Physiology & Behavior 85 (25) 265 27 Abstract Effect of different illumination levels on rat behavior in the elevated plus-maze Andrea Milena Becerra Garcia, Fernando Parra Cardenas, Silvio Morato Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto-USP, Av Bandeirantes, 39, 1449-91 Ribeirão Preto-SP, Brazil Received 11 February 25; received in revised form 31 March 25; accepted 4 April 25 The present study addressed the role of environmental light intensity on the exploratory behavior of rats in the elevated plus-maze, with the specific goal of determining the light intensity threshold for triggering the aversion to the open arms. Male Wistar-derived rats were tested in the elevated plus-maze under different illumination levels:, 1, 3, 1, 3, 1 and 3 lx. Exploratory behavior occurring in the open arms (e.g., entries and time spent in these arms) was more intense under and 1 lx than under the other illumination levels, which did not differ among themselves; on the other hand, locomotor behavior (as indicated by frequency of entries and distance run in the closed arms) was not altered under all illumination conditions. The data indicated that vision is important in triggering aversion to the open arms of the elevated plus-maze. They also indicated that the threshold of such aversion was found between 1 and 3 lx environmental illumination and that the phenomena is not intensity-dependent but rather of an all-or-none type. It should be emphasized that these conclusions only stand for unfamiliar environments. The role of light in familiar environments is currently under investigation in our laboratory. D 25 Elsevier Inc. All rights reserved. Keywords: Elevated plus-maze; Environmental illumination; Aversion; Aversion threshold; Rats; Light triggered aversion 1. Introduction The elevated plus-maze is a widely used anxiety animal model [12,26]. The test is a modification of a procedure introduced five decades ago by Montgomery [2]. Pharmacological, behavioral and physiological research has shown the test to be a reliable model for the study of animal anxiety [24]. Briefly, the test consists of a plus-shaped maze elevated above the floor level, with two wall-closed arms and two opposite open arms. A rat is allowed to freely explore the apparatus and the frequency of entries and the time spent in each type of arm and the frequency and time spent in several other behaviors are then analyzed; while exploring the maze, a rat will enter into both the closed and the open arms but will typically prefer to enter into the closed arms and remain inside them for longer periods (e.g., Corresponding author. E-mail address: smorato@ffclrp.usp.br (S. Morato). 31-9384/$ - see front matter D 25 Elsevier Inc. All rights reserved. doi:1.116/j.physbeh.25.4.7 [7]). Thus, there is a negative correlation between anxiety levels and the exploration of the open arms: lesser anxiety levels allow longer and more frequent exploration periods of the open arms [13,23]. This has been used to define the anxiolytic or anxiogenic properties of substances or behavioral procedures [23,24]. In addition, the test became more sensitive to detect anxiolytic or anxiogenic properties of drugs with the inclusion of the analysis of behaviors other than entering the arms, such as head-dipping, end-arm exploration, stretching or rearing [6,7,27]. In spite of the apparent simplicity of this test, the aversion to the open arms seems to be influenced by many factors [14]. Some of them are inherent to the subjects, such as sex [16] or age [15], while others are related to the experimental procedure, such as single or multiple pre-exposures to the maze [9,11,3] or moment of the day at which testing occurs [1,11]. Still others concern the test situation itself, such as the level of illumination in the test room, as can be seen in the next paragraph.
266 A.M.B. Garcia et al. / Physiology & Behavior 85 (25) 265 27 It has been reported that low levels of environmental illumination lead to increased exploration of the open arms [5,1,11,21]. Despite the broad use of this animal model of anxiety, little is known about the event(s) triggering the aversion that ultimately will cause rodents to avoid the open arms of the maze. It has been demonstrated that the innate aversion of rats to open spaces seems to be linked to rodent thigmotaxis [3] rather than to the fear of heights as previously supposed [23]. Cardenas et al. [5] submitted rats to acute bilateral removal of mystacial vibrissae at different lengths from the follicle, interfering with the normal thigmotaxis. Their results showed that the vibrissal sense is not the main sensory modality in the exploration of the elevated plus-maze. On the other hand, previous reports [28] suggest that when possible, rats use vision as the main perceptual system to obtain relevant information from the environment. Others [17] have reported that rats tested in the elevated plus-maze in the absence of light increased locomotor activity without decreasing open-arm avoidance. However, another report using three different levels of high illumination (3, 3 and 9 lx) [2] concluded that exploratory behavior of Wistar rats in the elevated plusmaze is not changed as a function of light levels. Previous reports from our laboratories showed that rats tested under low (2 lx) light intensity increased the number of entries and the time spent in open arms in contrast to rats tested under high (12 lx) intensities [21]. In the same vein, others [11] have found that high illumination levels significantly reduced the percentage of entries into the open arms, the percentage of time spent there and the total amount of general activity, in comparison to low illumination levels. Some authors [22] showed that rats submitted to sudden darkness while exploring the elevated plus-maze exhibited an increase in the number of entries into the open arms, the total amount of entries, the percentage of entries into the open arms, and the time spent exploring them. The same increments in locomotion were observed when animals were either tested in the open-field in the dark or after sudden darkness when initially tested in the light [31]. Using a modified version of the elevated plus-maze with four enclosed arms (two with transparent Plexiglas and two wooden walls) Martinez et al. [19] showed that rats preferred to stay in the wood surrounded arms rather than in the transparent enclosed areas, a behavior similar to that of rats avoiding the open arms in a standard elevated plusmaze. Such an avoidance disappeared when the transparent walls were covered with black opaque paper. These results suggest that light is a more powerful trigger for anxiety than the absence of thigmotaxis. All the above indicates that environmental illumination levels, that is the presence of light, is an important stimulus triggering aversion to open spaces in rats. Thus, the present study addresses the influence of different light levels on the exploratory behavior of rats in the elevated plus-maze, with the specific goal of determining the light intensity threshold for triggering the aversion to the open arms. 2. Methods 2.1. Animals Male Wistar-derived rats from the animal house of the University of São Paulo at Ribeirão Preto, weighing 21T1 g, were housed in polypropylene cages (43417 cm) in groups of six under a 12:12 dark/light cycle (light on at 7: h provided by a 1-W bulb 2.75 m above the center of the room-approximately 1 lx at central floor level) and temperatures kept between 24 and 27 -C, with free access to food and water throughout the experiment. All subjects were submitted to a habituation period to our animal room conditions and light cycle for 3 days before the beginning of the experiment. During this period they were not handled. The experiments reported in this paper were performed in compliance with the recommendations of the Brazilian Society of Neuroscience and Behavior which, in turn, are based on the US National Institutes of Health Guide for Care and Use of Laboratory Animals. 2.2. Apparatus An elevated plus-maze, described in detail elsewhere [18] was used. It consisted of two open arms (51 cm) crossed at right angles with two opposed arms of the same size. Two of the opposed arms were enclosed by wooden walls 4 cm high, except for the central part where the arms crossed. The whole apparatus was elevated 5 cm above the floor. To prevent the rats from falling, a rim of Plexiglas (.5 cm high) surrounded the perimeter of the open arms. The experimental sessions were recorded by a video camera interfaced with a TV monitor and a VCR in an adjacent room. In order to record displacements from one place to the other in the plus-maze and to specifically locate where other behaviors occurred, the image of the elevated plus-maze was divided into 1-cm squares in a transparent mask placed on the TV screen. This allowed the recording of the number of squares entered by an animal (which made possible to estimate the distance run) as well as the exact place of occurrence of the recorded behaviors. Displacements and behaviors were recorded with the use of a software (X-Plo- Rat 23) specifically designed in our laboratory for the recording of behavior in the elevated plus-maze. 2.3. Procedure After the 3-day habituation period, the rats were tested in the elevated plus-maze. Each rat was tested only once and had never been exposed to the apparatus before. They were divided into seven groups according to the illumination level in the test room:, 1, 3, 1, 3, 1 and 3 lx (N =8,
A.M.B. Garcia et al. / Physiology & Behavior 85 (25) 265 27 267 Time in the open arms Entries into the open arms (%) 16 14 12 1 8 6 4 2 7 6 5 4 3 2 1 8, 9, 1, 9, 9 and 9, respectively). Illumination levels were provided by three 1-W bulbs connected to a commercial dimmer which allowed any light intensity variations between and 5 lx. For the intensities of 1 and 3 lx, two of the bulbs had to be disconnected. Light intensities were previously measured with a luximeter (Lutron LX 13) placed in the central square. Each rat was gently placed in the central area with its nose facing one of the closed arms and allowed to freely explore the maze for 5 min. Before the next rat was tested, the maze was cleaned with a 5% ethanol solution and dried with a cloth. The number of entries and time spent into each kind of arm were recorded. The number of entries and the 1 3 1 3 1 3 Lux Fig. 1. Time spent (top) and percentage of entries (bottom) into the open arms. Bars represent the averages and vertical lines indicate S.E.M. Significantly different from the and 1 lux group (Tukey, P <.5). time spent at the distal parts (the two outer squares) of the open arms were also recorded. The total distance run in closed and open arms were estimated from the number of squares entered. In addition, the frequency and time spent in other behaviors were measured: (1) head dipping: dipping the head below the level of the maze floor; (2) stretching: when the animal stretches to its full length with the forepaws (keeping the hind paws in the same place) and turns back to the previous position; (3) rearing: partial or total raising on the hind limbs; and (4) grooming: species-specific behavioral sequences including cleaning of any part of the body surface or fur with the tongue, teeth, and/or forepaws. These categories were defined in accordance to previous works [4,7,27]. 2.4. Statistical analysis Data are reported as mean TS.E.M. and were analyzed with one-way analysis of variance (ANOVA). Whenever appropriate, post-hoc comparisons were made using the Tukey multiple comparison test. All tests were used with significance set at P <.5. 3. Results Fig. 1 shows the time spent in the open arms and the percentage of entries into the open arms for animals tested in the elevated plus-maze with the seven illumination conditions (, 1, 3, 1, 3, 1 and 3 lx). ANOVA showed significant differences for the time spent in the open arms between groups ( F [6, 61] =8.714, P <.1) and the posthoc comparisons between the group means indicated that rats tested under 3, 1, 3, 1 and 3 lx explored the open arms for a lesser time than those tested under and 1 lx. For the percentage of entries into the open arms, ANOVA also showed significant differences between the groups ( F [6, Table 1 Frequency and time (meants.e.m.) spent in behaviors performed by rats while exploring the elevated plus-maze under different illumination levels and the corresponding statistical results (one-way ANOVA) Parameters Lux ANOVA 1 3 1 3 1 3 F [6, 61] P Mean duration of open arm entries (s) 9.7T.6 1.5T1. 9.4T1.1 12.5T1.8 1.3T.8 1.5T.8 1.2T.9.845.541 Distance run in the open arms 7.9T1. 7.7T1.2 3.5T.5 a 3.3T.4 a 4.T.5 a 3.4T.5 a 3.1T.5 a 7.955 <.1 Entries into the open extremities 8.8T.8 7.9T1.2 3.4T.6 a 3.4T.4 a 4.1T.5 a 3.4T.5 a 3.3T.5 a 8.59 <.1 Time spent in the open arm extremities(s) 57.6T6. 6.9T8.5 21.5T4.5 a 22.7T3.7 a 31.3T4.7 a 25.4T4.2 a 21.8T3.6 a 7.833 <.1 Head-dipping frequency 13.T1.8 14.9T1.9 12.2T1.8 12.3T2.3 14.3T2.7 14.1T3.6 11.1T1.9.911.49 Time dipping head (s) 17.2T2.7 26.6T3.9 13.9T2.2 16.2T2.9 19.7T3.1 16.9T3.4 14.9T2.4 1.961.9 Stretching frequency 2.5T.3 1.8T.4 4.2T1. 3.4T.7 3.9T.8 1.8T3.9 2.8T.6.997.44 Time stretching (s) 2.3T.3 2.9T.7 6.T1.7 5.8T1.7 5.7T1.5 3.2T.7 3.6T.8 1.38.27 Rearing frequency 22.4T2.3 12.6T1.4 21.6T2.6 21.1T2.8 2.8T.8 12.8T3. 19.9T3.2 1.389.24 Time rearing (s) 31.9T2.3 26.6T3.5 36.T3.6 35.8T4.6 36.8T2.6 36.2T5.3 29.2T3..856.53 Grooming frequency 5.T1.1 3.4T.7 6.8T1.2 5.4T1.2 4.1T.6 3.2T.6 4.7T.9 1.483.24 Grooming total time (s) 24.9T5.9 15.7T4.5 26.4T5.9 25.5T6.3 15.7T2.5 13.9T2.1 23.8T5. 1.116.37 a Different from and 1.
268 A.M.B. Garcia et al. / Physiology & Behavior 85 (25) 265 27 Entries into the closed arms Distance run in the closed arms 12 1 8 6 4 2 1 9 8 7 6 5 4 3 2 1 61]=5.83, P <.1) and the post-hoc comparisons indicated that the animals tested under 3, 1, 3,1 and 3 lx entered the open arms less than the ones tested under and 1 lx. The fact that the mean duration of open arm entries did not alter (Table 1) indicates that the decrease in the two measures was due to decreases in the frequency of entries. Fig. 2 shows the frequency of entries and the distance run in the closed arms for all groups. There were no statistical differences in either the frequency of entries ( F [6, 61] = 1.287, P =.278) or the distance run (F [6, 61] =1.42, P =.48) in the closed arms. Table 1 shows the results for all other behaviors and a summary of statistics. ANOVA showed significant differences between the groups in the number of entries and the time spent exploring the extremities of the open arms. Posthoc comparisons between the means of the groups showed that animals tested under 3, 1, 3, 1 and 3 lx explored the open extremities less than the ones tested under and 1 lx. ANOVA did not show any significant effects neither in the frequency nor in the time spent in the behaviors of dipping the head, stretching, grooming and rearing. 4. Discussion 1 3 1 3 1 3 Lux Fig. 2. Frequency of entries (top) and distance run in the closed arms (bottom). Bars represent the averages and vertical lines the S.E.M. (Tukey, P <.5). Considering the great majority of reports in the literature investigate the elevated plus-maze under some degree of illumination, one of our results is that its lack increases exploratory behavior. This finding supports many others in the literature [5,1,11,17,21,22]; but see Ref. [2]. In the elevated plus-maze, this effect seems to be more specific to the open arms, since we observed no changes in exploratory behavior in the closed arms under no (or almost no) illumination. This has also been previously reported [5]. The above results mean illumination triggers the avoidance to the open arms since, in the darkness, rats do not avoid these arms. The analyses of number of entries and time spent in the open arms and open arms extremities suggest that animals tested under very low levels of illumination ( and 1 lx) behave as expected of animals with low levels of anxiety [23,29]. This anxiolytic-like effect of lower levels of illumination was confirmed by the significantly higher distance run in the open arms shown by animals tested in very low illumination conditions. This greater exploration of the open areas is not a consequence of a general increased level of activity. In fact, the distance run inside the closed arms, as well as the total entries into them, remained unchanged for all illumination conditions. An intriguing aspect of the data obtained in this experiment is that aside from displacements measures, no other naturalistic behavior was affected by the different illumination levels. Curiously, the literature discusses changes in these behaviors only when they do occur, mainly by drug action [18,23,27,32]. Little or nor mention is made when these behaviors are not altered (e.g., 3,7). One possible explanation is that these responses are modulated by thigmotactic cues rather than visual. The effects obtained with drugs on these behaviors [18,23,27,32] may be due to their action on mechanisms other that the ones involving vision. It is important to notice that our data indicated there is a threshold of environmental illumination for triggering anxiety-like behaviors which is somewhere between 1 and 3 lx. Above 3 lx of environmental illumination the behavior of the rats show avoidance of the open arms. However, the avoidance of the open arms (indicating anxiety) was not light intensity-dependent, since exploratory behavior did not decrease in a manner proportional to the increases in environmental illumination, indicating it is an all-or-none type of effect. Once the threshold is reached, the rats exhibit anxiety-like behaviors in a variety of environmental light intensities, at least in the range we used. It is difficult to compare these results with the literature since, to our knowledge, parametrical experiments, like using light intensities like this, were never performed. Nonetheless, there are reports on the behavioral effects of different environmental light intensities comparing 2 and 12 lx [21] or 3, 3 and 9 lx [2] but, aside from being carried out in different laboratories, they both used supra-threshold light intensities much superior to the threshold value we obtained and produced contradictory results. Our data do not allow the conclusion that environmental illumination, originated in the light bulbs and diffuse because of reflections, is the sole and direct stimulus triggering aversion, with its characteristic anxiety-like behaviors. In fact, another study from our
A.M.B. Garcia et al. / Physiology & Behavior 85 (25) 265 27 269 laboratory [19] carried out with fixed vertical illumination of about 1 lx and varying horizontal illumination levels suggests that two processes may produce aversion to open spaces: One triggered by increasing light levels reaching the retina horizontally... And the other triggered by image formation in the retina... (p. 24). This hypothesis is supported by another work [1] showing that horizontal visual information increases the aversiveness in an elevated plus-maze with transparent closed arms, which is fully supported by our data [19]. The hypothesis is further supported by a report [25] showing that the different parts of the retina convey different information about the environment: visual information reaching a specific area may trigger anxiety-like behaviors while this does not happen in other retinal areas. This is made more plausible in another report [8] demonstrating the existence of thalamic nuclei receiving visual input from the superior colliculi connected to the amygdala and providing it with rapid visual information, allowing for fast behavioral responses to threatening visual stimuli. Thus, it is probable that light entering the retina is able to modify the functioning of brains areas constituting the circuits which, when active, mediate fear and anxiety. More studies are necessary in order to clarify the role of horizontal visual information, since the present work dealt mainly with the determination of an illumination threshold for the appearance of anxiety-like behaviors such as the avoidance of the open arms of an elevated plus-maze. Above all, it should be remembered that all testing reported here occurred in unfamiliar environments and the conclusions are limited to them. 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