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Rocki Stenger
The objective of the present study was to evaluate the interaction of two kinds of external stimuli (long-term tactile stimulation and underwater trauma) on the exploratory behavior of malnourished rats in an elevated plus maze (EPM). The results showed that tactile stimulation partially recovered body weight deficits produced by malnutrition and decreased EPM exploration. Malnutrition increased EPM exploration while underwater trauma decreased it, thus reducing differences between well nourished and malnourished animals. These data show that only one of the two external stimuli (underwater trauma) interacted with diet condition, suggesting that the stress caused by trauma was efficient in decreasing higher EPM exploration, equalizing the response of malnourished animals to that of non-traumatized well-nourished animals.
Previous studies (Almeida, Arajo, Moreira, Paiva, & De Oliveira, 1998; Franolin-Silva & Almeida, 2004) showed that anxiogenic or stressful procedures (shortterm social isolation or immobilization) differentially affected the behavior of well-nourished and malnourished animals when tested in the EPM. These data suggest that the emotional changes produced by early protein malnutrition possibly interact with emotional changes induced by social isolation or immobilization applied just before the test, resulting in differences in exploratory behavior in the EPM. The exact way these two stressful situations (early malnutrition and isolation/immobilization) interact to produce behavioral alterations in the EPM is not known, but it has been suggested that stressful experiences early in life can interfere with the management of stressful experiences later in adulthood (Almeida et al., 1998; Cabib, Puglisi-Allegra, & D'Amato, 1993; Franolin-Silva & Almeida, 2004; Matthews & Robbins, 2003).
Environmental stimulation has been suggested to reduce the effects of the so-called functional isolation produced by early malnutrition (Levitsky & Barnes, 1972). The concept of functional isolation refers to a situation of low energy caused by protein malnutrition where animals prefer to isolate themselves from the environment to save energy in order to survive. Functional isolation results in a reduction of social and environmental exploratory behaviors during critical periods of development.
Thus, the objective of the present study was to evaluate the effects of two kinds of external stimuli, one believed to be anxiogenic (underwater trauma) and the other believed to be anxiolytic (tactile stimulation), on the behavior of early protein-malnourished rats submitted to the EPM test.
A total of 99 male Wistar rats from the animal colony of the Ribeiro Preto Campus of the University of So Paulo were used. Each litter was culled to 6 male and 2 female pups on the day of birth. The dams and the pups were housed in transparent plastic cages (40 x 33 x 20 cm) and randomly assigned to a 6% or 16% protein diet ad libitum during the lactation phase (0 21 days). The two diets have been described in detail elsewhere (Almeida et al., 1994). Briefly, the protein-deficient diet contained approximately 8% casein (6% protein), a 5% salt mixture, a 1% vitamin mixture, 8% corn oil, 0.2% choline, and 77.8 % cornstarch. The regular protein diet contained approximately 20% casein (16% protein), 60.8% cornstarch and the same percentage of the other constituents as the protein-deficient diet. The two diets were supplemented with L-methionine (2.0 g/kg protein), since casein is deficient in this amino acid. Only male rats were used in this study. After weaning (21 days), the animals continued to receive the same diets until 49 days of age, when all animals started to receive a regular lab chow diet until the end of the experiment. The animals were maintained on a 12L:12D cycle (lights on at 6 A.M.) with room temperature kept at 23-25o C, and free access to water and food throughout the experiment. Behavioral tests were conducted during the light period (1 to 5 PM). The experiments were performed in compliance with the recommendations of the Brazilian Society of Neuroscience and Behavior (SBNeC), which are based on the US National Institutes of Health Guide for Care and Use of Laboratory Animals.
A perforated metal cage (17 x 23 x 19 cm) and a round polyethylene container with 20-liter capacity were used for the underwater trauma. After the trauma, animals were tested in the EPM, which consisted of two open arms (50x10 cm) and two enclosed arms (50 x 10 x 40 cm) with an open roof, arranged in such a way that the two open arms were opposite to each other. The maze was elevated to a height of 50 cm from the floor. The behavior in the EPM was recorded with a video camera connected to a monitor and to a video cassette recorder in an adjacent room.
Tactile stimulation. Half the pups in both diet groups were exposed to daily 3-minute individual handlin g from birth to 49 days of age. The other half received no stimulation. The handling consisted of holding the animal in one hand and making cranio-sacral movements on the dorsal region with the thumb of the other hand.
Underwater trauma. At 70 days of age, the animals were exposed to the underwater trauma consisting of individually placing them in a perforated metal cage (17x23x19 cm), which was submerged in a round polyethylene container with a 20-liter capacity of water for a period of 30 seconds (Richter-Levin, 1998). The metal cage was then removed from the water and the rat was carefully dried with an absorbent cloth for a period of 60 seconds.
Body weight data are reported as means SEM and were analyzed by three-way (diet condition, stimulation and age) analysis of variance (ANOVA) with repeated measures based on age. Behavioral data are reported as means SEM and were analyzed by four-way ANOVA (diet, stimulation, trauma and session) with repeated measure based on session. When appropriate, post hoc comparisons were made using the Newman-Keuls test. The level of significance was set at .05.
The percentage of open-arm entries for all groups is illustrated in Figure 2. There was a significant effect of diet, F(1, 91) = 10.14, p < .01, indicating a higher percentage of open-arm entries for M than for W animals. There was also a significant effect of chronic stimulation, F(1, 91) = 4.99, p < .05, indicating a lower percentage of open-arm entries for stimulated animals compared with non-stimulated animals. A significant effect of session was also found, F(2, 182) = 64.28, p < .001, indicating decreases in the percentage of openarm entries across sessions. A post-hoc analysis showed a significantly lower number of open-arm entries in sessions 2-7 (p < .005) compared with the first exposure. Finally, there was a significant effect of session by trauma interaction, F(2, 182) = 3.51, p < .05, indicating a greater reduction of the percentage of open-arm entries in traumatized than in non-traumatized animals. The results for the time spent in the open-arms was similar to that of open-arm entries except for two aspects: a) there was a significant effect of trauma, F(1, 91) = 4.68, p < .05, indicating that traumatized animals spent less time in the open arms than non-traumatized animals; and b) there was a significant diet by trauma interaction, F(1, 91) = 4.17, p < .05, indicating that trauma significantly reduced the time spent in open-arms in M animals, with no effect on W animals. The reduction of the time spent in the open-arms by M animals was sufficient to equalize this time to that spent by non-traumatized W animals.
The present results showed that early postnatal protein malnutrition significantly reduced the body weight of the animals as reported in previous studies (Fukuda, Franolin-Silva, & Almeida, 2002; Franolin-Silva & Almeida, 2004). However, it was also demonstrated that chronic tactile stimulation produced an increase in the body weight of both W and M animals. It is possible that chronic tactile stimulation changed the eating behavior of dams and/or pups during lactation and of pups after weaning, resulting in increased food ingestion resulting in higher weight gain. Increases in brain weight, DNA and RNA levels after chronic tactile stimulation have also been previously reported by Lima, De-Oliveira, & Almeida, 1999, suggesting that environmental stimulation can be an advantageous external stimulus for weight gain.
Early malnourished animals increased behaviors believed to indicate lower anxiety in the EPM, a result consistent with previously reported data (Almeida et al., 1991; 1994). This increase in the exploration of the maze was not due to alterations in locomotor activity produced by malnutrition, since there were no differences between W and M animals in the number of closed arm entries, which is a measure indicative of locomotion.
Re-exposure to the EPM produced an increase in anxiety as indicated by significant reductions of exploration across sessions, a result consistent with previously reported studies on rats (Almeida et al., 1993) and mice (Espejo, 1997). This significant reduction in maze exploration has been interpreted as a different kind of fear after the first exposure to the maze. It is suggested that the anxiety generated by the first exposure changes to phobia in subsequent exposures (File & Zangrossi Jr, 1993). Since the present study showed there was no difference due to diet by session interactions, we conclude that if a different form of fear develops across sessions, it is not influenced by early malnutrition.
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