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Anxiolytic-like effects of short-term postnatal protein malnutrition in the elevated plus-maze test
Behavioural Brain Research 173 (2006) 310–314
Research report
Anxiolytic-like effects of short-term postnatal protein malnutrition in the elevated plus-maze test Ana Laura Franc¸olin-Silva a , Andr´ea da Silva Hernandes a , Marisa Tomoe Hebihara Fukuda b , Camila Tavares Valadares a , Sebasti˜ao Sousa Almeida a,∗ a
Laboratory of Nutrition and Behavior, University of S˜ao Paulo, Avenida dos Bandeirantes, 3900, 14040-901 Ribeir˜ao Preto, SP, Brazil b Department of Ophthalmology, Otorhinolaryngology and Head and Neck Surgery, School of Medicine of Ribeir˜ ao Preto, University of S˜ao Paulo, Avenida dos Bandeirantes, 3900, 14049-900 Ribeir˜ao Preto, SP, Brazil Received 27 April 2006; received in revised form 29 June 2006; accepted 30 June 2006
Abstract Given that protein malnutrition induces structural, neurochemical and functional changes in the CNS, the present study aimed to investigate the effects of short-term early protein malnutrition on the behavior and reactivity to diazepam (DZ) in the elevated plus-maze test (EPM). Male Wistar rats (n = 176) from well-nourished (16%-protein) or malnourished litters (6%-protein) were distributed in five different groups: W (wellnourished), M7 (malnourished for 7 days), M14 (malnourished for 14 days), M21 (malnourished for 21 days) and M28 (malnourished for 28 days) since birthday. EPM results showed that the longer the exposition to the deficient diet, the lower the anxiety of malnourished animals, a result similar to that produced by the treatment with DZ. This anxiolytic-like effect suggested that short-term malnutrition may affect neural and/or neurochemical systems believed to underlie behavioral expression in anxiogenic experimental situations. © 2006 Elsevier B.V. All rights reserved. Keywords: Short-term postnatal protein malnutrition; Anxiety; Elevated plus-maze; Diazepam; Rats
1. Introduction Protein or protein-calorie malnutrition imposed early in life is a well-known environmental factor producing brain alterations comprising morphological, neurochemical, neurophysiological, and functional aspects [5,7,8,18,29–31,44]. Moreover, it is also well known that protein malnutrition during the 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 light–dark transition [11,39], elevated plus-maze (EPM) [2,6] and elevated T-maze [4,23] showed behavioral alterations indicative of lower anxiety and/or higher impulsiveness. Although there is substantive information about the deleterious effects of short-term postnatal malnutrition on physical and motor development in rats [43,41], studies investigating behavioral effects of short-term malnutrition in the beginning of life are scarce in the literature. Thus, a substan-
∗
Corresponding author. Tel.: +55 16 36023663; fax: +55 16 36335015. E-mail address: [email protected] (S.S. Almeida).
0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.06.042
tive portion of the studies in this area uses prenatal malnutrition with nutritional recovery at birth [29–32], contrasting with few works using the procedure of short-term postnatal protein malnutrition. The relevance attributed to studies of short-term postnatal protein malnutrition is due to two main reasons. First, there is a theoretical importance to know if short-term malnutrition can affect brain development and behavior of rats. Second, several early life medical intercurrences during child development such as diarrhoea, infections and cystic fribrosis may expose children to short periods of malnutrition resulting body and brain injuries, as well as, impairments of cognitive function. It has been shown that short-term malnutrition affects homeorienting behaviors [15]; maternal behavior [16,26]; and learning in the Hebb–Williams maze [12]. However, the effects of short-term malnutrition in experimental models of anxiety as well as in the reactivity of malnourished animals to anxiolytic drugs on these models [2,39] are not clear. It is hypothesized that if even short-term postnatal protein malnutrition produces a lower anxiety in the elevated plus-maze, then the behaviors believed to express anxiety in this model should be modified
A.L. Fran¸colin-Silva et al. / Behavioural Brain Research 173 (2006) 310–314
in malnourished animals in a similar way as that induced by treatment with anxiolytic doses of diazepam (DZ). Thus, the objective of the present study was to investigate the similarities between the effects of short-term early protein malnutrition and the anxiolytic effects of DZ in animals exposed to the elevated plus-maze model. 2. Methods and materials
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2.3. Statistical analysis Body weight data are reported as means ± S.E.M. and were analyzed by oneway analysis of variance (ANOVA) on the day 70. Behavioral data are reported as means ± S.E.M. A two-way ANOVA (diet condition and drug condition) was initially conducted showing significant effects of nutrition and drug conditions. Thus, we conducted a one-way (dose of the drug) ANOVA for each diet condition to investigate the reactivity to the anxiolytic effects of the drug on each group. When appropriate, post hoc comparisons were made using the Newman–Keuls test. The level of significance was set at 0.05.
2.1. Subjects
3. Results Male Wistar rats from the animal colony of Ribeir˜ao Preto Campus, University of S˜ao Paulo were used. Each litter was culled to six male and two female pups on the day of birth. The dams and the pups were housed in transparent plastic cages (40 cm × 30 cm × 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 [2]. 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. The regular protein diet contained approximately 20% casein (16% protein), 60.8% corn starch and the 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 started receiving a regular lab chow diet until the end of the experiment and they were maintained on a 12:12-h light/dark cycle (lights on at 6:00 a.m.) with room temperature kept at 23–25 ◦ C, and with free access to water and food throughout the experiment. The behavioral tests were conducted during the light period (1:00–6:00 p.m.). 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. The rats from well-nourished (16%-protein) or malnourished litters (6%protein) were distributed in five different groups: W (well-nourished, n = 32), M7 (malnourished for 7 days, since day 0, n = 38), M14 (14 days, n = 36), M21 (21 days, n = 33) and M28 (28 days, n = 37). Furthermore, the animals were also subdivided in accordance with the dose of DZ utilized (0, 1, 2 or 4 mg/kg).
2.2. Apparatus and procedure The EPM was made of wood and consisted of two open arms (50 cm × 10 cm) opposite to each other, crossed by two enclosed arms (50 cm × 10 cm × 40 cm), with an open roof [34]. The maze was elevated 50 cm from the ground. Fluorescent ceiling lights (2 × 60 W) provided the only illumination in the experimental room. At 70 days of age, 30 min before being placed in the EPM, the rats were injected intraperitoneally with saline, 1, 2 or 4 mg/kg DZ. The animals were placed individually in the center of the maze, facing an enclosed arm and allowed to explore it 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 analyzed by an experimenter blind to the nutritional treatment, and the following behavioral categories were identified and recorded as previously described [2,3,6,13,14,37,38]: (1) the percent of open-arm entries (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 the first open-arm entry (latency to enter an open arm timed from the start of the test), (6) rearing (rising on the hind paws), (7) head-dipping (scanning over the sides of the maze toward the floor) and (8) stretch-attend posture (forward elongation of head and shoulders followed by retraction to the original position). The behavioral categories stretch-attend posture and headdips have been previously described as risk-assessment behaviors [2,37,38]. The rearing category was included as a classical measure of exploration in a novel environment [2,6]. The other behavioral categories have been behaviorally and pharmacologically validated for rats [14,34] and mice [13,25,37,38] as reliable measures of fear/anxiety levels.
3.1. Body weight On day 70, the animals of malnourished groups (M7: 444.04 ± 10.2; M14: 373.92 ± 7.8; M21: 369.45 ± 9.4; M28: 342.02 ± 6.9) weighed less than well-nourished animals (W: 476.09 ± 10.8) as indicated by a significant effect of diet treatment [F(4, 171) = 37.86; p < 0.05]. Post hoc analysis showed significant statistical differences in the weight of W as compared with all the other malnourished groups (p < 0.05). 3.2. Behavioral measures 3.2.1. Nutritional effects ANOVA showed a significant effect of diet condition on the behavioral categories believed to express anxiety in the elevated plus-maze such as percentage of open arm entries [F(4, 156) = 3.00, p < 0.05], percentage of time in open arms [F(4, 156) = 3.10, p < 0.05], head-dips [F(4, 156) = 3.80, p < 0.05]. Post hoc analysis showed that W groups differs significantly from M14, M21 and M28 groups in all behavioral categories (p < 0.05). 3.2.2. Drug effects The percentage of open-arm entries for all groups is represented in Fig. 1. There was a significant effect of drug dose for groups: W [F(3, 28) = 10.91; p < 0.001], M7 [F(3, 34) = 9.42; p < 0.001] and M14 [F(3, 32) = 4.25; p < 0,05], but no significant effects for groups M21 and M28. Post hoc analysis showed a significant increase in the open-arm entries after the treatment with 1, 2 and 4 mg/kg of DZ in W group (p < 0.05). However, post hoc analysis showed significant increases in the open-arm entries only after treatments with 2 and 4 mg/kg of DZ in the M7 group and with 4 mg/kg in the M14 group (p < 0.05). The percentage of time spent into open arms for all groups is represented in Fig. 1. There was a significant effect of drug dose for groups: W [F(3, 28) = 8.51; p < 0.001], M7 [F(3, 34) = 12.57; p < 0.001] and M21 [F(3, 29) = 3.13; p < 0.05], but no significant effects for groups M14 and M28. Post hoc analysis showed a significant increase in the time spent into open arms after the treatment with 1, 2 and 4 mg/kg of DZ in W group (p < 0.05). However, post hoc analysis showed significant increases in the time spent into open arms only after treatments with 2 and 4 mg/kg of DZ in the M7 group. The closed-arm entries for all groups are represented in Fig. 2. There was a significant effect of dose drug only on M7 [F(3,
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Fig. 1. Effect of DZ on the number of entries (top) and duration of time spent (bottom) in the open arms of the elevated plus-maze by well-nourished (W) and malnourished (M7, M14, M21 and M28) animals, in the doses 0, 1, 2 and 4 mg. Data are reported as mean ± S.E.M. percent of total number of entries or total time for 6–10 rats in each group. *p < 0.05 compared to dose of 0 mg (saline) in the same group (Newman–Keuls test).
34) = 5.15; p < 0.05] and M14 [F(3, 32) = 4,44; p < 0.05] groups. In the M7 group the post hoc analysis showed a significant decrease in the closed-arm entries only after the treatment with 4 mg/kg of DZ (p < 0.05). There were no significant effects on W, M21 and M28 groups. The attempts to enter open arms for all groups are represented in Fig. 2. There was a significant effect of dose drug on W [F(3, 28) = 3.64; p < 0.05], M7 [F(3, 34) = 11.56; p < 0.001] and M14 [F(3, 32) = 3.55; p < 0.05] groups. Post hoc analysis showed a significant decrease of attempts to enter open arms after the treatment with 2 and 4 mg/kg of DZ for M7 (p < 0.05) and after the treatment with 4 mg/kg for M14 (p < 0.05). There were no significant effects on W, M21 and M28 groups. The latencies of first open-arm entry for all groups are represented in Fig. 2. There was a significant effect of drug dose only on W [F(3, 28) = 6.19; p < 0.05]. Post hoc analysis showed a significant decrease in the latency of first open-arm entry after treatment with 1, 2 and 4 mg/kg (p < 0.05). There were no significant effects on M7, M14, M21 and M28 groups. The frequencies of head-dips for all groups are represented in Fig. 3. There was a significant effect of drug dose on W [F(3, 28) = 4.85; p < 0.05], M7 [F(3, 34) = 6.07; p < 0.05], M14 [F(3, 32) = 4.65; p < 0.05] and M21 [F(3, 29) = 3.65; p < 0.05]. Post hoc analysis showed a significant increase in the frequency of head-dips after 2 and 4 mg/kg of DZ on W and M21 groups and after 1, 2 and 4 mg/kg on M7 and M14 groups. There was no significant effect on M28 group. The frequencies of rearings for all groups are represented in Fig. 3. There was a significant effect of drug dose on M7 [F(3, 34) = 6.36; p < 0.05], M14 [F(3, 32) = 4,20; p < 0.05] and M21 [F(3, 29) = 3.78; p < 0.05] groups. Post hoc analysis showed a
Fig. 2. Effect of DZ on the number of closed-arm entries (top), attempts to enter into open arms (middle) and latencies for the first open-arm entry (bottom) by well-nourished (W) and malnourished (M7, M14, M21 and M28) animals, in the doses 0, 1, 2 and 4 mg. Data are reported as mean ± S.E.M. percent of total number of entries or total time for 6–10 rats in each group. *p < 0.05 compared to dose of 0 mg (saline) in the same group (Newman–Keuls test).
significant decrease in the frequency of rears after treatments with 4 mg/kg of DZ on M7 and M14 groups (p < 0.05), but significant decreases after 1, 2 and 4 mg/kg on M21 group (p < 0.05). There were no significant effects on W and M28 groups. 4. Discussion The present results showed that early postnatal protein malnutrition significantly reduced the body weight of the animals, as previously reported by our [20,21,36,39] and other groups [9,19,33,35], even when imposed for short periods early in life. Short-term early postnatal malnutrition also produced a lower anxiety level according to the EPM test, as indicated by high frequencies of entries and duration of time spent in the open arms, high frequencies of head-dips and low latencies to first open-arm entry. The lower anxiety of animals malnourished for long periods has been previously described in both prenatal [4,6] and postnatal protein malnutrition [1,3,39], suggesting that early life nutritional deficits change the responsiveness of rats in a pain-free animal model of anxiety later in adult life. This result cannot be attributed to changes produced by malnutrition in locomotor or exploratory activities since the frequency
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[40] and (c) the response to stressful stimuli possibly through changes in the GABAergic and/or serotonergic systems [28]. In accordance with these results, it can be concluded that early protein malnutrition, even imposed for short periods, altered the behavior of the animals in the EPM test, suggesting that the longer the exposition to the deficient diet, the lower the anxiety and/or higher the impulsiveness of the animals. The proteindeficient diet, imposed to the animals for short periods, seems to be enough to produce long lasting anxiolytic-like effects very similar to those presented by procedures in which the animals are exposed to protein-deficient diet for long periods. Acknowledgements
Fig. 3. Effect of DZ on the number of head-dips (top) and rears (bottom) by well-nourished (W) and malnourished (M7, M14, M21 and M28) animals, in the doses 0, 1, 2 and 4 mg. Data are reported as mean ± S.E.M. percent of total number of entries or total time for 6–10 rats in each group. *p < 0.05 compared to dose of 0 mg (saline) in the same group (Newman–Keuls test).
of closed-arms entries and rearings was not affected by diet conditions. The majority of data in literature regards protein malnutrition imposed for long periods (malnutrition until 49 days of life, and nutritional rehabilitation until day 70). Data of the present study also add the information that even short-term protein malnutrition after birth is sufficient to produce a consistent reduction in the behaviors indicative of anxiety in the elevated plus-maze. Moreover, short-term malnutrition showed, in the present study, an anxiolytic-like effect comparable to treatments with the classic bendizodiazepine diazepam. This anxiolytic-like effect suggests that short-term protein malnutrition may affect neural and/or neurochemical systems believed to underlie behavioral response in anxiogenic situations, such limbic structures as hippocampus and amygdala [10,27]. It has been reported that early malnutrition changes the structure [22,24,42] and the neurochemistry [17,28] of the hippocampus. Regarding the structural effects it has been showed that malnutrition decreased the mossy fiber system of the hippocampal formation [22], decreased the number of neurons in the principal cell layers of the adult rat hippocampal formation [24] and decreased GABAA receptor alpha1 and beta2 mRNA levels of hippocampal formation [42]. In addition, it has been shown that malnutrition changes both the GABAergic and the serotonergic systems of the hippocampal formation [17,28]. Thus, those structural and neurochemical changes in the GABAergic hippocampal system may be underlying the anxiolytic-like effects observed in previously malnourished animals tested in the elevated plus-maze. Previous reported data also showed that prenatal protein malnutrition alters: (a) the amnestic response to chlordiazepoxide in the Morris water maze [45], (b) the sensitivity to the stimulus properties of the chlordiazepoxide in a discriminative task
This work was supported by research grants from FAPESPBrazil (02/05674-5) and CNPq-Brazil (470415/2003-7 and 411029/2003-7). ALFS and ASH were the recipients of a research scholarship from FAPESP-Brazil (00/06330-2 and 00/01675-1, respectively). SSA was a recipient of a research scholarship from CNPq-Brazil (351507/1996-5). The authors thank Dalmo C.P. Nicola for technical assistance. References [1] Almeida SS, De Oliveira LM, Graeff FG. Early life protein malnutrition changes exploration of the elevated plus-maze and reactivity to anxiolytics. Psychopharmacology (Berl) 1991;103(4):513–8. [2] Almeida SS, Garcia RA, Cibien MMR, De Ara´ujo M, Moreira GMS, De Oliveira LM. The ontogeny of exploratory behaviors in early malnourished rats exposed to elevated plus-maze. Psychobiology 1994;22(4):283–8. [3] Almeida SS, Garcia RA, De Oliveira LM. Effects of early protein malnutrition on repeated testing upon locomotor and exploratory behaviors in the elevated plus-maze. Physiol Behav 1993;54(4):749–52. [4] Almeida SS, Tonkiss J, Galler JR. Prenatal protein malnutrition affects avoidance but not escape behavior in the elevated T-maze test. Physiol Behav 1996;60(1):191–5. [5] Almeida SS, Tonkiss J, Galler JR. Malnutrition and reactivity to drugs acting in the central nervous system. Neurosci Biobehav Rev 1996;20(3):389–402. [6] Almeida SS, Tonkiss J, Galler JR. Prenatal protein malnutrition behavior of female rats in elevated plus-maze. Physiol Behav 1996;60(2):675–80. [7] Bedi KS. Effects of undernutrition during early life on granule cell numbers in the rat dentate gyrus. J Comp Neurol 1991;311(3):425–33. [8] Bedi KS. Lasting neuroanatomical changes following undernutrition during early life. In: Dobbing J, editor. Early malnutrition and later achievement. London: Academic Press; 1987. p. 2–49. [9] Bedi KS. Undernutrition of rats during early life does not affect the number of cortical neurons. J Comp Neurol 1994;342(4):596–602. [10] Bremner JD. Brain imaging in anxiety disorders. Expert Rev Neurother 2004;4(2):275–84. [11] Brioni JD, C´ordoba N, Orsingher OA. Decreased reactivity to the anticonflict effect of diazepam in perinatally undernourished rats. Behav Brain Res 1989;34(1/2):159–62. [12] Celedon JM, Colombo M. Effects of chlordiazepoxide on maze performance of rats subjected to undernutrition in early life. Psychopharmacology 1979;63(1):29–32. [13] Cole JC, Rodgers RJ. Ethological evaluation of the effects of acute and chronic buspirone treatment in the murine elevated plus-maze: comparison with haloperidol. Psychopharmacology (Berl) 1994;114:288–96. [14] Cruz AP, Frei F, Graeff FG. Ethopharmacological analysis of rat behavior on the elevated plus-maze. Pharmacol Biochem Behav 1994;49(1): 171–6.
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Research report
Anxiolytic-like effects of short-term postnatal protein malnutrition in the elevated plus-maze test Ana Laura Franc¸olin-Silva a , Andr´ea da Silva Hernandes a , Marisa Tomoe Hebihara Fukuda b , Camila Tavares Valadares a , Sebasti˜ao Sousa Almeida a,∗ a
Laboratory of Nutrition and Behavior, University of S˜ao Paulo, Avenida dos Bandeirantes, 3900, 14040-901 Ribeir˜ao Preto, SP, Brazil b Department of Ophthalmology, Otorhinolaryngology and Head and Neck Surgery, School of Medicine of Ribeir˜ ao Preto, University of S˜ao Paulo, Avenida dos Bandeirantes, 3900, 14049-900 Ribeir˜ao Preto, SP, Brazil Received 27 April 2006; received in revised form 29 June 2006; accepted 30 June 2006
Abstract Given that protein malnutrition induces structural, neurochemical and functional changes in the CNS, the present study aimed to investigate the effects of short-term early protein malnutrition on the behavior and reactivity to diazepam (DZ) in the elevated plus-maze test (EPM). Male Wistar rats (n = 176) from well-nourished (16%-protein) or malnourished litters (6%-protein) were distributed in five different groups: W (wellnourished), M7 (malnourished for 7 days), M14 (malnourished for 14 days), M21 (malnourished for 21 days) and M28 (malnourished for 28 days) since birthday. EPM results showed that the longer the exposition to the deficient diet, the lower the anxiety of malnourished animals, a result similar to that produced by the treatment with DZ. This anxiolytic-like effect suggested that short-term malnutrition may affect neural and/or neurochemical systems believed to underlie behavioral expression in anxiogenic experimental situations. © 2006 Elsevier B.V. All rights reserved. Keywords: Short-term postnatal protein malnutrition; Anxiety; Elevated plus-maze; Diazepam; Rats
1. Introduction Protein or protein-calorie malnutrition imposed early in life is a well-known environmental factor producing brain alterations comprising morphological, neurochemical, neurophysiological, and functional aspects [5,7,8,18,29–31,44]. Moreover, it is also well known that protein malnutrition during the 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 light–dark transition [11,39], elevated plus-maze (EPM) [2,6] and elevated T-maze [4,23] showed behavioral alterations indicative of lower anxiety and/or higher impulsiveness. Although there is substantive information about the deleterious effects of short-term postnatal malnutrition on physical and motor development in rats [43,41], studies investigating behavioral effects of short-term malnutrition in the beginning of life are scarce in the literature. Thus, a substan-
∗
Corresponding author. Tel.: +55 16 36023663; fax: +55 16 36335015. E-mail address: [email protected] (S.S. Almeida).
0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.06.042
tive portion of the studies in this area uses prenatal malnutrition with nutritional recovery at birth [29–32], contrasting with few works using the procedure of short-term postnatal protein malnutrition. The relevance attributed to studies of short-term postnatal protein malnutrition is due to two main reasons. First, there is a theoretical importance to know if short-term malnutrition can affect brain development and behavior of rats. Second, several early life medical intercurrences during child development such as diarrhoea, infections and cystic fribrosis may expose children to short periods of malnutrition resulting body and brain injuries, as well as, impairments of cognitive function. It has been shown that short-term malnutrition affects homeorienting behaviors [15]; maternal behavior [16,26]; and learning in the Hebb–Williams maze [12]. However, the effects of short-term malnutrition in experimental models of anxiety as well as in the reactivity of malnourished animals to anxiolytic drugs on these models [2,39] are not clear. It is hypothesized that if even short-term postnatal protein malnutrition produces a lower anxiety in the elevated plus-maze, then the behaviors believed to express anxiety in this model should be modified
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in malnourished animals in a similar way as that induced by treatment with anxiolytic doses of diazepam (DZ). Thus, the objective of the present study was to investigate the similarities between the effects of short-term early protein malnutrition and the anxiolytic effects of DZ in animals exposed to the elevated plus-maze model. 2. Methods and materials
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2.3. Statistical analysis Body weight data are reported as means ± S.E.M. and were analyzed by oneway analysis of variance (ANOVA) on the day 70. Behavioral data are reported as means ± S.E.M. A two-way ANOVA (diet condition and drug condition) was initially conducted showing significant effects of nutrition and drug conditions. Thus, we conducted a one-way (dose of the drug) ANOVA for each diet condition to investigate the reactivity to the anxiolytic effects of the drug on each group. When appropriate, post hoc comparisons were made using the Newman–Keuls test. The level of significance was set at 0.05.
2.1. Subjects
3. Results Male Wistar rats from the animal colony of Ribeir˜ao Preto Campus, University of S˜ao Paulo were used. Each litter was culled to six male and two female pups on the day of birth. The dams and the pups were housed in transparent plastic cages (40 cm × 30 cm × 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 [2]. 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. The regular protein diet contained approximately 20% casein (16% protein), 60.8% corn starch and the 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 started receiving a regular lab chow diet until the end of the experiment and they were maintained on a 12:12-h light/dark cycle (lights on at 6:00 a.m.) with room temperature kept at 23–25 ◦ C, and with free access to water and food throughout the experiment. The behavioral tests were conducted during the light period (1:00–6:00 p.m.). 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. The rats from well-nourished (16%-protein) or malnourished litters (6%protein) were distributed in five different groups: W (well-nourished, n = 32), M7 (malnourished for 7 days, since day 0, n = 38), M14 (14 days, n = 36), M21 (21 days, n = 33) and M28 (28 days, n = 37). Furthermore, the animals were also subdivided in accordance with the dose of DZ utilized (0, 1, 2 or 4 mg/kg).
2.2. Apparatus and procedure The EPM was made of wood and consisted of two open arms (50 cm × 10 cm) opposite to each other, crossed by two enclosed arms (50 cm × 10 cm × 40 cm), with an open roof [34]. The maze was elevated 50 cm from the ground. Fluorescent ceiling lights (2 × 60 W) provided the only illumination in the experimental room. At 70 days of age, 30 min before being placed in the EPM, the rats were injected intraperitoneally with saline, 1, 2 or 4 mg/kg DZ. The animals were placed individually in the center of the maze, facing an enclosed arm and allowed to explore it 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 analyzed by an experimenter blind to the nutritional treatment, and the following behavioral categories were identified and recorded as previously described [2,3,6,13,14,37,38]: (1) the percent of open-arm entries (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 the first open-arm entry (latency to enter an open arm timed from the start of the test), (6) rearing (rising on the hind paws), (7) head-dipping (scanning over the sides of the maze toward the floor) and (8) stretch-attend posture (forward elongation of head and shoulders followed by retraction to the original position). The behavioral categories stretch-attend posture and headdips have been previously described as risk-assessment behaviors [2,37,38]. The rearing category was included as a classical measure of exploration in a novel environment [2,6]. The other behavioral categories have been behaviorally and pharmacologically validated for rats [14,34] and mice [13,25,37,38] as reliable measures of fear/anxiety levels.
3.1. Body weight On day 70, the animals of malnourished groups (M7: 444.04 ± 10.2; M14: 373.92 ± 7.8; M21: 369.45 ± 9.4; M28: 342.02 ± 6.9) weighed less than well-nourished animals (W: 476.09 ± 10.8) as indicated by a significant effect of diet treatment [F(4, 171) = 37.86; p < 0.05]. Post hoc analysis showed significant statistical differences in the weight of W as compared with all the other malnourished groups (p < 0.05). 3.2. Behavioral measures 3.2.1. Nutritional effects ANOVA showed a significant effect of diet condition on the behavioral categories believed to express anxiety in the elevated plus-maze such as percentage of open arm entries [F(4, 156) = 3.00, p < 0.05], percentage of time in open arms [F(4, 156) = 3.10, p < 0.05], head-dips [F(4, 156) = 3.80, p < 0.05]. Post hoc analysis showed that W groups differs significantly from M14, M21 and M28 groups in all behavioral categories (p < 0.05). 3.2.2. Drug effects The percentage of open-arm entries for all groups is represented in Fig. 1. There was a significant effect of drug dose for groups: W [F(3, 28) = 10.91; p < 0.001], M7 [F(3, 34) = 9.42; p < 0.001] and M14 [F(3, 32) = 4.25; p < 0,05], but no significant effects for groups M21 and M28. Post hoc analysis showed a significant increase in the open-arm entries after the treatment with 1, 2 and 4 mg/kg of DZ in W group (p < 0.05). However, post hoc analysis showed significant increases in the open-arm entries only after treatments with 2 and 4 mg/kg of DZ in the M7 group and with 4 mg/kg in the M14 group (p < 0.05). The percentage of time spent into open arms for all groups is represented in Fig. 1. There was a significant effect of drug dose for groups: W [F(3, 28) = 8.51; p < 0.001], M7 [F(3, 34) = 12.57; p < 0.001] and M21 [F(3, 29) = 3.13; p < 0.05], but no significant effects for groups M14 and M28. Post hoc analysis showed a significant increase in the time spent into open arms after the treatment with 1, 2 and 4 mg/kg of DZ in W group (p < 0.05). However, post hoc analysis showed significant increases in the time spent into open arms only after treatments with 2 and 4 mg/kg of DZ in the M7 group. The closed-arm entries for all groups are represented in Fig. 2. There was a significant effect of dose drug only on M7 [F(3,
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Fig. 1. Effect of DZ on the number of entries (top) and duration of time spent (bottom) in the open arms of the elevated plus-maze by well-nourished (W) and malnourished (M7, M14, M21 and M28) animals, in the doses 0, 1, 2 and 4 mg. Data are reported as mean ± S.E.M. percent of total number of entries or total time for 6–10 rats in each group. *p < 0.05 compared to dose of 0 mg (saline) in the same group (Newman–Keuls test).
34) = 5.15; p < 0.05] and M14 [F(3, 32) = 4,44; p < 0.05] groups. In the M7 group the post hoc analysis showed a significant decrease in the closed-arm entries only after the treatment with 4 mg/kg of DZ (p < 0.05). There were no significant effects on W, M21 and M28 groups. The attempts to enter open arms for all groups are represented in Fig. 2. There was a significant effect of dose drug on W [F(3, 28) = 3.64; p < 0.05], M7 [F(3, 34) = 11.56; p < 0.001] and M14 [F(3, 32) = 3.55; p < 0.05] groups. Post hoc analysis showed a significant decrease of attempts to enter open arms after the treatment with 2 and 4 mg/kg of DZ for M7 (p < 0.05) and after the treatment with 4 mg/kg for M14 (p < 0.05). There were no significant effects on W, M21 and M28 groups. The latencies of first open-arm entry for all groups are represented in Fig. 2. There was a significant effect of drug dose only on W [F(3, 28) = 6.19; p < 0.05]. Post hoc analysis showed a significant decrease in the latency of first open-arm entry after treatment with 1, 2 and 4 mg/kg (p < 0.05). There were no significant effects on M7, M14, M21 and M28 groups. The frequencies of head-dips for all groups are represented in Fig. 3. There was a significant effect of drug dose on W [F(3, 28) = 4.85; p < 0.05], M7 [F(3, 34) = 6.07; p < 0.05], M14 [F(3, 32) = 4.65; p < 0.05] and M21 [F(3, 29) = 3.65; p < 0.05]. Post hoc analysis showed a significant increase in the frequency of head-dips after 2 and 4 mg/kg of DZ on W and M21 groups and after 1, 2 and 4 mg/kg on M7 and M14 groups. There was no significant effect on M28 group. The frequencies of rearings for all groups are represented in Fig. 3. There was a significant effect of drug dose on M7 [F(3, 34) = 6.36; p < 0.05], M14 [F(3, 32) = 4,20; p < 0.05] and M21 [F(3, 29) = 3.78; p < 0.05] groups. Post hoc analysis showed a
Fig. 2. Effect of DZ on the number of closed-arm entries (top), attempts to enter into open arms (middle) and latencies for the first open-arm entry (bottom) by well-nourished (W) and malnourished (M7, M14, M21 and M28) animals, in the doses 0, 1, 2 and 4 mg. Data are reported as mean ± S.E.M. percent of total number of entries or total time for 6–10 rats in each group. *p < 0.05 compared to dose of 0 mg (saline) in the same group (Newman–Keuls test).
significant decrease in the frequency of rears after treatments with 4 mg/kg of DZ on M7 and M14 groups (p < 0.05), but significant decreases after 1, 2 and 4 mg/kg on M21 group (p < 0.05). There were no significant effects on W and M28 groups. 4. Discussion The present results showed that early postnatal protein malnutrition significantly reduced the body weight of the animals, as previously reported by our [20,21,36,39] and other groups [9,19,33,35], even when imposed for short periods early in life. Short-term early postnatal malnutrition also produced a lower anxiety level according to the EPM test, as indicated by high frequencies of entries and duration of time spent in the open arms, high frequencies of head-dips and low latencies to first open-arm entry. The lower anxiety of animals malnourished for long periods has been previously described in both prenatal [4,6] and postnatal protein malnutrition [1,3,39], suggesting that early life nutritional deficits change the responsiveness of rats in a pain-free animal model of anxiety later in adult life. This result cannot be attributed to changes produced by malnutrition in locomotor or exploratory activities since the frequency
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[40] and (c) the response to stressful stimuli possibly through changes in the GABAergic and/or serotonergic systems [28]. In accordance with these results, it can be concluded that early protein malnutrition, even imposed for short periods, altered the behavior of the animals in the EPM test, suggesting that the longer the exposition to the deficient diet, the lower the anxiety and/or higher the impulsiveness of the animals. The proteindeficient diet, imposed to the animals for short periods, seems to be enough to produce long lasting anxiolytic-like effects very similar to those presented by procedures in which the animals are exposed to protein-deficient diet for long periods. Acknowledgements
Fig. 3. Effect of DZ on the number of head-dips (top) and rears (bottom) by well-nourished (W) and malnourished (M7, M14, M21 and M28) animals, in the doses 0, 1, 2 and 4 mg. Data are reported as mean ± S.E.M. percent of total number of entries or total time for 6–10 rats in each group. *p < 0.05 compared to dose of 0 mg (saline) in the same group (Newman–Keuls test).
of closed-arms entries and rearings was not affected by diet conditions. The majority of data in literature regards protein malnutrition imposed for long periods (malnutrition until 49 days of life, and nutritional rehabilitation until day 70). Data of the present study also add the information that even short-term protein malnutrition after birth is sufficient to produce a consistent reduction in the behaviors indicative of anxiety in the elevated plus-maze. Moreover, short-term malnutrition showed, in the present study, an anxiolytic-like effect comparable to treatments with the classic bendizodiazepine diazepam. This anxiolytic-like effect suggests that short-term protein malnutrition may affect neural and/or neurochemical systems believed to underlie behavioral response in anxiogenic situations, such limbic structures as hippocampus and amygdala [10,27]. It has been reported that early malnutrition changes the structure [22,24,42] and the neurochemistry [17,28] of the hippocampus. Regarding the structural effects it has been showed that malnutrition decreased the mossy fiber system of the hippocampal formation [22], decreased the number of neurons in the principal cell layers of the adult rat hippocampal formation [24] and decreased GABAA receptor alpha1 and beta2 mRNA levels of hippocampal formation [42]. In addition, it has been shown that malnutrition changes both the GABAergic and the serotonergic systems of the hippocampal formation [17,28]. Thus, those structural and neurochemical changes in the GABAergic hippocampal system may be underlying the anxiolytic-like effects observed in previously malnourished animals tested in the elevated plus-maze. Previous reported data also showed that prenatal protein malnutrition alters: (a) the amnestic response to chlordiazepoxide in the Morris water maze [45], (b) the sensitivity to the stimulus properties of the chlordiazepoxide in a discriminative task
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