred and inbred Roman rats as observed by Fernández-Teruel et al. (1991, 1994) and Escorihuela et al. (1997). On the other hand, the manifestation of sex differences in the exploration box is dependent on the test conditions, and tends to increase over measurement duration (Pisula, 1997). The response to novelty seems to be one of such situations in which sex differences are very clearly delineated. In this study, it was the males that approached the source of novelty more than females. In conclusion, genetic and gender differences in the processing of novel environments possessing varied degrees of stimulation may provide insight into the cognitive traits of a given species. The psychogenetically selected Roman rat sublines appear to be attractive and promising subjects for further studies along these lines.
Acknowledgements I am especially grateful to Dr. Thierry Steimer, Dr. Peter Driscoll, and Prof. Jerome Siegel for their help in preparation of this study and this manuscript. This study was funded by Polish Committee for Scientific Research Grant # 1H01F03117.
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