The interaction of housing condition and acute immobilization stress on the elevated plus-maze behaviors of protein-malnourished rats

Protein malnutrition induces structural, neurochemical and functional alterations in the central nervous system, leading to behavioral alterations. In the present study, we used the elevated plus-maze (EPM) as a measure of anxiety to evaluate the interaction between acute immobilization and housing conditions on the behavior of malnourished rats. Pups (6 males and 2 females) were fed by Wistar lactating dams receiving a 6% (undernourished) or 16% (well-nourished) protein diet. After weaning, the animals continued to receive the same diets ad libitum until 49 days of age when they started to receive a regular lab chow diet. From weaning to the end of the tests on day 70, the animals were housed under two different conditions, i.e., individual or in groups of three. On the 69th day, half of the animals were submitted to immobilization for 2 h, while the other half were undisturbed, and both groups were tested 24 h later for 5 min in the EPM. Independent of other factors, protein malnutrition increased, while immobilization and social isolation per se decreased, EPM exploration. Analysis of the interaction of diet vs immobilization vs housing conditions showed that the increased EPM exploration presented by the malnourished group was reversed by acute immobilization in animals reared in groups but not in animals reared individually. The interaction between immobilization and housing conditions suggests that living for a long time in social isolation is sufficiently stressful to reduce the responses to another anxiogenic procedure (immobilization), while living in groups prompts the animals to react to acute stress. Thus, it is suggested that housing condition can modulate the effects of an anxiogenic procedure on behavioral responses of malnourished rats in the EPM. Correspondence


Introduction
Protein or protein-calorie malnutrition imposed early in life is a well-known environmental factor that produces alterations in the brain.These alterations can affect morphological, neurochemical, neurophysiological, and functional aspects of the developing brain (1)(2)(3)(4)(5)(6)(7)(8).Moreover, it is also well known that protein malnutrition during gestation and/or lactation can modify the behavior of rats in several animal models of anxiety.Malnourished animals submitted to experimental models of anxiety such as light-dark transition (9,10), elevated plus-maze (EPM) (11,12) and elevated T-maze (13) tests showed behavioral alterations that have been interpreted as lower anxiety levels and/or higher impulsiveness.
A study by our group showed that using an anxiogenic or stressful procedure (shortterm social isolation) reduces open-arm exploration by control animals but does not change the behavior of animals malnourished early in life (14).In contrast, social isolation for 1, 2 or 24 h reduces exploration in the EPM (15).This suggests that the emotional alteration produced by early protein malnutrition is resistant to changes even when the animals are exposed to a stressful procedure before being tested in the EPM, thus showing a robust long-lasting emotional alteration produced by the nutritional deficit at the beginning of life.
In addition, immobilization is another classic stressful procedure used to alter exploratory behavior in the EPM.Animals submitted to immobilization stress present physiological, sensory or neurochemical alterations, as well as behavioral changes when submitted to the EPM, open-field, tail-flick and hole-board tests.Immobilization, when applied 24 h before the test, produced anxiogenic-like responses in the EPM (16)(17)(18)(19)(20) and in the open-field tests (21)(22)(23).In the EPM, immobilization for 2 h causes reduction in both the percent of entries and time spent in the open arms (16)(17)(18)(19)(20).
Since only long-term social isolation was not effective in reducing the increased exploration of the open arms in previously malnourished animals, we thought it would be useful to investigate the effects of two stressful procedures: social isolation followed by acute immobilization before the EPM test.Thus, our main objective was to determine if acute immobilization stress differentially affects open-arm exploration by wellnourished and malnourished animals reared individually or in groups from weaning to adulthood.

Subjects and Methods
Male Wistar rats from the animal colony of the Ribeirão Preto Campus, University of São 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 assigned randomly to a 6% or a 16% protein ad libitum diet during the lactation phase (0-21 days).The diets have been described elsewhere (11).The protein-deficient diet contained approximately 8% casein (6% protein), 5% salt mixture, 1% vitamin mixture, 8% corn oil, 0.2% choline, and 77.8% corn starch (w/w).The regular protein diet contained approximately 20% casein (16% protein), 60.8% corn starch 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 receiving a regular lab chow diet ad libitum until the end of the experiment.The animals were maintained on a 12:12-h light:dark cycle (lights on at 6:00 am) with room temperature kept at 23-25ºC, and with free access to water and food throughout the experiment.
The behavioral tests were conducted during the light period (1:00 to 5:00 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.

Housing conditions
After weaning, the animals submitted to both nutritional conditions were reared under two different housing conditions: individually in 30 x 19 x 14 cm plastic cages, or in groups of three animals per plastic cage (40 x 33 x 17 cm).Before the behavioral tests, animals in each housing condition were divided into immobilized and non-immobilized subjects generating four groups: nonimmobilized reared individually (NII, N = 67); non-immobilized reared in groups (NIG, N = 22); immobilized reared individually (II, N = 42), and immobilized reared in groups (IG, N = 24).The different number of animals was due to higher mortality of animals exposed to protein malnutrition, especially animals reared in groups.

Apparatus
The rats were placed on a wood base and wrapped with a hospital bandage secured with adhesive tape for the immobilization procedure.The wood bases were constructed according to the mean size of the animals on the day of immobilization (day 69): 24.5 x 9.5 x 1.5 cm for well-nourished animals and 19.5 x 6.8 x 1.5 cm for malnourished animals.The EPM was made of wood and consisted of two open arms (50 x 10 cm) opposite to each other, crossed by two enclosed arms (50 x 10 x 40 cm) with an open roof (24).The maze was elevated 50 cm from the ground.Fluorescent ceiling lights (2 x 60 W) provided the only illumination in the experimental room.

Procedure
On the 69th day, animals were placed on the wood base, hand-held and wrapped with the bandage except for the nose and the tail.The animals remained immobilized for 2 h before being returned to their home cages.The non-immobilized animals were also transported to the same room and kept undisturbed in their home cages during the same period or time (2 h).Twenty-four hours later, the animals were tested in the EPM.For the EPM test the animals were placed individually in the center of the maze facing an enclosed arm and allowed to explore for 5 min.The test session was recorded with a vertically mounted video-camera (Sony, Tokyo, Japan) linked to a monitor and VCR in an adjacent room.The video-tapes were later analyzed by an experimenter, and the following behavioral categories were identified and recorded, as previously described (11,12 ) rearing (rising on the hind paws), 7) head-dipping (scanning over the sides of the maze toward the floor), 8) stretch-attend posture (forward elongation of head and shoulders followed by retraction to the original position).The behavioral categories stretch-attend posture and head-dips have been previously described as risk-assessment behaviors (11,25,26).The rearing category was included as a classical measure of exploration in a novel environment (11,12).The other behavioral categories have been behaviorally and pharmacologically validated for rats (24,27) and mice (25,26,28,30) as reliable measures of fear/ anxiety levels.

Statistical analysis
Data are reported as means ± SEM and were analyzed by three-way (diet, housing condition and immobilization) analysis of variance (ANOVA).When appropriate, post hoc comparisons were made using the Newman-Keuls test.The level of significance was set at 0.05.

Body weight
On day 70 the malnourished animals weighed less than the well-nourished animals (F(1,151) = 738.60;P < 0.001), indicat-ing that even a nutritional rehabilitation procedure did not restore the body weight deficits produced by early protein malnutrition.In addition, the weight of control and malnourished animals was differentially affected by housing conditions (F(1,151) = 12.66; P < 0.001).Control animals living in groups increased their body weights as compared with animals living individually, while malnourished animals living in groups decreased their body weights compared with animals living individually (Table 1).

Behavioral measures
As illustrated in Figure 1   As illustrated in Figure 3, rearings were not affected by any factor studied.However, early malnutrition produced an increase in the head-dips as indicated by a significant main effect of diet (F(1,147) = 8.47; P < 0.05).Animals in the immobilized groups showed a decrease in head-dip frequencies as indicated by a significant main effect of the immobilization procedure (F(1,147) = 5.56; P < 0.05), and the animals reared in groups showed an increase in head-dip frequencies as indicated by a significant main effect of housing condition (F(1,147) = 31.08;P < 0.001).Furthermore, ANOVA indicated a significant interaction between immobilization and housing conditions (F(1,147) = 4.40; P < 0.05), indicating that in the nonimmobilized condition animals living in groups had higher head-dip frequencies compared to the same condition in the immobilized condition.In addition, ANOVA also indicated a significant diet vs immobilization vs housing condition interaction (F(1,147) = 4.99; P < 0.05).Only malnour- * ished animals in the grouped housing condition had their performance significantly affected by the immobilization procedure.There was a decrease in head-dip frequency.Finally, ANOVA revealed that animals reared in groups showed a decrease in stretch-attend posture frequency as indicated by a significant effect of housing condition (F(1,147) = 9.68; P < 0.05).

Discussion
The results presented here show that early postnatal protein malnutrition significantly reduced the body weight of the animals, as previously reported by our group (10,(31)(32)(33)(34) and in the literature (35)(36)(37)(38).In addition, social stimulation (rearing animals in groups) increased the body weight of well-nourished animals while reducing the body weight of malnourished animals.It is not easy to provide a satisfactory explanation for this result.However, it is possible that malnourished animals spent much more energy on competition for food than well-nourished animals when living in groups.
Early postnatal protein malnutrition also produced a lower anxiety level according to the EPM test, as indicated by high frequency of entries and duration of time spent in the open arms, high frequencies of head-dips, low frequencies of attempts to enter open arms, and low latencies to the first open-arm entry.Stretch-attend posture was not affected by malnutrition.The lower anxiety of malnourished animals has been described in our studies on both pre- (12,13) and post-natal protein malnutrition (10,29,32), suggesting that an early nutritional deficit changes the responsiveness of rats in pain-free animal models of anxiety.This result cannot be attributed to changes in locomotor activity or environmental exploration produced by protein malnutrition since the frequency of closed-arm entries and rearings was not affected by any of the factors studied (diet, immobilization or housing condition).
Both immobilization and social isolation stress, the two other independent variables, produced changes consistent with other studies (15)(16)(17)(18)(19)(20).Thus, acute immobilization and housing condition reduced all the classical behavioral categories investigated in the EPM (frequency and duration of open-arm exploration).It was also shown that housing animals individually was more effective than acute immobilization in producing increased anxiety in the EPM, as observed in the other behavioral categories, since housing condition affected all behaviors except closedarm entries and rearings (behaviors more related to locomotor activity and exploration), while immobilization did not affect closed-arm entries, attempts to enter open arms, latency to first open-arm entry, rearings, or stretch-attend posture.Thus, while both factors increased anxiety it appears that social condition has a stronger effect on riskassessment behaviors in the EPM compared to acute immobilization stress.
The new information reported in the present study concerns the interaction of protein malnutrition and the other two variables, i.e., housing and immobilization.Thus, while in the non-immobilized condition the malnourished animals showed a higher frequency of exploration of open arms compared to well-nourished animals, the immobilization procedure reduced the exploration of open arms (% time in the open arms and headdips) of malnourished animals to values similar to those presented by well-nourished animals.These data suggest that the introduction of an acute stress was efficient in reversing differences due to diet in the level of EPM exploration.Moreover, this influence was more evident for animals reared in groups than for animals living individually, leading to a significant diet vs immobilization vs housing condition interaction.These data also showed that the anxiogenic effects of acute immobilization, responsible for a higher reduction in EPM exploration in malnourished than in well-nourished rats, depended on housing condition, as indicated by a significant housing condition vs immobilization interaction.Thus, it seems clear that the effectiveness of an acute stress depends on the housing conditions of the animals.It appears that living individually for a long time (from weaning to adult age) is sufficiently stressful to reduce the responses to a different stressful procedure (immobilization).However, living in a group prompts an animal to exhibit a greater reaction to an acute stress procedure.This result contrasts with our previous report showing that shortterm social isolation reduced EPM exploration in well-nourished rats but did not affect early postnatal protein-malnourished rats (14).Thus, we suggest that housing condition is an important factor to consider when choosing stressful intervention to study behavioral responses in malnourished animals.
,25-29): 1) the percent of open-arm entries (an arm entry defined as the rat having all four paws into an arm), 2) the duration of time spent in the open arms, 3) closed-arm entries, 4) attempts to enter open arms (entering an open arm with only the forepaws and returning to the central platform or closed arm), 5) latency to first open-arm entry (latency to enter an open arm timed from the start of the test), 6 , malnourished animals showed increased percent of exploration of open-arms when compared to wellnourished animals, as indicated by a significant effect of diet on entries (F(1,147) = 21.02;P < 0.001) and duration of time spent in the open arms (F(1,147) = 26.58;P < 0.001).The immobilization procedure reduced the percent of entries (F(1,147) = 6.17;P < 0.05) and the duration of time spent in the open arms (F(1,147) = 10.03;P < 0.05).There was a significant main effect of housing conditions as indicated by an increased percent of entries (F(1,147) = 27.68;P < 0.001) and the duration of time spent in the open arms (F(1,147) = 56.01;P < 0.001) by animals raised in groups compared with individually housed animals.ANOVA also showed a significant diet vs immobilization interaction in duration of time spent in the open arms (F(1,147) = 9.19; P < 0.05), showing that malnourished immobilized animals decreased the duration of time spent in the open arms in relation to non-immobilized animals, as compared with no effects on well-nourished animals.Furthermore, ANOVA indicated a significant immobilization vs housing interaction in duration of time spent in the open arms (F(1,147) = 5.17; P < 0.05), indicating that the duration of time spent in the open arms was decreased by the immobilization procedure in grouped ani- condition and immobilization mals as compared with no effect in the animals reared individually.Finally, ANOVA indicated a significant diet vs immobilization vs housing condition interaction in the duration of time spent in the open arms (F(1,147) = 12.36; P < 0.001).Only the malnourished animals in the grouped condition had their performance significantly affected by the immobilization procedure (P < 0.05).They presented a decreased duration of time spent in the open arms.As illustrated in Figure2, closed-arm entries were not affected by any of the variables studied.However, early protein malnutrition decreased the attempts to enter open arms (F(1,147) = 9.06; P < 0.05) as well as the latency to first entry into the open arms (F(1,147) = 8.89; P < 0.05).Furthermore, rearing the animals in groups reduced their attempts to enter open arms (F(1,147) = 8.43; P < 0.05) and the latency to first entry into open arms (F(1,147) = 19.01;P < 0.001) compared to animals reared individually.

Figure 2 .Figure 3 .
Figure 2. Number of closed-arm entries (top), attempts to enter into open arms (middle) and latencies for the first open-arm entry (bottom) by non-immobilized animals reared individually (NII) or in groups (NIG) and immobilized animals reared individually (II) or in groups (IG).Data are reported as means ± SEM for 17-57 rats in each group.*P < 0.05 compared to well-nourished animals (Newman-Keuls test).

Table 1 .
Body weight of rats at the age of 70 days when behavioral tests were carried out.