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Keywords:

  • Anxiety;
  • gene–environment interaction;
  • inbred rats;
  • laboratory environment;
  • Lewis;
  • SHR

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

The behavioral characterization of rodent strains in different studies and laboratories can provide unreplicable results even when genotypes are kept constant and environmental control is maximized. In the present study, the influence of common laboratory environmental variables and their interaction with genotype on the results of behavioral tests of anxiety/emotionality were investigated. To this end, the inbred rat strains Lewis (LEW) and spontaneously hypertensive rats (SHR), which are known to differ for numerous emotionality-related behaviors, were tested in the open field (OF), elevated plus maze (EPM) and black/white box (BWB), while three environmental factors were systematically controlled and analyzed: (1) the experimenter handling the animal (familiar or unfamiliar); (2) the position of the home cage (top or bottom shelf of the rack) and (3) the behavioral state of the animal immediately before the test (arousal or rest). Experimenter familiarity did not alter the behavior of rats in the OF. Cage position, on the other hand, influenced the behavior in the OF and BWB, with rats housed in top cages appearing less anxious like than those housed in the bottom. In the BWB (but not in the OF), these effects were genotype dependent. Finally, the behavioral state of the animals prior to testing altered the results of the EPM in a strain-dependent manner, with some anxiety-related genotypic differences being found only among rats that were aroused in their home cages. This study showed that common variations in the laboratory environment interact with genotype in behavioral tests of anxiety/emotionality. Recognizing and understanding such variations can help in the design of more effective experiments.

During the past decade, massive and systematic behavioral testing of multiple strains of laboratory rodents became a key step in neuroscience research, particularly in the field of behavior genetics. When populations of animals developed through inbreeding, selective breeding, transgenesis or gene targeting started to be characterized, it became evident that behavioral data from different laboratories were often not reproducible. Such discrepancies could result not only from differences in the genetic background of the animals but also from variations in the testing and/or laboratory environments (Chesler et al. 2002; Van der Staay & Steckler 2002; Wahlsten et al. 2003; Würbel 2002). A seminal work by Crabbe et al. (1999) has shown that major efforts to standardize both the test situation and the rearing environment do not guarantee the consistency of results across laboratories. For example, the mouse strains A/J and 129/SvEvTac, when compared to each other, displayed either higher or lower locomotor response to cocaine, depending on the laboratory where they were tested, in spite of environmental factors having been rigorously controlled. On the one hand, these data revealed that subtle, uncontrollable environmental variations between and within laboratories can influence behavior genetic studies. On the other hand, the effects of such an environmental background remained largely undefined.

Identifying and describing environmental factors that are potentially relevant to a large number of studies and laboratories are of foremost importance. This knowledge will contribute to improve the consistency of results, the interpretation of genotypic differences and the design of behavioral experiments. This problem is certainly not restricted to genetic studies and is not limited to mice, because behavioral testing is also essential in pharmacology, for example, and numerous studies using inbred and transgenic strains are carried out with rats.

The aim of the present study was thus to investigate the effects of some laboratory environmental variants which are commonly present in many, if not all, rodent behavioral studies through the use of a genetic model that has been well validated for the study of emotionality/anxiety. To this end, the inbred rat strains Lewis (LEW) and spontaneously hypertensive rats (SHR) were tested in three behavioral models widely used in the study of anxiety, namely the open field (OF), elevated plus maze (EPM) and black/white box (BWB). The LEW and SHR strains were chosen, because they have previously shown contrasting anxiety-related behaviors in all the aforementioned tests, with the former avoiding the aversive stimulus of each test more markedly than the latter (Ramos et al. 1997, 1998, 2002). Moreover, such strain differences seem reasonably robust, as they were confirmed in different substrains, laboratories and countries (Ramos et al. 2002) and they were shown to resist to variations in the test situation, such as day/night testing in the BWB, first/second trial in the EPM (unpublished) and strong/dim light in the OF (Ramos et al. 2002).

Several biological and environmental variables are known to influence behavioral tests. They are therefore equated and/or listed in most publications in the field. Among them, one could mention age (Imhof et al. 1993), sex (Imhof et al. 1993; Johnston & File 1991), time of testing (Gentsch et al. 1982), illumination level (Cardenas et al. 2001), floor surface (Morato & Castrechini 1989), transportation to the test room (Morato & Brandão 1997), previous experience in the apparatus (Bertoglio & Carobrez 2000; File et al. 1992; Treit et al. 1993), handling (Henderson 1970) and group housing (Maisonnette et al. 1993). Other factors, however, in spite of being potentially relevant, have rarely been considered and/or investigated. Herein, LEW and SHR rats were tested using the same protocols and apparatuses that were used in previous studies, but three environmental factors that had never been considered for these strains and tests have had their variations systematically controlled and analyzed.

The first of these factors was the experimenter performing the test, which has been shown to affect the output of nociception tests (Chesler et al. 2002). In the present work, this factor varied between one subject who was familiar to the animals and had large experience with all practices of the laboratory and another who had never entered the housing facilities. The second variable was the position of the cages inside the housing room, which has been shown to influence sensitivity to d-amphetamine in rats (Exner & Clark 1993). Like in many laboratories, this factor could vary between the highest (top) and the lowest (bottom) of five-rack shelves. Among other things, the illumination level was very different between these two housing situations.

Finally, the behavior of the animal immediately before testing was monitored and analyzed. Arousal has been proposed to be one of the two dimensions of human affective experience and, according to Heller's model of brain activity and affect, anxiety could result from the association between unpleasant affect and high arousal or activation (Schmidtke & Heller 2004). Behavioral tests of anxiety are typically short in time but are carried out during sessions that last several hours and involve many different animals. Even if these individuals do not to differ in the psychological trait being inferred, it can be expected that their behavioral (e.g. sleeping, eating and playing) and hence their arousal status at the moment preceding the test will vary. Yet, as pointed out by Lister (1990), most models of anxiety provide measures of state anxiety, which is thought to be a temporary and not a permanent feature of the individual. We have thus hypothesized that the ‘anxious state’ inferred during a specific test can be influenced by the arousal state of the animal immediately before the trial. We approached this problem in the EPM by testing rats that had been in a state of either ‘rest’ or ‘arousal’ in their home cages for at least 5 min continuously before the test.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Animals

The colony of SHR rats, originally acquired from Harvard University (Boston, MA), was obtained from UNESP (Botucatu, SP, Brazil). The LEW strain, originally acquired from Harlan Spreague Dowley Inc. (Indianapolis, IN), was obtained from UNICAMP (Campinas, SP, Brazil). At the time this study was carried out, both these strains had been maintained in our laboratory for eight (SHR) and nine (LEW) generations under a system of brother–sister mating, as it is generally recommended for inbred strains (http://www.dels.nas.edu/ilar/jour_online/34_4/definitionandnomenrat.asp). The animals were weaned and separated by sex at 4 weeks of age and, thereafter, kept in collective plastic cages (five rats/cage) with food and water available ad libitum under a 12-h/12-h light/dark cycle (lights on at 0700 h) at 22 ± 2 °C. Seventy-eight animals (9–10/strain/sex/treatment) were tested in the OF, EPM and BWB at 9, 10 and 11 weeks of age, respectively, with male and female rats being tested in different days. Rats were transported, just before tests, from the housing room to an adjacent testing room inside a container that allowed free movements to the animals. This was a transparent plastic cylinder with 25 cm of height and 21 cm of diameter with no lid. Transportation did not last more than 10 seconds and was done one rat at a time. All tests were performed between 1400 h and 1800 h, with adequate measures being taken to minimize pain or discomfort. The present experiments were in accordance with the local regulations for the ethical use of animals in research (CEUA/UFSC) and covered by the valid permission no. 23080.002412/2001-26.

Behavioral tests

Open field

The apparatus was made of wood covered with white impermeable Formica, had a floor of 100 × 100 cm (divided by black lines into 25 squares of 20 × 20 cm) and four white walls 40 cm high. The illumination in the test room provided 7 lx in the center of the apparatus. Each rat was placed in the center of the OF, which was novel to the animal, and the following variables were scored for 5 min: the number of peripheral (adjacent to the walls) and central (away from the walls) squares crossed with all four paws and total number of fecal boli. The behavior of each animal was recorded by a video camera positioned above the apparatus, which allowed monitoring in another room via a closed TV circuit. The floor of the apparatus was cleaned with a sponge wetted with water and a dry paper towel between rats. Recording and cleaning methods were kept unaltered for the other two behavioral tests.

Elevated plus maze

The apparatus was made of wood covered with a layer of black formica and had four elevated arms (52 cm from the floor) 50 cm long and 10 cm wide. The arms were arranged in a cross-like disposition, with two opposite arms being enclosed (by 40-cm high opaque walls) and two being open, having at their intersection a central platform (10 × 13.5 cm) that gave access to any of the four arms. The open arms were surrounded by a raised ledge (1 mm thick and 5 mm high) to avoid rats falling off the arms. The illumination had an intensity of 65 lx at the central platform, 70–90 lx along the open arms and 20 lx inside the closed arms. Each rat was placed in the central platform facing an open arm, and the following behaviors were registered for 5 min: the number of entries and the time spent (with all four paws) inside each type of arm and the percentage of open-arm entries in relation to the total number of arm entries.

Black/white box

The apparatus was made of wood covered with formica and presented two compartments. One was larger (27 × 27 × 27 cm) and white, with the floor divided by black lines in nine squares (9 × 9 cm), being strongly illuminated by a 40-W white bulb. The other was smaller (27 × 18 × 27 cm high) and black, with the floor divided by white lines into six squares (9 × 9 cm), being illuminated by a 40-W red bulb. Both white and red bulbs were located 30 cm above the floor of apparatus, thus providing 1000 lx inside the white compartment and 25 lx inside the black compartment. The two compartments, separated by a wall, were connected by a small square opening (7 × 7 cm). Each rat was placed in the center of the white compartment, and the following variables were registered for 5 min: the number of squares crossed and time spent with all four paws in each compartment and the number of transitions between compartments (one entry in the white and one return to the black). The time spent in the white compartment did not include the initial latency to the first entry into the black compartment.

Environmental factors

Experimenter

Half of the animals tested in the OF were manipulated (i.e. picked up from their home cages, transported to the testing room and placed inside the apparatus) by a familiar experimenter who entered the housing room every day and was the only person to handle the rats during cage cleaning (performed three times per week) between weaning and the day of test. An unfamiliar experimenter who had never entered the housing room before the beginning of the tests manipulated the other half of the animals. Both experimenters were non-smoking men. They wore white laboratory coats, latex gloves and no perfume during the days of test. Because our objective was to evaluate the influence of familiarity with the person performing the test rather than the effects of context/procedure differences, the movements were highly standardized between experimenters, who practiced beforehand with other animals in another laboratory room. All the objects to be moved before testing (e.g. cages, lids and drinking bottles) were moved in the same order and placed in the same positions, which were defined by marks on the top of the bench. One single trained observer, located in an adjacent room, performed observation and registration of all behavioral measures.

Cage position

Animals were housed in cages located in regular open racks containing five shelves each. The rats used in the present study were housed, from weaning to the day of test, in the same animal room, either on the highest (top) or the lowest (bottom) rack shelves, with both strains and sexes being equally distributed between these two conditions. Top and bottom shelves were located at 155 and 18 cm from the floor, respectively. The three intermediate shelves were also occupied with rat cages that were not being used in this experiment. Several environmental factors (e.g. light intensity and ammonia concentration) are expected to vary between these two conditions. For example, the illumination measured in the middle area of both shelf levels showed 240 and 12 lx for top and bottom cages, respectively. Temperature, on the other hand, did not vary between these two conditions. It is assumed here that variation in cage position is likely to be found (but not controlled) in many behavioral laboratories. The effects of home-cage position were thus evaluated in the OF, EPM and BWB tests.

Behavioral state

Thirty minutes before starting to test the animals in the EPM, at 1330 h, one cage of each strain was removed from the cage rack and placed on the top of the bench inside the housing room. While left undisturbed, the behavior of the rats in both home cages was continuously registered through a video camera positioned above the cages, being monitored by a trained observer through a TV set located in an adjacent room. The behavioral state of each animal was classified into two broad categories, namely ‘arousal’ and ‘rest’. Arousal included active behaviors where body movement could be observed (e.g. walking, eating, drinking and playing) and also the states of immobility where the eyes were open, thus letting no doubt about the fact that the animal was awake. Rest, on the contrary, was considered when the animal was immobile, with a relaxed muscle tonus and closed eyes, i.e. in an apparent state of sleep. When an individual rat remained in one of these two states for 5 min continuously, as registered by the observer, another familiar experimenter entered the housing room, gently picked up this animal from its cage and immediately transported it to be tested in the EPM located in another adjacent room. Once tested, the rat was marked with ink on its back (so that it could be recognized by the observer through the video) and was returned to its home cage but was no longer considered for EPM testing. All male rats were tested before female rats with 4 days of test being spent for each sex. Within each day, rats of the two strains and the two behavioral states had their order balanced throughout time.

Statistics

The results were analyzed, separately for each environmental factor, by a three-way anova for the factors strain (LEW/SHR), sex (male/female) and environmental variables (familiar/unfamiliar experimenter, top/bottom cage and arousal/rest). In cases where significant interactions were identified, the method of contrasts (Norman & Streiner 1998) was used for planned comparisons among subgroups. All analyses were carried out with the software statistica (1998; Statsoft, Tulsa, OK).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Experimenter

No significant differences (P > 0.05) between experimenters were observed for any behavioral measure of the OF test, but highly significant strain effects (F1,70 = 78.0 and P < 0.001) were found for central locomotion in the OF, with LEW rats showing overall lower scores than SHRs (Fig. 1a). A significant overall sex effect (F1,70 = 9.61 and P < 0.01) was found for total locomotion in the OF, with female rats being more active than male rats (Fig. 1b). No interactions (P > 0.05) were found between factors.

image

Figure 1. Central(a) and total locomotion(b) in the open-field test for the strains Lewis (LEW) and spontaneously hypertensive rats (SHR) (male and female rats) handled by either of the two different experimenters, one familiar (F) and the other unfamiliar (U) to the animals. Bars represent mean ± SEM values. Significant effects revealed by the three-way anova are shown in the boxes.

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Cage position

Because no influence of the experimenter was detected in the OF, data of both experimenters were pooled and analyzed for strain, sex and cage-position effects. A general effect of cage position (F1,70 = 4.35 and P < 0.05) was found for the locomotion in the central area of the OF, regardless of strain or sex, with rats housed on the top having higher scores than those housed in the bottom (Fig. 2a). Cage position did not affect any other measure of the OF or any measure of the EPM.

image

Figure 2. Central locomotion in the open field(a), time spent(b) and locomotion(c) in the white compartment of the black/white box for the strains Lewis (LEW) and spontaneously hypertensive rats (SHR) (male and female rats) housed either in top (T) or bottom (B) rack shelves from weaning to the day of test. Bars represent mean ± SEM values. Significant effects revealed by the three-way anova are shown in the boxes. When significant interactions were found, an asterisk (*) represents significant (P < 0.01) differences between top- and bottom-housed rats within each strain and sex.

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Different measures of the BWB were affected by cage position. For the time spent in the white compartment (Fig. 2b), there was a significant interaction (F1,70 = 10.65 and P < 0.01) between this environmental factor and rat strain. Contrast analyses revealed that SHR (but not LEW) rats of both sexes were affected by cage position, with animals housed in the bottom showing higher avoidance of the white compartment than those housed on the top. Because of this interaction, strain differences (LEW < SHR) within sex and treatment, which were highly significant (P < 0.001) for the other subgroups, were not detected (P > 0.05) in male rats housed in the bottom. For this same variable, there was also a significant overall sex effect (F1,70 = 7.19 and P < 0.01), with female rats spending more time in the white compartment than male rats. A significant interaction (F1,70 = 7.26 and P < 0.01) between strain, sex and cage position was found for the locomotion in the white compartment. Here, only SHR male rats and LEW female rats had their scores significantly (P < 0.01) changed by position (top > bottom), whereas strain differences were significant (P < 0.05) for all sex/treatment subgroups (Fig. 2c). For the number of transitions (data not shown), there was also an interaction between strain, sex and cage position (F1,70 = 5.84 and P < 0.05), with group comparisons showing the same differences as those found for the locomotion in the white compartment.

Behavioral state

There was an overall effect of strain (F1,70 = 19.49 and P < 0.0001) but also of the behavioral state the animal was in just before testing (F1,70 = 11.72 and P < 0.01) on the closed-arm entries in the EPM, the main measure of locomotor activity in this test (Fig. 3a). LEW rats did in general more closed entries than SHRs, and aroused rats were overall more active than resting rats. There was a significant interaction between behavioral state and strain for the two main anxiety-related behaviors of the EPM, namely the time spent (F1,70 = 6.92 and P < 0.05) and the percentage of entries (F1,70 = 6.03 and P < 0.05) in the open arms. Mean comparisons revealed that LEW and SHR male rats differed from each other for both behavioral variables (LEW < SHR; P < 0.05) only when they were aroused and not when they were resting before the test (Fig. 3b,c). For female rats, strain differences in the time spent in the open arms were significant (P < 0.05) under both arousal and rest states, whereas for the percentage of open-arm entries these differences appeared only in aroused animals. Arousal changed (i.e. increased) the time spent in the open arms only in SHR male rats but decreased the percentage of open-arm entries in LEW female rats (P < 0.05).

image

Figure 3. Number of closed-arm entries(a), time spent in the open arms(b) and percentage of entries in the open arms(c) in the elevated plus maze for the strains Lewis (LEW) and spontaneously hypertensive rats (SHR) (male and female rats) divided according to their behavioral state before the test. Each rat was classified as being either in a rest (R) or an arousal (A) state for 5 min before the test. Bars represent mean ± SEM values. Significant effects revealed by the three-way anova are shown in the boxes. When significant interactions were found, an asterisk (*) represents significant (P < 0.05) differences between resting and aroused rats within each strain and sex.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

The results of the present work showed that some common environmental factors which are expected to vary within and between laboratories devoted to behavioral testing can alter not only the baseline levels of anxiety-related behaviors but also the manifestation of genotypic differences between rodent strains. The location of the animals' home cage from weaning to the day of test was shown to influence fearfulness in the OF test regardless of strain or sex. In the BWB, on the other hand, this same variable affected fear-related behaviors in a genotype-dependent manner, in such a way that some strain differences appeared only in rats housed in top shelves. The behavioral state of the animal observed immediately before testing has also been shown to affect the output of the EPM test, with some anxiety-related differences appearing between strains only for rats that were aroused before testing. The familiarity of the animals with the experimenter, however, was found not to influence any of the strain's behavior in the OF test.

Previous studies had shown that LEW rats of both sexes, when compared with their SHR counterparts, avoid more the aversive stimuli of different tests such as the central area of the OF, the open arms of the EPM and the white compartment of the BWB (Berton et al. 1998; Ramos et al. 1997, 1998, 2002). The analysis of an intercross between these strains showed that some of the behavioral contrasts between them were highly (>50%) heritable (Ramos et al. 1998). In spite of the apparent robustness of this genetic model, some interstudy variability has been observed. For instance, strain differences in the percentage of open-arm entries, one of the main indices of anxiety from the EPM, were sometimes but not always significant, depending on the study (Ramos et al. 1997, 1998, 2002; Vendruscolo et al. 2003). We have therefore hypothesized that environmental factors may not only alter the baseline behavioral scores of LEW and SHR rats but also interact with genotype. However, when we varied environmental aspects of the testing situation itself such as day/night in the BWB, previous experience in the EPM (unpublished) and illumination in the OF (Ramos et al. 2002), the strain differences remained unchanged. These findings suggested that factors acting before the test might be more relevant for strain–environment interactions, an idea that is partially supported by the results of the present study.

Being handled by an unfamiliar experimenter before the OF test was not enough to change the behavioral profile of the two strains. Crabbe et al. (1999) suggested that the experimenter was one of the causes of variable results when mouse strains were compared at three different laboratories. Accordingly, Chesler et al. (2002) found that the experimenter was an important cause of interstudy variability in nociception data. In the present study, differently from these previous reports, only two experimenters were compared and all efforts were made to standardize their procedures, movements and clothing. Had all these factors not been controlled or had more experimenters been compared, then a significant effect might have been observed. The limited number of experimenters used herein impedes strong conclusions at this point, but the present data suggest that the control of all pretest procedures and of some experimenter traits and habits may help to minimize data variability that arises due to different staff members having to perform the same tests in a given laboratory.

The home-cage position in the housing rack (top or bottom) was shown to affect the results of two behavioral tests. In the OF, rats housed on top shelves displayed more central locomotion than those housed in the bottom, with this effect being similar across strains and sexes. Rodents tend to avoid exploring the central area of a novel large arena. Yet, such an avoidance can be reversed by anxiolytic drugs (Ramos & Mormède 1998; Vendruscolo et al. 2003) and is therefore thought to be anxiety related. The present results suggest thus that rats housed in top shelves are less fearful in this test that is performed under dim-light conditions. In the BWB, which has high luminosity as its main aversive component, a similar influence of cage position was observed. This effect, however, was strain and sex specific. Interestingly, strain differences in the time spent in the white compartment, a highly contrasting measure for rats from top cages, were not significant for male rats housed in the bottom. Thus, any study not controlling such an environmental variable and having a bias on cage location (e.g. most rats housed in bottom shelves) might totally miss important genotypic differences that could be otherwise revealed.

Overall, the present results suggest that being housed in either higher or lower cages, the rats exhibit changes in their emotional reactivity, which is in agreement with one previous report on responses to d-amphetamine (Exner & Clark 1993). The control of cage location has also been the object of concern in one study on carcinogenicity (Herzberg & Lagakos 1992). But, to our knowledge, this is the first study to demonstrate the effect of cage location on strain comparisons in behavioral tests. It is not possible to determine what environmental factor(s) was (were) responsible for such a phenomenon herein, but we can suggest that luminosity played a central role, because it varied between 240 (top) and 12 lx (bottom). Nevertheless, other factors not controlled herein could be expected to vary between the two housing situations such as odor, air quality and visual stimuli (other than light).

The EPM is a model of anxiety which sometimes produces unreplicable results (Hogg 1996). The present data showed that the arousal state of the animals immediately before this test can contribute to decrease replicability. For the two most important indices of anxiety (i.e. time spent and percentage of entries in the open arms), LEW and SHR male rats differed from each other only when tested after being aroused. The present results corroborate our hypothesis that the arousal status of an animal prior to testing can influence its ‘state anxiety’. Moreover, the observation that most genetic differences in experimental anxiety were found only in aroused rats is compatible with Heller's proposition that human anxiety, differently from depression, is expressed in a situation of high arousal or activation (Schmidtke & Heller 2004). One can suppose that the arousal status before testing is very likely to vary among individual animals within and between studies and that such a variation is not necessarily random. If an individual's state of arousal is affected by the cage order at which it is tested, for example, then one could expect that cage order would influence the output of a test. Such an influence has in fact been observed in nociception studies (Chesler et al. 2002) and, in the present study, it has been shown to act on measures of the OF and BWB. In general (data not shown), the first rat to be tested in a given cage was more anxious–like and less active than rats tested afterwards.

In summary, the results of this study showed that at least two different laboratory environmental factors have relevant influence on the results of behavioral tests and that some of this influence is genotype dependent. These findings are relevant for studies that aim at comparing animals with different genotypes but also have a potential impact on other types of neurobiological experiments. Further studies should investigate the causes of such environmental effects and their impact on gene expression and use this knowledge to propose changes in pretesting procedures. Meanwhile, an effort to randomize these effects would be advisable. For example, balancing strains/treatments across different housing situations (Herzberg & Lagakos 1992) and/or manipulating each cage a few minutes before starting the tests, in such a way that all rats get initially aroused, might help to increase the reproducibility of results in the OF, BWB and EPM tests.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

A. Ramos had a fellowship from CNPq, G. S. Izídio had a scholarship from CAPES and D. M. Lopes and L. Spricigo Jr had scholarships from CNPq.