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Vibrissal sense is not the main sensory modality in rat exploratory behavior in the elevated plus-maze
Behavioural Brain Research 122 (2001) 169– 174 www.elsevier.com/locate/bbr
Research report
Vibrissal sense is not the main sensory modality in rat exploratory behavior in the elevated plus-maze Fernando Cardenas, Marisol R. Lamprea, Silvio Morato * Faculdade de Filosofia Cieˆncias e Letras de Ribeira˜o Preto, Uni6ersidade de Sa˜o Paulo, A6. Bandeirantes 3900, Ribeira˜o Preto-SP 14040 -901, Brazil Received 7 April 2000; received in revised form 30 January 2001; accepted 30 January 2001
Abstract Four groups of male Wistar rats were submitted to acute bilateral removal of mystacial vibrissae at different lengths from the follicle. Each group was divided into two subgroups, tested under high (150 Lux) and low environmental illumination (2 Lux). All the subjects were allowed to freely explore an elevated plus-maze for 5 min. Results indicated that rats tested under low illumination tended to explore the open arms more frequently and longer then rats tested under high illumination. When tested under low illumination, rats in the group that suffered whole vibrissa removal stayed longer in the open arms than those in the other groups but did not differ in the number of entries. The average increase in the length of open arm entries, rather than a decrease in aversion to the open arms, may be due to the need of more time to obtain information about the environment since there is no light and the vibrissae were removed. This effect was not seen with rats tested under high illumination, possibly because vision could be used to obtain relevant information. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Elevated plus-maze; Exploratory behavior; Sensory systems; Thigmotaxis; Vibrissa removal; Information search; Aversion; Rats
1. Introduction To date there is not a thorough understanding of the role played by different aversive stimuli on rodent exploratory behavior in some behavioral tasks like the open field or the elevated plus-maze. For a long time, it has been assumed that the innate fear of open spaces was the most important anxiety trigger. However, the mechanisms by means of which rodents perceive the openness of an environment are still unclear. According to Becker and Grecksch [9], the visual system has no role in the genesis of the elevated plus-maze-evoked anxiety. On the other hand, there is some evidence for a potential role for a thigmotactic mediation by perioral vibrissae as a powerful determinant of open spaceinduced fear [45,48]. * Corresponding author. Tel.: + 55-16-6023662; fax: +55-166023632. E-mail address: [email protected] (S. Morato).
Since its pharmacological validation in 1985, by Pellow et al. [37], the elevated plus-maze has been widely used as a tool in the study of rodent anxiety [10,11,34,36,37,50]. It consists of four opposed arms, two of them enclosed by walls and two with no enclosure, the whole set elevated above the floor. The cited reports stress that rodents show a preference for entering and staying more in the closed arms than in the open ones. The total number of entries into each kind of arm, as well as the total time spent exploring the open arms, vary according to the type of treatment: for example, pre-treating animals with anxiolytic drugs leads to increases in both the number of entries and the time spent in the open arms [10,15,16,21,33]. On the other hand, pre-treating them with anxiogenic drugs decreases both the number of entries and the time spent in the open arms [5,6,40,52]. Similarly, behavioral manipulations such as individual housing [27,31], handling [4,22,44], malnutrition [3], aversiveness of the experimental situation [2,17,50] or behavioral restriction [28,30], have been shown to pro-
0166-4328/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 1 ) 0 0 1 8 0 - 2
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F. Cardenas et al. / Beha6ioural Brain Research 122 (2001) 169–174
duce effects according to their motivational properties on this test. The elevated plus-maze has been used for the study and elucidation of the role of different cerebral structures in anxiety [7,12– 14,16,19,24,25,38, 39,41,43,50]. Despite the broad use of this and other behavioral tests based on the same principle, much remains to be unraveled about the stimuli responsible for the avoidance from the open spaces shown by rats, mice and
other rodents. Some authors have proposed that environmental illumination is an important aversive stimulus in these behavioral tests [4,32]. Others have proposed that height was the main aversive characteristic [35]. However, Treit et al. [49] have demonstrated that height is not important for the aversiveness of the open arms. Instead, it is the open arm’s lack of cues for thigmotaxis (trend to remain near or in contact with vertical surfaces) that is an important aversive factor [1,45,49,51]. In fact, it has been suggested [8] that rodents in the wild show this behavior probably as a defense against aerial predators [18]. In light of these studies, it is clear that any modification in thigmotactic perception would lead to changes in the normal way with which the rat approaches novel environments. What is not clear, however, is by what means the animal senses the vertical surfaces. Vision might be used to this end, but so could the vibrissae. In order to investigate their possible role, we submitted rats to the plus-maze under two illumination conditions, combined with partial or total removal of mystacial vibrissae.
2. Materials and methods
2.1. Animals Seventy-eight adult male Wistar rats, weighing 208.49 14.9 g (mean9S.E.M.) and with free access to food and water were housed in groups of six under a 12-h light/dark cycle (lights on at 07:00 h). The subjects came from the animal house of the campus of the University of Sao Paulo at Ribeira˜ o Preto and had a habituation period of 3 days before testing. During this period, they were handled daily for 5 min.
2.2. Apparatus
Fig. 1. Percentage of entries (top), time spent in the open arms (mid) and mean duration of open-arm entries (bottom). I, Intact control group (n = 20); T, without the tip of each vibrissa (n= 18); H, without half of each vibrissa (n= 20); W, without the whole vibrissa (n=20). Light, 150 Lux; dark, 2 Lux. * Different from both rats with intact vibrissae in the respective group and from the correspondent high illumination group. As a general effect, both time spent and percent of entries into the open arms in darkness were significantly higher than in brightness.
An elevated plus-maze made of dark brown wood with a dark blue Formica floor was used. The apparatus consisted of two opposed open arms (50×10 cm) crossed at right angles with other two opposed arms of the same size but enclosed by 40-cm high walls. The whole structure was elevated 50 cm above the floor. A 1-cm rim of transparent Plexiglas surrounded the open arms. Mystacial vibrissae were cut using normal lab scissors. One-half of the rats were tested under a 100-W white lamp placed 1.2 m above the maze (high illumination, 150 Lux in the central square). The other half were tested using a 20-W red lamp bulb at the same height (low illumination, 2 Lux in the central square). The experimental sessions were recorded using a video camera, linked to a VCR and a TV monitor located in an adjacent room.
F. Cardenas et al. / Beha6ioural Brain Research 122 (2001) 169–174
171
Table 1 Results of the two-way ANOVA applied to the behavioral measures Behavioral measure
Entries into the closed arms Total entries Percent of entries into the open arms Mean duration of open arm entry Time spent in the open arms Time spent in the closed arms Time spent in the central square
Light level
Length removed
Interaction
F[1,70]
P
F[3,70]
P
F[3,70]
P
15.219 62.113 41.476 7.354 95.206 71.878 31.560
B0.001 B0.001 B0.001 0.008 B0.001 B0.001 B0.001
1.020 1.389 0.332 3.594 0.614 0.571 0.92
0.389 0.253 0.802 0.018 0.608 0.636 0.436
0.769 2.053 0.910 6.67 2.258 0.282 0.61
0.515 0.114 0.441 B0.001 0.089 0.838 0.611
2.3. Procedure Rats were randomly assigned to one of four groups. One-half of each group was tested under high illumination and the other half under low illumination. A control group was tested without any treatment, while the other three groups had all their mystacial vibrissae cut at three different lengths from the follicle (just the tip, half or the whole vibrissa). Cuts were made 5 min before the test. In order to ensure equal conditions to all animals, the vibrissae of control rats were gently touched with the side of the scissors used for removal, and clipping sounds were made by just opening and closing the scissors without cutting anything. After that, each rat was gently placed in the central square of the maze facing one of the closed arms and allowed to freely explore for 5 min. After removing an animal, the maze was cleaned up with a solution of 20% ethanol and dried. The number of entries and the total amount of time spent in each arm were measured. An entry was recorded every time the rat placed all four paws on one arm.
while the amount of vibrissa removal did not have any effect on these two measures; in both cases, there were no interactions (Table 1). It can be seen in Fig. 1 and Table 1 that there were differences in the mean duration of the open arm entries. ANOVA showed a general effect for level of light, amount of vibrissa removal and significant interactions between the two. Tukey’s test showed that, under low illumination, rats with the whole vibrissae removed stayed longer in each open arm entry than either rats tested under low illumination with intact vibrissae or rats tested under high illumination with the whole vibrissa removed.
2.4. Data analysis Comparisons between the different levels of ablation of the vibrissae (none — or intact, tip, half, or whole) were performed using two-way analysis of variance (ANOVA) followed by post-hoc comparisons of group means (Tukey’s test for comparisons of group means versus a control). Significance levels were set at PB 0.05.
3. Results Fig. 1 shows the percentage of entries into the open arms, time spent in the open arms and mean duration of each entry into the open arms. ANOVA showed that, as a general effect, low light levels significantly increased both the average percentage of entries into the open arms and the time spent in the open arms,
Fig. 2. Frequency of entries (top) and time (bottom) spent in the closed arms. I, Intact control group (n = 20); T, without the tip of each vibrissa (n = 18); H, without half of each vibrissa (n =20); W, without the whole vibrissa (n= 20). Light, 150 Lux; dark, 2 Lux. As a general effect, the two measures in darkness were significantly larger than those in brightness.
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F. Cardenas et al. / Beha6ioural Brain Research 122 (2001) 169–174
Fig. 3. Total entries (top) into both kinds of arms and time (bottom) spent in the center of the elevated plus-maze. I, Intact control group (n=20); T, without the tip of each vibrissa (n = 18); H, without half of each vibrissa (n =20); W, without the whole vibrissa (n= 20). Light, 150 Lux; dark, 2 Lux. As a general effect, the two measures in darkness were significantly larger than those in brightness.
Fig. 2 shows the frequency of entries and the time spent in the closed arms. ANOVA followed by the Tukey test showed that, as a general effect, low light levels significantly increased the frequency of entries into the closed arms and decreased the time spent in the closed arms, while the amount of vibrissa removed did not have any effect on these two measures (Table 1). Fig. 3 shows the total amount of entries into both kinds of arms and the time spent in the central square. ANOVA followed by the Tukey test showed that, as a general effect, low light levels significantly increased both total entries and the time spent in the central square, while the amount of vibrissa removed did not have any effect on these two measures (Table 1).
4. Discussion It has been demonstrated that disturbances of vibrissal tactile perception affect general exploratory behavior patterns in the rat [1,45,48,49,51]. It is also known that the illumination level is another important determinant of rodent exploratory behavior [4,17,23,32]. However, the effect of simultaneous disturbances in both sensory systems has not been studied before. Here, we
investigated the exploratory behavior of rats submitted to different kinds of vibrissal removal in the elevated plus-maze under two very different illumination conditions. Two main results were found. On the one hand, it can be seen that rats exploring the plus-maze under low illumination do so in a different manner than those exploring it under high illumination. This result supports others investigating the influence of light levels on exploratory behavior in the plus-maze [17,32] or even in the open-field [51]. However, there is a report that illumination has no effect on rat’s behavior in the elevated plus-maze, although the light intensities used in that experiment might not have been so different for the animals to react as in light versus dark [9]. The other result was the increase in the mean time of each open arm entry. It has been proposed that abnormal functioning of the vibrissae enhances visual and other sensory modalities [1,42,46]. In fact, Schiffman et al. [42] proposed that the rat’s performance on some tasks involving visual discrimination could be improved by removing the vibrissae. In the present experiment, whole vibrissae removal may have prevented the animal from obtaining information about the environment under low illumination and caused the rats to stay longer in the open arms in order to compensate for the abnormal information processing. This would not have happened under high illumination, a situation in which the rats could use vision instead. In fact, there are reports relating exploration and information seeking [20,26,29,47]. From this view, it is the lack of information that triggers exploratory behavior, which is then seen as a means for obtaining more information. We propose that, in brightness, the impairment of vibrissal perception does not alter the aversion to the open spaces while under low illumination it would cause the animal to explore longer in order to obtain the information, since it would not be affected by the vision of open spaces and consequently would not avoid the open arms. Thus, rats apparently do not use the vibrissae as the main source of information in an elevated plus-maze. This sensory modality is probably a part of a more comprehensive system for information gathering. It remains to be discovered what are the relevant sensory modalities for rat exploratory behavior in the elevated plus-maze and what are their connections with the defensive repertoire exhibited in such experimental situations.
Acknowledgements This research was supported by grant number 523094/95-7, from CNPq and number 98/11187-2, from FAPESP to S.M.
F. Cardenas et al. / Beha6ioural Brain Research 122 (2001) 169–174
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Research report
Vibrissal sense is not the main sensory modality in rat exploratory behavior in the elevated plus-maze Fernando Cardenas, Marisol R. Lamprea, Silvio Morato * Faculdade de Filosofia Cieˆncias e Letras de Ribeira˜o Preto, Uni6ersidade de Sa˜o Paulo, A6. Bandeirantes 3900, Ribeira˜o Preto-SP 14040 -901, Brazil Received 7 April 2000; received in revised form 30 January 2001; accepted 30 January 2001
Abstract Four groups of male Wistar rats were submitted to acute bilateral removal of mystacial vibrissae at different lengths from the follicle. Each group was divided into two subgroups, tested under high (150 Lux) and low environmental illumination (2 Lux). All the subjects were allowed to freely explore an elevated plus-maze for 5 min. Results indicated that rats tested under low illumination tended to explore the open arms more frequently and longer then rats tested under high illumination. When tested under low illumination, rats in the group that suffered whole vibrissa removal stayed longer in the open arms than those in the other groups but did not differ in the number of entries. The average increase in the length of open arm entries, rather than a decrease in aversion to the open arms, may be due to the need of more time to obtain information about the environment since there is no light and the vibrissae were removed. This effect was not seen with rats tested under high illumination, possibly because vision could be used to obtain relevant information. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Elevated plus-maze; Exploratory behavior; Sensory systems; Thigmotaxis; Vibrissa removal; Information search; Aversion; Rats
1. Introduction To date there is not a thorough understanding of the role played by different aversive stimuli on rodent exploratory behavior in some behavioral tasks like the open field or the elevated plus-maze. For a long time, it has been assumed that the innate fear of open spaces was the most important anxiety trigger. However, the mechanisms by means of which rodents perceive the openness of an environment are still unclear. According to Becker and Grecksch [9], the visual system has no role in the genesis of the elevated plus-maze-evoked anxiety. On the other hand, there is some evidence for a potential role for a thigmotactic mediation by perioral vibrissae as a powerful determinant of open spaceinduced fear [45,48]. * Corresponding author. Tel.: + 55-16-6023662; fax: +55-166023632. E-mail address: [email protected] (S. Morato).
Since its pharmacological validation in 1985, by Pellow et al. [37], the elevated plus-maze has been widely used as a tool in the study of rodent anxiety [10,11,34,36,37,50]. It consists of four opposed arms, two of them enclosed by walls and two with no enclosure, the whole set elevated above the floor. The cited reports stress that rodents show a preference for entering and staying more in the closed arms than in the open ones. The total number of entries into each kind of arm, as well as the total time spent exploring the open arms, vary according to the type of treatment: for example, pre-treating animals with anxiolytic drugs leads to increases in both the number of entries and the time spent in the open arms [10,15,16,21,33]. On the other hand, pre-treating them with anxiogenic drugs decreases both the number of entries and the time spent in the open arms [5,6,40,52]. Similarly, behavioral manipulations such as individual housing [27,31], handling [4,22,44], malnutrition [3], aversiveness of the experimental situation [2,17,50] or behavioral restriction [28,30], have been shown to pro-
0166-4328/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 1 ) 0 0 1 8 0 - 2
170
F. Cardenas et al. / Beha6ioural Brain Research 122 (2001) 169–174
duce effects according to their motivational properties on this test. The elevated plus-maze has been used for the study and elucidation of the role of different cerebral structures in anxiety [7,12– 14,16,19,24,25,38, 39,41,43,50]. Despite the broad use of this and other behavioral tests based on the same principle, much remains to be unraveled about the stimuli responsible for the avoidance from the open spaces shown by rats, mice and
other rodents. Some authors have proposed that environmental illumination is an important aversive stimulus in these behavioral tests [4,32]. Others have proposed that height was the main aversive characteristic [35]. However, Treit et al. [49] have demonstrated that height is not important for the aversiveness of the open arms. Instead, it is the open arm’s lack of cues for thigmotaxis (trend to remain near or in contact with vertical surfaces) that is an important aversive factor [1,45,49,51]. In fact, it has been suggested [8] that rodents in the wild show this behavior probably as a defense against aerial predators [18]. In light of these studies, it is clear that any modification in thigmotactic perception would lead to changes in the normal way with which the rat approaches novel environments. What is not clear, however, is by what means the animal senses the vertical surfaces. Vision might be used to this end, but so could the vibrissae. In order to investigate their possible role, we submitted rats to the plus-maze under two illumination conditions, combined with partial or total removal of mystacial vibrissae.
2. Materials and methods
2.1. Animals Seventy-eight adult male Wistar rats, weighing 208.49 14.9 g (mean9S.E.M.) and with free access to food and water were housed in groups of six under a 12-h light/dark cycle (lights on at 07:00 h). The subjects came from the animal house of the campus of the University of Sao Paulo at Ribeira˜ o Preto and had a habituation period of 3 days before testing. During this period, they were handled daily for 5 min.
2.2. Apparatus
Fig. 1. Percentage of entries (top), time spent in the open arms (mid) and mean duration of open-arm entries (bottom). I, Intact control group (n = 20); T, without the tip of each vibrissa (n= 18); H, without half of each vibrissa (n= 20); W, without the whole vibrissa (n=20). Light, 150 Lux; dark, 2 Lux. * Different from both rats with intact vibrissae in the respective group and from the correspondent high illumination group. As a general effect, both time spent and percent of entries into the open arms in darkness were significantly higher than in brightness.
An elevated plus-maze made of dark brown wood with a dark blue Formica floor was used. The apparatus consisted of two opposed open arms (50×10 cm) crossed at right angles with other two opposed arms of the same size but enclosed by 40-cm high walls. The whole structure was elevated 50 cm above the floor. A 1-cm rim of transparent Plexiglas surrounded the open arms. Mystacial vibrissae were cut using normal lab scissors. One-half of the rats were tested under a 100-W white lamp placed 1.2 m above the maze (high illumination, 150 Lux in the central square). The other half were tested using a 20-W red lamp bulb at the same height (low illumination, 2 Lux in the central square). The experimental sessions were recorded using a video camera, linked to a VCR and a TV monitor located in an adjacent room.
F. Cardenas et al. / Beha6ioural Brain Research 122 (2001) 169–174
171
Table 1 Results of the two-way ANOVA applied to the behavioral measures Behavioral measure
Entries into the closed arms Total entries Percent of entries into the open arms Mean duration of open arm entry Time spent in the open arms Time spent in the closed arms Time spent in the central square
Light level
Length removed
Interaction
F[1,70]
P
F[3,70]
P
F[3,70]
P
15.219 62.113 41.476 7.354 95.206 71.878 31.560
B0.001 B0.001 B0.001 0.008 B0.001 B0.001 B0.001
1.020 1.389 0.332 3.594 0.614 0.571 0.92
0.389 0.253 0.802 0.018 0.608 0.636 0.436
0.769 2.053 0.910 6.67 2.258 0.282 0.61
0.515 0.114 0.441 B0.001 0.089 0.838 0.611
2.3. Procedure Rats were randomly assigned to one of four groups. One-half of each group was tested under high illumination and the other half under low illumination. A control group was tested without any treatment, while the other three groups had all their mystacial vibrissae cut at three different lengths from the follicle (just the tip, half or the whole vibrissa). Cuts were made 5 min before the test. In order to ensure equal conditions to all animals, the vibrissae of control rats were gently touched with the side of the scissors used for removal, and clipping sounds were made by just opening and closing the scissors without cutting anything. After that, each rat was gently placed in the central square of the maze facing one of the closed arms and allowed to freely explore for 5 min. After removing an animal, the maze was cleaned up with a solution of 20% ethanol and dried. The number of entries and the total amount of time spent in each arm were measured. An entry was recorded every time the rat placed all four paws on one arm.
while the amount of vibrissa removal did not have any effect on these two measures; in both cases, there were no interactions (Table 1). It can be seen in Fig. 1 and Table 1 that there were differences in the mean duration of the open arm entries. ANOVA showed a general effect for level of light, amount of vibrissa removal and significant interactions between the two. Tukey’s test showed that, under low illumination, rats with the whole vibrissae removed stayed longer in each open arm entry than either rats tested under low illumination with intact vibrissae or rats tested under high illumination with the whole vibrissa removed.
2.4. Data analysis Comparisons between the different levels of ablation of the vibrissae (none — or intact, tip, half, or whole) were performed using two-way analysis of variance (ANOVA) followed by post-hoc comparisons of group means (Tukey’s test for comparisons of group means versus a control). Significance levels were set at PB 0.05.
3. Results Fig. 1 shows the percentage of entries into the open arms, time spent in the open arms and mean duration of each entry into the open arms. ANOVA showed that, as a general effect, low light levels significantly increased both the average percentage of entries into the open arms and the time spent in the open arms,
Fig. 2. Frequency of entries (top) and time (bottom) spent in the closed arms. I, Intact control group (n = 20); T, without the tip of each vibrissa (n = 18); H, without half of each vibrissa (n =20); W, without the whole vibrissa (n= 20). Light, 150 Lux; dark, 2 Lux. As a general effect, the two measures in darkness were significantly larger than those in brightness.
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Fig. 3. Total entries (top) into both kinds of arms and time (bottom) spent in the center of the elevated plus-maze. I, Intact control group (n=20); T, without the tip of each vibrissa (n = 18); H, without half of each vibrissa (n =20); W, without the whole vibrissa (n= 20). Light, 150 Lux; dark, 2 Lux. As a general effect, the two measures in darkness were significantly larger than those in brightness.
Fig. 2 shows the frequency of entries and the time spent in the closed arms. ANOVA followed by the Tukey test showed that, as a general effect, low light levels significantly increased the frequency of entries into the closed arms and decreased the time spent in the closed arms, while the amount of vibrissa removed did not have any effect on these two measures (Table 1). Fig. 3 shows the total amount of entries into both kinds of arms and the time spent in the central square. ANOVA followed by the Tukey test showed that, as a general effect, low light levels significantly increased both total entries and the time spent in the central square, while the amount of vibrissa removed did not have any effect on these two measures (Table 1).
4. Discussion It has been demonstrated that disturbances of vibrissal tactile perception affect general exploratory behavior patterns in the rat [1,45,48,49,51]. It is also known that the illumination level is another important determinant of rodent exploratory behavior [4,17,23,32]. However, the effect of simultaneous disturbances in both sensory systems has not been studied before. Here, we
investigated the exploratory behavior of rats submitted to different kinds of vibrissal removal in the elevated plus-maze under two very different illumination conditions. Two main results were found. On the one hand, it can be seen that rats exploring the plus-maze under low illumination do so in a different manner than those exploring it under high illumination. This result supports others investigating the influence of light levels on exploratory behavior in the plus-maze [17,32] or even in the open-field [51]. However, there is a report that illumination has no effect on rat’s behavior in the elevated plus-maze, although the light intensities used in that experiment might not have been so different for the animals to react as in light versus dark [9]. The other result was the increase in the mean time of each open arm entry. It has been proposed that abnormal functioning of the vibrissae enhances visual and other sensory modalities [1,42,46]. In fact, Schiffman et al. [42] proposed that the rat’s performance on some tasks involving visual discrimination could be improved by removing the vibrissae. In the present experiment, whole vibrissae removal may have prevented the animal from obtaining information about the environment under low illumination and caused the rats to stay longer in the open arms in order to compensate for the abnormal information processing. This would not have happened under high illumination, a situation in which the rats could use vision instead. In fact, there are reports relating exploration and information seeking [20,26,29,47]. From this view, it is the lack of information that triggers exploratory behavior, which is then seen as a means for obtaining more information. We propose that, in brightness, the impairment of vibrissal perception does not alter the aversion to the open spaces while under low illumination it would cause the animal to explore longer in order to obtain the information, since it would not be affected by the vision of open spaces and consequently would not avoid the open arms. Thus, rats apparently do not use the vibrissae as the main source of information in an elevated plus-maze. This sensory modality is probably a part of a more comprehensive system for information gathering. It remains to be discovered what are the relevant sensory modalities for rat exploratory behavior in the elevated plus-maze and what are their connections with the defensive repertoire exhibited in such experimental situations.
Acknowledgements This research was supported by grant number 523094/95-7, from CNPq and number 98/11187-2, from FAPESP to S.M.
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