- Email: [email protected]
Effects of postweaning undernutrition on exploratory behavior, memory and sensory reactivity in rats: Implication of the dopaminergic system
Physiology & Behavior 86 (2005) 195 – 202
Effects of postweaning undernutrition on exploratory behavior, memory and sensory reactivity in rats: Implication of the dopaminergic system Meryem Alamy a, Mohammed Errami b, Khalid Taghzouti c, Fatima Saddiki-Traki c, Wail A. Bengelloun c,* a
Faculty of Science, Casablanca (Ain Chock), Morocco b Faculty of Science, Rabat, Morocco c Faculty of Science, Tetouan, Morocco
Received 30 March 2005; received in revised form 5 July 2005; accepted 12 July 2005
Abstract The effects of early undernutrition on behavior and brain biochemistry were examined in rats. At weaning, rats were provided either an ad lib diet (control group) or maintained at 80% of the weight of their control littermates (undernourished group). Three weeks into the diet they were tested in an open field. After 6 weeks of diet, HPLC analyses were conducted on sample brains from each group to assess levels of dopamine and metabolites, respectively dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum. At seven weeks of diet, remaining rats were trained in an 8-arm radial maze, and a retention test conducted 72 h after attaining the learning criterion. At fourteen weeks of diet, sensory reactivity was measured by tail-immersion in a water bath maintained at constant temperature 50 T 1 -C. Undernourished rats exhibited hyperactivity and increased exploratory behavior in the open field, as well as increased sensory reactivity in the tail flick test. In the radial maze, however, undernourished rats did not differ from controls in either learning or retention. Haloperidol (i. p. injection) impaired retention by control but not undernourished animals. HPLC analyses showed an increase in dopamine turnover in the striatum of undernourished rats. Our results suggest that, unlike its effects when induced immediately at birth or in adulthood, undernutrition at weaning does not appear to influence learning and retention but induced an hyperactivity and alterations in striatal DA turnover which was associated with a decrease in responsiveness to i. p. haloperidol injection. D 2005 Elsevier Inc. All rights reserved. Keywords: Undernutrition; Open field activity; Radial maze; Learning; Memory; Dopamine; Rat
1. Introduction Undernutrition is a form of malnutrition where all nutriments required by the species are available in the diet but the amounts are insufficient [1]. In Africa, the weaning period seems to be especially critical because access to maternal nutriments ceases at this time [2]. Yet, few investigators have addressed malnutrition at weaning. In animal models, the behavioral effects of malnutrition induced either prenatally [3], immediately postnatally [4], or in adulthood [5] have been largely described (for a * Corresponding author. Department of Biology, Faculty of Science, BP 1014, Rabat, Morocco. Tel.: +212 6 116 6277; fax: +212 3 777 4261. E-mail address: [email protected] (W.A. Bengelloun). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2005.07.008
review, see [6]). Furthermore, prenatal, postnatal and adult malnutrition also induce changes in brain cholinergic, noradrenergic, dopaminergic, serotoninergic and GABAergic systems as well as changes in sensitivity to psychoactive drugs (for review see [7]). Prenatal protein malnutrition was reported to impair acquisition of Differential Reinforcement of Low rates of responding (DRL) performance [8] and visual discrimination learning [9] in adulthood. It has been demonstrated that malnourished rats exhibit deficits in memory tests (e.g., T-maze [10]), but not in spatial alternation [8] or in Morris water maze [11]. Prenatal malnutrition has also been shown to influence the development of the hippocampal formation [12 –15] as well as DA and DOPAC levels in both hypothalamus and hippocampus [16].
196
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
Impairments of learning and spatial memory were described by some authors after early postnatal protein malnutrition in radial maze [17] and in distal but not in proximal cue versions of the Morris water maze [18,19]. A decrease in granule cell numbers in the dentate gyrus was reported following early postnatal malnutrition [20]. Similarly, the number of striatal dopamine receptors were decreased in animals undernourished during suckling [21], as was the haloperidol-induced catatonic response [22]. When prolonged protein malnutrition was instituted both pre and postnatally in dam and offspring, biochemical findings were contradictory. While Almeida et al. [7] reported a lower reactivity of malnourished rats to drugs mediated through the catecholaminergic system, Brioni et al. [4] showed that repeated amphetamine (AMPH) treatment led to sensitization, as demonstrated by increases in both locomotor activity and binding of dopaminergic receptors in the striatum. Moreover, nutritional state did not however influence the behavioral stereotypy normally produced by AMPH activation of catecholaminergic function [23]. Protein deprivation in adult rats led to reduced emotional reactivity and impaired Morris maze performance: malnourished rats were slower to learn an efficient search strategy [5]. The behavioral deficits induced by malnutrition in adulthood were associated in large measure with structural abnormalities in the hippocampal formation including a marked loss of hippocampal neurons [5,24] and synapses [5]. Further, exposure to low-protein diet in adulthood produced changes in hippocampal cholinergic and GABAergic systems [25] in rats and an enhanced locomotor response to d-amphetamine in mice [26]. The present study was designed to determine the behavioral effects of undernutrition (protein-calorie malnutrition) induced at weaning, and its relationship to striatal neurochemical systems. Both the age at which malnutrition occurs and its duration are critical factors in determining its effects on the brain (cf. [1]). Few previous studies have induced malnutrition or undernutrition at weaning in spite of the fact that in humans, it has been shown to represent a stage of particular vulnerability [2]. Moreover, Ogura et al. [27] have recently reported an impairment in the acquisition of certain behavioral tasks after striatal dopamine depletion. We therefore examined the performance of rats in learning, memory and sensory reactivity paradigms, and assessed DA, DOPAC and HVA concentrations in striatum.
2. Materials and methods 2.1. Animals Wistar rats from the animal colony of Mohammed V University (Rabat, Morocco) were used. After mating and prior to delivery, female rats were caged individually and housed in an animal room with natural light and temper-
ature conditions. During the study, temperature varied between 18 and 24 -C with humidity at 45 to 55%. The light / dark cycle was around 14 / 12. Litters were culled to a maximum of 8 pups. At 10 days of age, maternal retrieval tests were conducted in each litter, and determined that none of the pups were being rejected by the mother (and therefore malnourished). Weaning took place at 25 days of age: pups were sexed and only those litters containing a minimum of two males were retained. Thus, 46 male pups were used (23 paired littermates) in this experiment. They were weighed, and same weight male littermates were identified and paired. Depending on litter composition, 2, 4 or 6 male pups were used from the same litter in the experiment. Animals were placed in individual cages; the control rat had free access to food and water throughout the experimental period (except during maze testing) while the paired undernourished rat was maintained at 80% of the control littermate’s weight. For this purpose all subjects were weighed daily and fed a ration sufficient to maintain their undernourished state. This meant that the quantity of food consumed was reduced by about 50% during the first week and by 40% on subsequent weeks. The commercial (Cicalim, Casablanca) diet contained protein (17%), fat (3.2%), cellulose (5.2%), minerals (6%), vitamins A, E, D3, as well as calcium and phosphate, with 68.6% carbohydrates. Water was available to all animals ad lib. All procedures in this experiment conformed to international guidelines on the ethical use of animals and every effort was made to minimize the number of animals used. 2.2. General protocol Three weeks into the diet, all animals were tested for five minutes in an open field on 3 consecutive days in order to evaluate their levels of activity, emotional state and capacity to habituate to novel environments. At the end of the sixth week of diet, 16 rats (8 control rats with their 8 undernourished littermate) were randomly selected for sacrifice, and their brains assayed by high-performance liquid chromatography (HPLC) for striatal dopamine and metabolites. One week later (week 7 of diet), the remaining animals started daily testing in a radial 8-arm maze to assess the effects of post-weaning undernutrition on learning ability. 72 h after attaining the acquisition criterion, retention was evaluated following an i. p. injection of either Haloperidol (dopamine antagonist) or saline. At the 14th week of diet, rats were tested in a tail-flick test to assess temperature reactivity. Of the 46 rats tested in the open field, 16 were sacrificed for HPLC. Subsequently, one animal died, leading us to eliminate its littermate from the study. 28 animals (14 undernourished and 14 controls) were thus tested in the radial arm maze, and later contributed to the temperature reactivity data. All experiments were performed at the same time of day between 1100 and 1600 h.
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
2.3. Open field The open field was a black wood arena measuring 75 45 35 cm high, previously described [28]. The floor of the apparatus was divided into 15 squares (15 15 cm). At the beginning of each trial the rat was placed in a corner square facing the wall. Each rat was tested during a 5-min session, on 3 consecutive days. Behavior was recorded by video system to assess activity (number of crossing between squares), and rearing. 2.4. Neurochemical assays for DOPAC, dopamine and HVA Rats (undernourished and paired control littermates) were sacrificed at 1100 h by decapitation and brains were immediately removed, the striatum dissected over ice. The extraction procedure was a modification of a method previously published [29]. Briefly, tissue samples were homogenized immediately after dissection with weak sonication in perchloric acid 0.1 M (1 / 10; W/V), the crude supernatant was diluted in the mobile phase after centrifugation (10 min; 15 000 g at 4-). The homogenate was filtered (Whatman; 0.22 Am) and 20 Al injected in a reversed phase chromatographic system. The HPLC system consisted of a Shimadzu L C-6A pump equipped with a 20 Al (Rheodyne 7125) injection loop. The Shimadzu L-ECD-6A amperometric detector was equipped with a 4 Al electrochemical cell fitted with a glass carbon electrode set at 0.7 V with respect to a Silver – Silver chloride reference electrode. Separation of monoamines and metabolites was achieved using a 150 4.6 mm reversed phase analytical column (ODS 5 Am, Varian), the guard column was a Varian (1 4 mm). The mobile phase consisted of sodium acetate (0.1M), Sodium Octyl Sulfate (10 5 M), EDTA (5.6 10 4) and methanol (8%). The pH was adjusted to 4.5 by acetic acid. The flow rate was set at 1 ml/min and the sensitivity was set between 50 and 100 pA full-scale detection. This method leads to a complete separation of dopamine and metabolites in less than 25 min. Identification of the compounds and quantification were performed against injections of external standard solutions containing 100 pg of each product and 3.4 dihydroxybenzylamine (DHBA) as an internal standard at a concentration of 50 pg using an integrator Shimadzu C-4A. Standard solutions were prepared daily by dilution of stock solutions. The sensitivity of the system was found to be about 6 pg for DA, DOPAC and HVA. Monoamine levels were expressed in terms of pg per mg of tissue. 2.5. Radial maze Radial maze tests started on week 7 of diet. The radial maze consisted of a central platform (35.5 cm diameter), with eight arms of 44 cm length and 25 cm width. The flat maze was elevated 75 cm off the floor, without enclosing
197
walls. A small plastic cup was fixed at the end of each of the eight arms to enclose a 0,1 mg reinforcement food pellet (Cicalim, Casablanca). The maze was placed in a softly lighted room with the same external cues throughout the experiment, and maze arms were numbered from 1 to 8 for identification. Animals in both groups were subjected to daily food restriction starting on the third day of week 6 of diet, until they completed testing in the maze. Control animals were restricted to 90% of their body weight. Body weights of undernourished animals were consequently reduced to maintain 80% of littermate control weight. This reduction was meant to motivate maze learning behavior. 2.6. Acquisition Rats were initially provided with a 10-min habituation session on each of three consecutive days, with a pellet placed in the cup at the end of each arm. At the start of each session the subject was placed in the middle of the central platform. On the fourth day, the acquisition phase began. The subject was placed in the center of the radial maze facing arm 1 and allowed a maximum of 15 min to find and eat the 8 pellets. During this and the following daily sessions, time spent in the maze and number of errors (entering an arm in which the pellet was consumed earlier during the session or entering an arm with all 4 paws and not consuming the food pellet) were recorded. Acquisition was considered complete when not more than 1 error was committed on two consecutive days (the criterion). We recorded the number of days to attain criterion, trial duration, total number of errors in the first day as well as the total number of errors during the entire acquisition phase. 2.7. Retention 72 h after acquisition, paired littermates were randomly assigned to receive an i. p. injection of 0.5 ml of either haloperidol (0.2 mg/kg of Haldol, Maphar, Casablanca) [30] or saline solution 60 min prior to undergoing a retention test in the maze. 2.8. Tail flick At the end of the experiment (14 weeks of diet), subjects were tested in a tail flick procedure. During each trial the rat was held vertically, its tail immersed (approximately 2 cm) in water at 50 -C. Reaction time (latency to withdrawal of tail from the water) was recorded during trials conducted at 11 am and 4 pm on three consecutive days. 2.9. Statistical analysis Unweighted means analyses of variance (ANOVA) were used to evaluate data from the open field (OF)
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
and tail flick (TF) tests. Diet group was a between factor, while Daily sessions were within factors. One way ANOVA analyses were used to treat all other data. Statistical tests were performed using SPSS software (Chicago, IL). Differences were considered significant at p < 0.05 level. Where the multivariate analysis indicated a significant effect, univariate analyses were applied. Body weights are presented as mean T standard deviation. Other data are presented in the figures as mean T standard error of the mean.
Striatal neurochemistry A. Striatal Dopamine and metabolites 1200 1000
pg/mg
198
C U
*
800 600
*
400 200 0 DA
DOPAC
HVA
3. Results
B. Striatal DOPAC/DA and HVA/DA ratios 3.1. Body weight
5
Fig. 1 shows the evolution of body weight for control and undernourished (maintained at 80% of control littermates) animals during the 6 weeks of diet. During week 1, mean body weight increased by 13 T 1.7 g in the control group and 5.28 T 0.30 g in the undernourished group. During the next 5 weeks, the average body weight increases were respectively 40.77 T 3.97 g/wk and 30.31 T 4.99 g/wk. At 14 weeks, the undernourished rats were still at 80% of control body weight.
4
3.2. Striatal neurochemistry Fig. 2A shows a significant decrease ( 22%) in DA in striatum of undernourished rats as compared to controls ( F(1,16) = 6.92, p = 0.019). Additionally, there is an increase in DOPAC (+ 61%, F(1,16) = 10.17, p = 0.0065), with a resulting increase of about 107% (Fig. 2B) in the DOPAC/DA ratio in undernourished rats ( F(1,16) = 8.59; p = 0.01). There was a tendency towards an increase in HVA levels, although this did not attain statistical significance ( p = 0.08). The increase in HVA / DA ratio did however reach significance ( F(1,16) = 7.64; p = 0.015). Body weight during six weeks of undernutrition 350 300
Control
weight (g)
Undernourished
250 200 150
C U
* 3
*
2 1 0 DOPAC/DA
HVA/DA
Fig. 2. Striatal neurochemistry. A. Tissue concentration of striatal DA, DOPAC and HVA in control (C) and undernourished (U) rats. B. Striatal DOPAC/DA and HVA/DA ratios. HPLC analysis was performed on brains of 16 randomly selected rats (8 control and 8 undernourished rats).
3.3. Behavior 3.3.1. Open field Analysis of crossing activity yielded significant main effects for Nutrition ( F(1,44) = 4.828, p = 0.033) and Day ( F(2,43) = 20.335, p = 0.0001). Total crossing activity during the three days of testing (Fig. 3A) was significantly higher in Undernourished animals, but this effect was primarily due to the difference in behavior on Day 1 ( F(1,27) = 5.13, p = 0.028). In fact, total crossing activity of control as well as undernourished rats was significantly higher on day 1 compared to days 2 or 3 (Fig. 3A). Undernutrition led to a significant overall increase in rearing activity ( F(1,44) = 13.405; p = 0.001), mainly due to results obtained on days 1 ( F(1,45) = 12.74, p = 0.0008) and 3 ( F(1,45) = 6.027, p = 0.01). A significant main effect was also found for Day ( F(2,43) = 6.765, p = 0.003); rearing activity decreased significantly on days 2 and 3 (Fig. 3B) relative to day 1 in control and in malnourished rats.
100
3.4. Radial maze
50 0 Weaning
WK2
WK4
WK6
Fig. 1. Mean body weight (TSD) of control (n = 23) and undernourished rats (n = 23) during 6 weeks of undernutrition.
No significant differences were found in the number of days to attain criterion when undernourished and control animals were compared ( F(1,25) = 0.2967, p = 0.59). Session duration also did not appear to be affected by
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
A. Total crossing activity Number
150 C U
* 100 50 0
Day 1
Day 2
Day 3
B. Total rearing activity Number
50 40
C U
*
30
*
20 10 0 Day 1
Day 2
Day 3
Fig. 3. Open field performance of control (C, n = 23) and undernourished (U, n = 23) rats. A. Total crossing activity. B. Total rearing activity. Histograms represent the mean number of squares entered with four paws or stands ups during 3 consecutive daily sessions.
nutritional state ( F(1,25) = 0.0096; p = 0.92). Further, no change was detected between the two groups in terms of either number of errors on the first day ( F(1,25) = 0.81;
Radial arm maze: Retention
p = 0.37) or during the entire acquisition phase ( F(1,25) = 1.37; p = 0.25). During the retention test 72 h later, the performance of saline-injected undernourished and control rats did not differ from their performance during the last day of acquisition, when measured in number of errors or in session duration. Saline also did not differentially influence the performance of undernourished and control animals during retention. Haloperidol injection led to an increase in mean number of errors (5.16 vs. 0.33: F(1,11) = 8.96, p = 0.01) and in mean session duration (5395 vs. 1398 s: ( F (1,11) = 6.03, p = 0.03) in control animals during retention in comparison to their performance during the last day of acquisition. This effect was not observed in undernourished animals. During the retention session (Fig. 4), undernourished haloperidol-injected animals exhibited fewer errors ( F (1,11) = 5.43, p = 0.04) and shorter session duration ( F (1,11) = 6.60, p = 0.02) than control animals receiving the same injection. In well-nourished controls, haloperidol injection led to an increase in mean number of errors ( F(1,11) = 6.57, p = 0.02) and in session duration ( F
Tail flick Day 1 Latency (s)
Open field performance
A. Number of errors 10
*
4 2 0 C
*
U
B. Session duration
400 200 0 C
*
*
14 12 10 8 6 4 2 0
4 p.m.
Day 2 C U
* *
U
14 12 10 8 6 4 2 0
C U
*
*
11 a.m. Fig. 4. Retention performance in control (C, n = 7) and undernourished rats (U, n = 7) after saline or Haloperidol injection. Histograms represent mean number of errors (A) and mean session duration (B).
4 p.m.
Day 3
Saline Haloperidol
600
*
11 a.m.
Latency (s)
Minutes
*
C U
11 a.m.
6
800
14 12 10 8 6 4 2 0
Saline Haloperidol
Latency (s)
Errors
8
199
4 p.m.
Fig. 5. Tail-flick latencies in control (C, n = 14) and undernourished rats (U, n = 14) during test sessions at 11 a.m. and 4 p.m. on 3 consecutive days.
200
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
(1,11) = 6.28, p = 0.03). Again, this effect was not observed in undersnourished rats. 3.5. Tail flick Undernutrition decreased the latency to tail withdrawal from hot water ( F(1,25) = 19.174, p = 0.0001) (Fig. 5). No effect of Day ( p = 0.166) or Time ( p = 0.699) was noted. The only significant interaction was Day Time ( F(2,25) = 5.026, p = 0.015, due to an increase in latency at 4 p.m. relative to 11 am on Day 2.
4. Discussion In the present study, undernutrition induced at weaning resulted in hyperactivity, manifested in the open field by an increase in square crossing and rearing. The increase in crossing activity was described by previous studies after postnatal (at weaning) protein malnutrition [2], chronic food restriction induced at birth [22] and after protein deprivation in adult rats [5]. The latter suggested that the elevated locomotion score (square crossing) at the beginning of the first testing session (day 1) in malnourished rats was due to a decrease in emotional reactivity to novel environments. In the present study, the increased activity observed during the first daily session but not apparent during the later sessions (days 2 and 3) seems to be in line with such an interpretation. Moreover, this seems to be in agreement with the findings of Almeida et al. [10] after prenatal protein malnutrition and Hernandes [31] after postnatal malnutrition. Both suggest that malnutrition results in a reduction in anxiety. Rearing activity of undernourished rats was more elevated throughout all three daily sessions. Our results were therefore in agreement with those obtained with prenatal malnutrition [32] but different from results observed after protein deprivation in adults: Lukoyanov and Andrade [5], found no differences between malnourished and control rats on this measure. Rearing on the hind legs was suggested to include a vertical component of exploration [32], thus indicating that prenatal protein calorie malnutrition as well as post-weaning undernutrition resulted in an increase in exploratory behavior. Finally, the increase in crossing and in rearing activity in malnourished rats may reflect not only a lower anxiety to novel environment [5] but also an active search for food (food seeking), linked to the nutritional treatment [2]. In the tail-flick test undernourished animals exhibited lower latencies to tail withdrawal, suggesting that they were more sensitive to heat than controls. Such changes in sensitivity threshold may be related to the hyperactivity observed in the open field test suggesting the possibility of a state of sensory hyperreactivity following undernutrition. Smart et al. [33] have shown that a mildly painful stimulus (foot-shock) produced greater reactivity in rats previously
undernourished prenatally and immediately postnatally. Our data, coupled with those of Smart et al. [33], suggest that undernourished animals are more reactive to unpleasant or aversive stimuli, irrespective of the age at which nutritional regime is initiated. Such changes in sensory reactivity may underlie the emotional changes suggested by some authors [34]. Further investigations are needed to elucidate these phenomena. Analysis of data from the radial maze showed that undernourished and control rats were similar in rate of acquisition of the task. No differences were observed on any of the behavioral measures. Previous reports using another maze (Morris water maze) yielded differing results, depending on the age at which malnutrition was introduced. Whereas Tonkiss et al. [11] found that prenatal malnutrition affected neither proximal nor distal cue navigation, other studies found an impairment in distal cue navigation after malnutrition from birth to 16 days of age [18] or from birth through 49 days of age [19]. Protein malnutrition induced in adulthood did not influence acquisition [5]. In the present study using a radial maze, both proximal and distal cues were available to our subjects; it is therefore possible that this facilitated the learning process. The retention test 72 h after initial acquisition showed that, under saline conditions, the performance of both undernourished and well-nourished rats did not differ from their performance recorded during the last day of acquisition. This indicates that undernutrition, under our conditions, did not influence the capacity to retain information. Although similar results were described after prenatal protein malnutrition [11], other studies involving early life malnutrition [18,19] or protein deprivation in adult rats [5] showed an impaired retention particularly in distal cue version of the Morris maze. The retention deficit exhibited by malnourished rats may be due to impaired consolidation of spatial information, as was suggested by Fukuda et al. [19]. Tonkiss et al. [11] also suggest that development of distal cue navigation may be more vulnerable to malnutrition experienced during the postnatal rather than the prenatal period. Further, the degree of nutritional rehabilitation prior to testing may be important (unlike those of Tonkiss, our rats did not benefit from nutritional rehabilitation prior to testing). Haloperidol led to a marked deterioration in control performance during retention, a phenomenon not observed in undernourished rats. Haloperidol injection induced a significant increase in number of errors and session duration in control when their performance compared with the last day of acquisition or with undernourished rats receiving the same injection. These results are in line with, and extend, previous studies which have shown a lower reactivity of malnourished rats to drugs acting through the catecholaminergic system [7], the GABA-ergic system and the Glutamatergic system [35,36]. In malnourished animals, alteration in the central catecholaminergic system following malnutrition may be
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
responsible for differences in response to drugs acting through this system. Thus, an altered cathecolamine metabolism was observed after protein restriction in adult mice [26], expressed by a prolonged hyperlocomotion after injection of d-amphetamine. Moreover, postnatal protein calorie malnutrition reduced the catatonic response to Haloperidol [22]. Such decreased responsiveness by malnourished animals to drugs acting through the catecholaminergic system was confirmed by our data showing an increase in dopamine turnover in striatum (decrease in DA and increase in DOPAC concentrations). Similar decreases in DA were described in the hypothalamus and hippocampus after prenatal protein malnutrition [16]. Increased turnover of DA system may thus induce an alteration in the number and/or in affinity of DA receptors as a down regulation in malnourished rats. It has been reported that protein deprivation decreases the numbers of dopaminergic receptors in the striatum of malnourished animals [21]. Hence, the antagonist effect of Haloperidol administration would be expected to be more effective in control than in malnourished rats. In our study, using 0.2 mg/Kg, we did not observe the catatonic responses to 0.6 mg/Kg Haloperidol injection described by Masur and Ribiero [22]. We did however note a severely impaired performance in well-nourished rats, manifested as an increase in number of errors and session duration. In summary, prenatal protein malnutrition as well as postweaning undernutrition in rats or protein restriction in adult mice, all led to an altered DA metabolism in the brain. This strongly suggests that the dopaminergic pathway may be involved in the behavioral changes observed following such treatments. It should be noted that undernutrition in the present study (80% of control littermate body weight) may be considered comparatively mild relative to that induced in other studies during postnatal protein malnutrition, where body weights were diminished to as little as 40% of controls [18,19]. Our model is however closer to clinical malnutrition profile generally encountered in developing countries. In fact even in rats, prenatal protein malnutrition led to body weight deficits only 6% to 20% at birth [10,11]. In conclusion, mild undernutrition introduced at weaning resulted in behavioral hyperactivity, possibly due, as has been suggested by some, to a decreased emotionality to novel environments. An alternative interpretation, taking into consideration data from the tail-flick task, suggests that open field hyperactivity, coupled with decreases in tail flick latencies, indicate a state of sensory hyperreactivity. Unlike protein malnutrition during the immediately postnatal period and in adulthood, undernutrition in the postweaning period did not impair acquisition and retention. However, as was observed after prenatal protein malnutrition, post-weaning undernutrition led to an increase in DA turnover in undernourished rats which may be responsible for the diminished responsiveness to i.p.
201
Haloperidol injection. An alteration in drug response may reflect changes in brain function [7], and may underly the emotional and/or sensory changes observed. Weaning may thus constitute a vulnerable period during which undernutrition influences neural systems, with consequent behavioral modifications.
Acknowledgements This study was supported by PROTARS II National Moroccan Research Support Program grant P31/02 to WB.
References [1] Morgane PJ, Mokler DJ, Galler JR. Effects of prenatal protein malnutrition on the hippocampal formation. Neurosci Biobehav Rev 2002;26(4):471 – 83. [2] Bengelloun WA. Kwashiorkor: behavioural indices of neurological sequelae. (Mal)Nutrition and the infant brain. Wiley-Liss, Inc.; 1990. p. 65 – 81. [3] Tonkiss J, Galler JR, Morgane PJ, Bronzino J, Austin-LaFrance RJ. Prenatal protein malnutrition and postnatal brain function. Ann N Y Acad Sci 1993;678:215 – 27. [4] Brioni JD, Keller EA, Levin LE, Cordoba N, Orsingher OA. Reactivity to amphetamine in perinatally undernourished rats: behavioral and neurochemical correlates. Pharmacol Biochem Behav 1986;24(3):449 – 54. [5] Lukoyanov NV, Andrade JP. Behavioral effects of protein deprivation and rehabilitation in adult rats: relevance to morphological alterations in the hippocampal formation. Behav Brain Res 2000; 112(1 – 2):85 – 97. [6] Galler RJ, Shumsky JS, Morgane PJ. Malnutrition and brain development. In: Walker WA, Decker BC, editors. Nutrition in pediatrics, Part II. London’ Hamilton; 1997. p. 196 – 212. [7] 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. [8] Tonkiss J, Galler JR, Formica RN, Shukitt-Hale B, Tim RR. Fetal protein malnutrition impairs acquisition of DRL task in adult rats. Physiol Behav 1990;48(1):73 – 7. [9] Tonkiss J, Galler JR, Shukitt-Hale B, Rocco FJ. Prenatal protein malnutrition impairs visual discrimination learning in adult rats. Psychobiology 1991;19:247 – 50. [10] 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. [11] Tonkiss J, Shultz P, Galler JR. An analysis of spatial navigation in prenatally protein malnourished rats. Physiol Behav 1994;55(2): 217 – 24. [12] Cintra L, Diaz-Cintra S, Galvan A, Kemper T, Morgane PJ. Effects of protein undernutrition on the dentate Gyrus in rats of three age groups. Brain Res 1990;532(1 – 2):271 – 7. [13] Cintra L, Granados L, Aguilar A, Kemper T, Debassio WA, Galler JR, et al. Effects of prenatal protein malnutrition on mossy fibers of the hippocampal formation in rats of four age groups. Hippocampus 1997;7(2):184 – 91. [14] Debassio WA, Kemper TL, Galler JR, Tonkiss J. Prenatal malnutrition effect on pyramidal and granule cell generation in the hippocampal formation. Brain Res Bull 1994;35(1):57 – 61. [15] Debassio WA, Kemper TL, Tonkiss J, Galler JR. Effect of prenatal protein deprivation on postnatal granule cell generation in the hippocampal dentate gyrus. Brain Res Bull 1996;41(6):379 – 83.
202
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
[16] Kehoe P, Mallinson K, Bronzino J, McCormick CM. Effects of prenatal protein malnutrition and neonatal stress on CNS responsiveness. Dev Brain Res 2001;132(1):23 – 31. [17] Jordan TC, Cane SE, Howells KF. Deficits in spatial memory performance induced by early undernutrition. Dev Psychobiol 1981;14(4):317 – 25. [18] Castro CA, Rudy JW. Early-life malnutrition selectively retards the development of distal – but not proximal – cue navigation. Dev Psychobiol 1987;20(5):521 – 37. [19] Fukuda MTH, Silva ALF, Almeida SS. Early postnatal protein malnutrition affects learning and memory in the distal but not in the proximal cue version of the Morris water maze. Behav Brain Res 2002;133(2):271 – 7. [20] Bedi KS. Effects of undernutrition during early life on granule cell numbers in the rat dentate gyrus. J Comp Neurol 1991;311:425 – 33. [21] Wiggins RC, Fuller G, Enna SJ. Undernutrition and the development of brain neurotransmitter systems. Life Sci 1984;35(21):2085 – 94. [22] Masur J, Ribiero MJ. Chronic starvation impairs the effect of depressant drugs on CNS of rats. Pharmacology 1981;23(2):64 – 8. [23] Leahy JP, Stern WC, Resnick O, Morgane PJ. A neuropharmacological analysis of central nervous system catecholamine systems in developmental protein malnutrition. Dev Psychobiol 1978;11(4): 361 – 70. [24] Paula-Barbosa MM, Andrade JP, Castedo JL, Azevedo FP, Comoes I, Vogt B, et al. Cell loss in the cerebellum and hippocampal formation of adult rats after long-term low protein diet. Exp Neurol 1989; 103(2):186 – 93. [25] Andrade JP, Paula-Barbosa MM. Protein malnutrition alters the cholinergic and GABAergic systems of the hippocampal formation of the adult rat: an immunocytochemical study. Neurosci Lett 1996;211(3):211 – 5. [26] Dietrich MO, Mantese CE, Anjos GM, Rotta LN, Perry MLS, Souza DO, et al. Increased locomotor response to amphetamine, but not other psychostimulants, in adult mice submitted to a low-protein diet. Physiol Behav 2004;83(1):129 – 33.
[27] Ogura T, Ogata M, Akita H, Jitsuki S, Akiba L, Noda K, et al. Impaired acquisition of skilled behavior in rotarod task by moderate depletion of striatal dopamine in a pre-symptomatic stage model of Parkinson’s disease. Neurosci Res 2005;51(3):299 – 308. [28] Bengelloun WA, Nelson DJ, Zent HM, Beatty WW. Behavior of male and female rats with septal lesions: influence of prior gonadectomy. Physiol Behav 1976;16(3):317 – 30. [29] El Ayadi A, Errami M. Interactions between neomycin and cerebral dopaminergic and serotoninergic transmission in rats. Therapie 1999;54:595 – 9. [30] Louilot A. Effet de divers traitements pharmacologiques sur le me´tabolisme de la dopamine dans le noyau accumbens et le tubercule olfactif, utilisation de la voltametrie differentielle in vivo. Unpublished thesis, Universite´ de Bordeau II; 1983. [31] Hernandes ADS, Franc¸olin-Silva AL, Valadares CT, Fukuda MTH, Almeida SS. Effects of different malnutrition techniques on the behavior of rats tested in the elevated T-maze. Behav Brain Res 2005;162(2):240 – 5. [32] Almeida SS, Tonkiss J, Galler JR. Prenatal protein malnutrition affects the social interactions of juvenile rats. Physiol Behav 1996;60(1): 197 – 201. [33] Smart JL, Whatson TS, Dobbing J. Thresholds of response to electric shock in previously undernourished rats. Br J Nutr 1975;34(3):511 – 6. [34] Trzcinska M, Tonkiss J, Galler JR. Influence of prenatal protein malnutrition on behavioral reactivity to stress in adult rats. Stress 1999;3(1):71 – 83. [35] Tonkiss J, Almeida SS, Galler JR. Prenatally malnourished female but not male rats show increased sensitivity to MK-801 in a differential reinforcement of low rates task. Behav Pharmacol 1998;9(1):49 – 60. [36] Tonkiss J, Trzcinska M, Shultz P, Vincitore M, Galler JR. Prenatally protein malnourished rats are less sensitive to the amnestic effects of medial septal infusions of chlordiazepoxide. Behav Pharm 2000; 11(6):437 – 46.
Effects of postweaning undernutrition on exploratory behavior, memory and sensory reactivity in rats: Implication of the dopaminergic system Meryem Alamy a, Mohammed Errami b, Khalid Taghzouti c, Fatima Saddiki-Traki c, Wail A. Bengelloun c,* a
Faculty of Science, Casablanca (Ain Chock), Morocco b Faculty of Science, Rabat, Morocco c Faculty of Science, Tetouan, Morocco
Received 30 March 2005; received in revised form 5 July 2005; accepted 12 July 2005
Abstract The effects of early undernutrition on behavior and brain biochemistry were examined in rats. At weaning, rats were provided either an ad lib diet (control group) or maintained at 80% of the weight of their control littermates (undernourished group). Three weeks into the diet they were tested in an open field. After 6 weeks of diet, HPLC analyses were conducted on sample brains from each group to assess levels of dopamine and metabolites, respectively dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum. At seven weeks of diet, remaining rats were trained in an 8-arm radial maze, and a retention test conducted 72 h after attaining the learning criterion. At fourteen weeks of diet, sensory reactivity was measured by tail-immersion in a water bath maintained at constant temperature 50 T 1 -C. Undernourished rats exhibited hyperactivity and increased exploratory behavior in the open field, as well as increased sensory reactivity in the tail flick test. In the radial maze, however, undernourished rats did not differ from controls in either learning or retention. Haloperidol (i. p. injection) impaired retention by control but not undernourished animals. HPLC analyses showed an increase in dopamine turnover in the striatum of undernourished rats. Our results suggest that, unlike its effects when induced immediately at birth or in adulthood, undernutrition at weaning does not appear to influence learning and retention but induced an hyperactivity and alterations in striatal DA turnover which was associated with a decrease in responsiveness to i. p. haloperidol injection. D 2005 Elsevier Inc. All rights reserved. Keywords: Undernutrition; Open field activity; Radial maze; Learning; Memory; Dopamine; Rat
1. Introduction Undernutrition is a form of malnutrition where all nutriments required by the species are available in the diet but the amounts are insufficient [1]. In Africa, the weaning period seems to be especially critical because access to maternal nutriments ceases at this time [2]. Yet, few investigators have addressed malnutrition at weaning. In animal models, the behavioral effects of malnutrition induced either prenatally [3], immediately postnatally [4], or in adulthood [5] have been largely described (for a * Corresponding author. Department of Biology, Faculty of Science, BP 1014, Rabat, Morocco. Tel.: +212 6 116 6277; fax: +212 3 777 4261. E-mail address: [email protected] (W.A. Bengelloun). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2005.07.008
review, see [6]). Furthermore, prenatal, postnatal and adult malnutrition also induce changes in brain cholinergic, noradrenergic, dopaminergic, serotoninergic and GABAergic systems as well as changes in sensitivity to psychoactive drugs (for review see [7]). Prenatal protein malnutrition was reported to impair acquisition of Differential Reinforcement of Low rates of responding (DRL) performance [8] and visual discrimination learning [9] in adulthood. It has been demonstrated that malnourished rats exhibit deficits in memory tests (e.g., T-maze [10]), but not in spatial alternation [8] or in Morris water maze [11]. Prenatal malnutrition has also been shown to influence the development of the hippocampal formation [12 –15] as well as DA and DOPAC levels in both hypothalamus and hippocampus [16].
196
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
Impairments of learning and spatial memory were described by some authors after early postnatal protein malnutrition in radial maze [17] and in distal but not in proximal cue versions of the Morris water maze [18,19]. A decrease in granule cell numbers in the dentate gyrus was reported following early postnatal malnutrition [20]. Similarly, the number of striatal dopamine receptors were decreased in animals undernourished during suckling [21], as was the haloperidol-induced catatonic response [22]. When prolonged protein malnutrition was instituted both pre and postnatally in dam and offspring, biochemical findings were contradictory. While Almeida et al. [7] reported a lower reactivity of malnourished rats to drugs mediated through the catecholaminergic system, Brioni et al. [4] showed that repeated amphetamine (AMPH) treatment led to sensitization, as demonstrated by increases in both locomotor activity and binding of dopaminergic receptors in the striatum. Moreover, nutritional state did not however influence the behavioral stereotypy normally produced by AMPH activation of catecholaminergic function [23]. Protein deprivation in adult rats led to reduced emotional reactivity and impaired Morris maze performance: malnourished rats were slower to learn an efficient search strategy [5]. The behavioral deficits induced by malnutrition in adulthood were associated in large measure with structural abnormalities in the hippocampal formation including a marked loss of hippocampal neurons [5,24] and synapses [5]. Further, exposure to low-protein diet in adulthood produced changes in hippocampal cholinergic and GABAergic systems [25] in rats and an enhanced locomotor response to d-amphetamine in mice [26]. The present study was designed to determine the behavioral effects of undernutrition (protein-calorie malnutrition) induced at weaning, and its relationship to striatal neurochemical systems. Both the age at which malnutrition occurs and its duration are critical factors in determining its effects on the brain (cf. [1]). Few previous studies have induced malnutrition or undernutrition at weaning in spite of the fact that in humans, it has been shown to represent a stage of particular vulnerability [2]. Moreover, Ogura et al. [27] have recently reported an impairment in the acquisition of certain behavioral tasks after striatal dopamine depletion. We therefore examined the performance of rats in learning, memory and sensory reactivity paradigms, and assessed DA, DOPAC and HVA concentrations in striatum.
2. Materials and methods 2.1. Animals Wistar rats from the animal colony of Mohammed V University (Rabat, Morocco) were used. After mating and prior to delivery, female rats were caged individually and housed in an animal room with natural light and temper-
ature conditions. During the study, temperature varied between 18 and 24 -C with humidity at 45 to 55%. The light / dark cycle was around 14 / 12. Litters were culled to a maximum of 8 pups. At 10 days of age, maternal retrieval tests were conducted in each litter, and determined that none of the pups were being rejected by the mother (and therefore malnourished). Weaning took place at 25 days of age: pups were sexed and only those litters containing a minimum of two males were retained. Thus, 46 male pups were used (23 paired littermates) in this experiment. They were weighed, and same weight male littermates were identified and paired. Depending on litter composition, 2, 4 or 6 male pups were used from the same litter in the experiment. Animals were placed in individual cages; the control rat had free access to food and water throughout the experimental period (except during maze testing) while the paired undernourished rat was maintained at 80% of the control littermate’s weight. For this purpose all subjects were weighed daily and fed a ration sufficient to maintain their undernourished state. This meant that the quantity of food consumed was reduced by about 50% during the first week and by 40% on subsequent weeks. The commercial (Cicalim, Casablanca) diet contained protein (17%), fat (3.2%), cellulose (5.2%), minerals (6%), vitamins A, E, D3, as well as calcium and phosphate, with 68.6% carbohydrates. Water was available to all animals ad lib. All procedures in this experiment conformed to international guidelines on the ethical use of animals and every effort was made to minimize the number of animals used. 2.2. General protocol Three weeks into the diet, all animals were tested for five minutes in an open field on 3 consecutive days in order to evaluate their levels of activity, emotional state and capacity to habituate to novel environments. At the end of the sixth week of diet, 16 rats (8 control rats with their 8 undernourished littermate) were randomly selected for sacrifice, and their brains assayed by high-performance liquid chromatography (HPLC) for striatal dopamine and metabolites. One week later (week 7 of diet), the remaining animals started daily testing in a radial 8-arm maze to assess the effects of post-weaning undernutrition on learning ability. 72 h after attaining the acquisition criterion, retention was evaluated following an i. p. injection of either Haloperidol (dopamine antagonist) or saline. At the 14th week of diet, rats were tested in a tail-flick test to assess temperature reactivity. Of the 46 rats tested in the open field, 16 were sacrificed for HPLC. Subsequently, one animal died, leading us to eliminate its littermate from the study. 28 animals (14 undernourished and 14 controls) were thus tested in the radial arm maze, and later contributed to the temperature reactivity data. All experiments were performed at the same time of day between 1100 and 1600 h.
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
2.3. Open field The open field was a black wood arena measuring 75 45 35 cm high, previously described [28]. The floor of the apparatus was divided into 15 squares (15 15 cm). At the beginning of each trial the rat was placed in a corner square facing the wall. Each rat was tested during a 5-min session, on 3 consecutive days. Behavior was recorded by video system to assess activity (number of crossing between squares), and rearing. 2.4. Neurochemical assays for DOPAC, dopamine and HVA Rats (undernourished and paired control littermates) were sacrificed at 1100 h by decapitation and brains were immediately removed, the striatum dissected over ice. The extraction procedure was a modification of a method previously published [29]. Briefly, tissue samples were homogenized immediately after dissection with weak sonication in perchloric acid 0.1 M (1 / 10; W/V), the crude supernatant was diluted in the mobile phase after centrifugation (10 min; 15 000 g at 4-). The homogenate was filtered (Whatman; 0.22 Am) and 20 Al injected in a reversed phase chromatographic system. The HPLC system consisted of a Shimadzu L C-6A pump equipped with a 20 Al (Rheodyne 7125) injection loop. The Shimadzu L-ECD-6A amperometric detector was equipped with a 4 Al electrochemical cell fitted with a glass carbon electrode set at 0.7 V with respect to a Silver – Silver chloride reference electrode. Separation of monoamines and metabolites was achieved using a 150 4.6 mm reversed phase analytical column (ODS 5 Am, Varian), the guard column was a Varian (1 4 mm). The mobile phase consisted of sodium acetate (0.1M), Sodium Octyl Sulfate (10 5 M), EDTA (5.6 10 4) and methanol (8%). The pH was adjusted to 4.5 by acetic acid. The flow rate was set at 1 ml/min and the sensitivity was set between 50 and 100 pA full-scale detection. This method leads to a complete separation of dopamine and metabolites in less than 25 min. Identification of the compounds and quantification were performed against injections of external standard solutions containing 100 pg of each product and 3.4 dihydroxybenzylamine (DHBA) as an internal standard at a concentration of 50 pg using an integrator Shimadzu C-4A. Standard solutions were prepared daily by dilution of stock solutions. The sensitivity of the system was found to be about 6 pg for DA, DOPAC and HVA. Monoamine levels were expressed in terms of pg per mg of tissue. 2.5. Radial maze Radial maze tests started on week 7 of diet. The radial maze consisted of a central platform (35.5 cm diameter), with eight arms of 44 cm length and 25 cm width. The flat maze was elevated 75 cm off the floor, without enclosing
197
walls. A small plastic cup was fixed at the end of each of the eight arms to enclose a 0,1 mg reinforcement food pellet (Cicalim, Casablanca). The maze was placed in a softly lighted room with the same external cues throughout the experiment, and maze arms were numbered from 1 to 8 for identification. Animals in both groups were subjected to daily food restriction starting on the third day of week 6 of diet, until they completed testing in the maze. Control animals were restricted to 90% of their body weight. Body weights of undernourished animals were consequently reduced to maintain 80% of littermate control weight. This reduction was meant to motivate maze learning behavior. 2.6. Acquisition Rats were initially provided with a 10-min habituation session on each of three consecutive days, with a pellet placed in the cup at the end of each arm. At the start of each session the subject was placed in the middle of the central platform. On the fourth day, the acquisition phase began. The subject was placed in the center of the radial maze facing arm 1 and allowed a maximum of 15 min to find and eat the 8 pellets. During this and the following daily sessions, time spent in the maze and number of errors (entering an arm in which the pellet was consumed earlier during the session or entering an arm with all 4 paws and not consuming the food pellet) were recorded. Acquisition was considered complete when not more than 1 error was committed on two consecutive days (the criterion). We recorded the number of days to attain criterion, trial duration, total number of errors in the first day as well as the total number of errors during the entire acquisition phase. 2.7. Retention 72 h after acquisition, paired littermates were randomly assigned to receive an i. p. injection of 0.5 ml of either haloperidol (0.2 mg/kg of Haldol, Maphar, Casablanca) [30] or saline solution 60 min prior to undergoing a retention test in the maze. 2.8. Tail flick At the end of the experiment (14 weeks of diet), subjects were tested in a tail flick procedure. During each trial the rat was held vertically, its tail immersed (approximately 2 cm) in water at 50 -C. Reaction time (latency to withdrawal of tail from the water) was recorded during trials conducted at 11 am and 4 pm on three consecutive days. 2.9. Statistical analysis Unweighted means analyses of variance (ANOVA) were used to evaluate data from the open field (OF)
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
and tail flick (TF) tests. Diet group was a between factor, while Daily sessions were within factors. One way ANOVA analyses were used to treat all other data. Statistical tests were performed using SPSS software (Chicago, IL). Differences were considered significant at p < 0.05 level. Where the multivariate analysis indicated a significant effect, univariate analyses were applied. Body weights are presented as mean T standard deviation. Other data are presented in the figures as mean T standard error of the mean.
Striatal neurochemistry A. Striatal Dopamine and metabolites 1200 1000
pg/mg
198
C U
*
800 600
*
400 200 0 DA
DOPAC
HVA
3. Results
B. Striatal DOPAC/DA and HVA/DA ratios 3.1. Body weight
5
Fig. 1 shows the evolution of body weight for control and undernourished (maintained at 80% of control littermates) animals during the 6 weeks of diet. During week 1, mean body weight increased by 13 T 1.7 g in the control group and 5.28 T 0.30 g in the undernourished group. During the next 5 weeks, the average body weight increases were respectively 40.77 T 3.97 g/wk and 30.31 T 4.99 g/wk. At 14 weeks, the undernourished rats were still at 80% of control body weight.
4
3.2. Striatal neurochemistry Fig. 2A shows a significant decrease ( 22%) in DA in striatum of undernourished rats as compared to controls ( F(1,16) = 6.92, p = 0.019). Additionally, there is an increase in DOPAC (+ 61%, F(1,16) = 10.17, p = 0.0065), with a resulting increase of about 107% (Fig. 2B) in the DOPAC/DA ratio in undernourished rats ( F(1,16) = 8.59; p = 0.01). There was a tendency towards an increase in HVA levels, although this did not attain statistical significance ( p = 0.08). The increase in HVA / DA ratio did however reach significance ( F(1,16) = 7.64; p = 0.015). Body weight during six weeks of undernutrition 350 300
Control
weight (g)
Undernourished
250 200 150
C U
* 3
*
2 1 0 DOPAC/DA
HVA/DA
Fig. 2. Striatal neurochemistry. A. Tissue concentration of striatal DA, DOPAC and HVA in control (C) and undernourished (U) rats. B. Striatal DOPAC/DA and HVA/DA ratios. HPLC analysis was performed on brains of 16 randomly selected rats (8 control and 8 undernourished rats).
3.3. Behavior 3.3.1. Open field Analysis of crossing activity yielded significant main effects for Nutrition ( F(1,44) = 4.828, p = 0.033) and Day ( F(2,43) = 20.335, p = 0.0001). Total crossing activity during the three days of testing (Fig. 3A) was significantly higher in Undernourished animals, but this effect was primarily due to the difference in behavior on Day 1 ( F(1,27) = 5.13, p = 0.028). In fact, total crossing activity of control as well as undernourished rats was significantly higher on day 1 compared to days 2 or 3 (Fig. 3A). Undernutrition led to a significant overall increase in rearing activity ( F(1,44) = 13.405; p = 0.001), mainly due to results obtained on days 1 ( F(1,45) = 12.74, p = 0.0008) and 3 ( F(1,45) = 6.027, p = 0.01). A significant main effect was also found for Day ( F(2,43) = 6.765, p = 0.003); rearing activity decreased significantly on days 2 and 3 (Fig. 3B) relative to day 1 in control and in malnourished rats.
100
3.4. Radial maze
50 0 Weaning
WK2
WK4
WK6
Fig. 1. Mean body weight (TSD) of control (n = 23) and undernourished rats (n = 23) during 6 weeks of undernutrition.
No significant differences were found in the number of days to attain criterion when undernourished and control animals were compared ( F(1,25) = 0.2967, p = 0.59). Session duration also did not appear to be affected by
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
A. Total crossing activity Number
150 C U
* 100 50 0
Day 1
Day 2
Day 3
B. Total rearing activity Number
50 40
C U
*
30
*
20 10 0 Day 1
Day 2
Day 3
Fig. 3. Open field performance of control (C, n = 23) and undernourished (U, n = 23) rats. A. Total crossing activity. B. Total rearing activity. Histograms represent the mean number of squares entered with four paws or stands ups during 3 consecutive daily sessions.
nutritional state ( F(1,25) = 0.0096; p = 0.92). Further, no change was detected between the two groups in terms of either number of errors on the first day ( F(1,25) = 0.81;
Radial arm maze: Retention
p = 0.37) or during the entire acquisition phase ( F(1,25) = 1.37; p = 0.25). During the retention test 72 h later, the performance of saline-injected undernourished and control rats did not differ from their performance during the last day of acquisition, when measured in number of errors or in session duration. Saline also did not differentially influence the performance of undernourished and control animals during retention. Haloperidol injection led to an increase in mean number of errors (5.16 vs. 0.33: F(1,11) = 8.96, p = 0.01) and in mean session duration (5395 vs. 1398 s: ( F (1,11) = 6.03, p = 0.03) in control animals during retention in comparison to their performance during the last day of acquisition. This effect was not observed in undernourished animals. During the retention session (Fig. 4), undernourished haloperidol-injected animals exhibited fewer errors ( F (1,11) = 5.43, p = 0.04) and shorter session duration ( F (1,11) = 6.60, p = 0.02) than control animals receiving the same injection. In well-nourished controls, haloperidol injection led to an increase in mean number of errors ( F(1,11) = 6.57, p = 0.02) and in session duration ( F
Tail flick Day 1 Latency (s)
Open field performance
A. Number of errors 10
*
4 2 0 C
*
U
B. Session duration
400 200 0 C
*
*
14 12 10 8 6 4 2 0
4 p.m.
Day 2 C U
* *
U
14 12 10 8 6 4 2 0
C U
*
*
11 a.m. Fig. 4. Retention performance in control (C, n = 7) and undernourished rats (U, n = 7) after saline or Haloperidol injection. Histograms represent mean number of errors (A) and mean session duration (B).
4 p.m.
Day 3
Saline Haloperidol
600
*
11 a.m.
Latency (s)
Minutes
*
C U
11 a.m.
6
800
14 12 10 8 6 4 2 0
Saline Haloperidol
Latency (s)
Errors
8
199
4 p.m.
Fig. 5. Tail-flick latencies in control (C, n = 14) and undernourished rats (U, n = 14) during test sessions at 11 a.m. and 4 p.m. on 3 consecutive days.
200
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
(1,11) = 6.28, p = 0.03). Again, this effect was not observed in undersnourished rats. 3.5. Tail flick Undernutrition decreased the latency to tail withdrawal from hot water ( F(1,25) = 19.174, p = 0.0001) (Fig. 5). No effect of Day ( p = 0.166) or Time ( p = 0.699) was noted. The only significant interaction was Day Time ( F(2,25) = 5.026, p = 0.015, due to an increase in latency at 4 p.m. relative to 11 am on Day 2.
4. Discussion In the present study, undernutrition induced at weaning resulted in hyperactivity, manifested in the open field by an increase in square crossing and rearing. The increase in crossing activity was described by previous studies after postnatal (at weaning) protein malnutrition [2], chronic food restriction induced at birth [22] and after protein deprivation in adult rats [5]. The latter suggested that the elevated locomotion score (square crossing) at the beginning of the first testing session (day 1) in malnourished rats was due to a decrease in emotional reactivity to novel environments. In the present study, the increased activity observed during the first daily session but not apparent during the later sessions (days 2 and 3) seems to be in line with such an interpretation. Moreover, this seems to be in agreement with the findings of Almeida et al. [10] after prenatal protein malnutrition and Hernandes [31] after postnatal malnutrition. Both suggest that malnutrition results in a reduction in anxiety. Rearing activity of undernourished rats was more elevated throughout all three daily sessions. Our results were therefore in agreement with those obtained with prenatal malnutrition [32] but different from results observed after protein deprivation in adults: Lukoyanov and Andrade [5], found no differences between malnourished and control rats on this measure. Rearing on the hind legs was suggested to include a vertical component of exploration [32], thus indicating that prenatal protein calorie malnutrition as well as post-weaning undernutrition resulted in an increase in exploratory behavior. Finally, the increase in crossing and in rearing activity in malnourished rats may reflect not only a lower anxiety to novel environment [5] but also an active search for food (food seeking), linked to the nutritional treatment [2]. In the tail-flick test undernourished animals exhibited lower latencies to tail withdrawal, suggesting that they were more sensitive to heat than controls. Such changes in sensitivity threshold may be related to the hyperactivity observed in the open field test suggesting the possibility of a state of sensory hyperreactivity following undernutrition. Smart et al. [33] have shown that a mildly painful stimulus (foot-shock) produced greater reactivity in rats previously
undernourished prenatally and immediately postnatally. Our data, coupled with those of Smart et al. [33], suggest that undernourished animals are more reactive to unpleasant or aversive stimuli, irrespective of the age at which nutritional regime is initiated. Such changes in sensory reactivity may underlie the emotional changes suggested by some authors [34]. Further investigations are needed to elucidate these phenomena. Analysis of data from the radial maze showed that undernourished and control rats were similar in rate of acquisition of the task. No differences were observed on any of the behavioral measures. Previous reports using another maze (Morris water maze) yielded differing results, depending on the age at which malnutrition was introduced. Whereas Tonkiss et al. [11] found that prenatal malnutrition affected neither proximal nor distal cue navigation, other studies found an impairment in distal cue navigation after malnutrition from birth to 16 days of age [18] or from birth through 49 days of age [19]. Protein malnutrition induced in adulthood did not influence acquisition [5]. In the present study using a radial maze, both proximal and distal cues were available to our subjects; it is therefore possible that this facilitated the learning process. The retention test 72 h after initial acquisition showed that, under saline conditions, the performance of both undernourished and well-nourished rats did not differ from their performance recorded during the last day of acquisition. This indicates that undernutrition, under our conditions, did not influence the capacity to retain information. Although similar results were described after prenatal protein malnutrition [11], other studies involving early life malnutrition [18,19] or protein deprivation in adult rats [5] showed an impaired retention particularly in distal cue version of the Morris maze. The retention deficit exhibited by malnourished rats may be due to impaired consolidation of spatial information, as was suggested by Fukuda et al. [19]. Tonkiss et al. [11] also suggest that development of distal cue navigation may be more vulnerable to malnutrition experienced during the postnatal rather than the prenatal period. Further, the degree of nutritional rehabilitation prior to testing may be important (unlike those of Tonkiss, our rats did not benefit from nutritional rehabilitation prior to testing). Haloperidol led to a marked deterioration in control performance during retention, a phenomenon not observed in undernourished rats. Haloperidol injection induced a significant increase in number of errors and session duration in control when their performance compared with the last day of acquisition or with undernourished rats receiving the same injection. These results are in line with, and extend, previous studies which have shown a lower reactivity of malnourished rats to drugs acting through the catecholaminergic system [7], the GABA-ergic system and the Glutamatergic system [35,36]. In malnourished animals, alteration in the central catecholaminergic system following malnutrition may be
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
responsible for differences in response to drugs acting through this system. Thus, an altered cathecolamine metabolism was observed after protein restriction in adult mice [26], expressed by a prolonged hyperlocomotion after injection of d-amphetamine. Moreover, postnatal protein calorie malnutrition reduced the catatonic response to Haloperidol [22]. Such decreased responsiveness by malnourished animals to drugs acting through the catecholaminergic system was confirmed by our data showing an increase in dopamine turnover in striatum (decrease in DA and increase in DOPAC concentrations). Similar decreases in DA were described in the hypothalamus and hippocampus after prenatal protein malnutrition [16]. Increased turnover of DA system may thus induce an alteration in the number and/or in affinity of DA receptors as a down regulation in malnourished rats. It has been reported that protein deprivation decreases the numbers of dopaminergic receptors in the striatum of malnourished animals [21]. Hence, the antagonist effect of Haloperidol administration would be expected to be more effective in control than in malnourished rats. In our study, using 0.2 mg/Kg, we did not observe the catatonic responses to 0.6 mg/Kg Haloperidol injection described by Masur and Ribiero [22]. We did however note a severely impaired performance in well-nourished rats, manifested as an increase in number of errors and session duration. In summary, prenatal protein malnutrition as well as postweaning undernutrition in rats or protein restriction in adult mice, all led to an altered DA metabolism in the brain. This strongly suggests that the dopaminergic pathway may be involved in the behavioral changes observed following such treatments. It should be noted that undernutrition in the present study (80% of control littermate body weight) may be considered comparatively mild relative to that induced in other studies during postnatal protein malnutrition, where body weights were diminished to as little as 40% of controls [18,19]. Our model is however closer to clinical malnutrition profile generally encountered in developing countries. In fact even in rats, prenatal protein malnutrition led to body weight deficits only 6% to 20% at birth [10,11]. In conclusion, mild undernutrition introduced at weaning resulted in behavioral hyperactivity, possibly due, as has been suggested by some, to a decreased emotionality to novel environments. An alternative interpretation, taking into consideration data from the tail-flick task, suggests that open field hyperactivity, coupled with decreases in tail flick latencies, indicate a state of sensory hyperreactivity. Unlike protein malnutrition during the immediately postnatal period and in adulthood, undernutrition in the postweaning period did not impair acquisition and retention. However, as was observed after prenatal protein malnutrition, post-weaning undernutrition led to an increase in DA turnover in undernourished rats which may be responsible for the diminished responsiveness to i.p.
201
Haloperidol injection. An alteration in drug response may reflect changes in brain function [7], and may underly the emotional and/or sensory changes observed. Weaning may thus constitute a vulnerable period during which undernutrition influences neural systems, with consequent behavioral modifications.
Acknowledgements This study was supported by PROTARS II National Moroccan Research Support Program grant P31/02 to WB.
References [1] Morgane PJ, Mokler DJ, Galler JR. Effects of prenatal protein malnutrition on the hippocampal formation. Neurosci Biobehav Rev 2002;26(4):471 – 83. [2] Bengelloun WA. Kwashiorkor: behavioural indices of neurological sequelae. (Mal)Nutrition and the infant brain. Wiley-Liss, Inc.; 1990. p. 65 – 81. [3] Tonkiss J, Galler JR, Morgane PJ, Bronzino J, Austin-LaFrance RJ. Prenatal protein malnutrition and postnatal brain function. Ann N Y Acad Sci 1993;678:215 – 27. [4] Brioni JD, Keller EA, Levin LE, Cordoba N, Orsingher OA. Reactivity to amphetamine in perinatally undernourished rats: behavioral and neurochemical correlates. Pharmacol Biochem Behav 1986;24(3):449 – 54. [5] Lukoyanov NV, Andrade JP. Behavioral effects of protein deprivation and rehabilitation in adult rats: relevance to morphological alterations in the hippocampal formation. Behav Brain Res 2000; 112(1 – 2):85 – 97. [6] Galler RJ, Shumsky JS, Morgane PJ. Malnutrition and brain development. In: Walker WA, Decker BC, editors. Nutrition in pediatrics, Part II. London’ Hamilton; 1997. p. 196 – 212. [7] 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. [8] Tonkiss J, Galler JR, Formica RN, Shukitt-Hale B, Tim RR. Fetal protein malnutrition impairs acquisition of DRL task in adult rats. Physiol Behav 1990;48(1):73 – 7. [9] Tonkiss J, Galler JR, Shukitt-Hale B, Rocco FJ. Prenatal protein malnutrition impairs visual discrimination learning in adult rats. Psychobiology 1991;19:247 – 50. [10] 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. [11] Tonkiss J, Shultz P, Galler JR. An analysis of spatial navigation in prenatally protein malnourished rats. Physiol Behav 1994;55(2): 217 – 24. [12] Cintra L, Diaz-Cintra S, Galvan A, Kemper T, Morgane PJ. Effects of protein undernutrition on the dentate Gyrus in rats of three age groups. Brain Res 1990;532(1 – 2):271 – 7. [13] Cintra L, Granados L, Aguilar A, Kemper T, Debassio WA, Galler JR, et al. Effects of prenatal protein malnutrition on mossy fibers of the hippocampal formation in rats of four age groups. Hippocampus 1997;7(2):184 – 91. [14] Debassio WA, Kemper TL, Galler JR, Tonkiss J. Prenatal malnutrition effect on pyramidal and granule cell generation in the hippocampal formation. Brain Res Bull 1994;35(1):57 – 61. [15] Debassio WA, Kemper TL, Tonkiss J, Galler JR. Effect of prenatal protein deprivation on postnatal granule cell generation in the hippocampal dentate gyrus. Brain Res Bull 1996;41(6):379 – 83.
202
M. Alamy et al. / Physiology & Behavior 86 (2005) 195 – 202
[16] Kehoe P, Mallinson K, Bronzino J, McCormick CM. Effects of prenatal protein malnutrition and neonatal stress on CNS responsiveness. Dev Brain Res 2001;132(1):23 – 31. [17] Jordan TC, Cane SE, Howells KF. Deficits in spatial memory performance induced by early undernutrition. Dev Psychobiol 1981;14(4):317 – 25. [18] Castro CA, Rudy JW. Early-life malnutrition selectively retards the development of distal – but not proximal – cue navigation. Dev Psychobiol 1987;20(5):521 – 37. [19] Fukuda MTH, Silva ALF, Almeida SS. Early postnatal protein malnutrition affects learning and memory in the distal but not in the proximal cue version of the Morris water maze. Behav Brain Res 2002;133(2):271 – 7. [20] Bedi KS. Effects of undernutrition during early life on granule cell numbers in the rat dentate gyrus. J Comp Neurol 1991;311:425 – 33. [21] Wiggins RC, Fuller G, Enna SJ. Undernutrition and the development of brain neurotransmitter systems. Life Sci 1984;35(21):2085 – 94. [22] Masur J, Ribiero MJ. Chronic starvation impairs the effect of depressant drugs on CNS of rats. Pharmacology 1981;23(2):64 – 8. [23] Leahy JP, Stern WC, Resnick O, Morgane PJ. A neuropharmacological analysis of central nervous system catecholamine systems in developmental protein malnutrition. Dev Psychobiol 1978;11(4): 361 – 70. [24] Paula-Barbosa MM, Andrade JP, Castedo JL, Azevedo FP, Comoes I, Vogt B, et al. Cell loss in the cerebellum and hippocampal formation of adult rats after long-term low protein diet. Exp Neurol 1989; 103(2):186 – 93. [25] Andrade JP, Paula-Barbosa MM. Protein malnutrition alters the cholinergic and GABAergic systems of the hippocampal formation of the adult rat: an immunocytochemical study. Neurosci Lett 1996;211(3):211 – 5. [26] Dietrich MO, Mantese CE, Anjos GM, Rotta LN, Perry MLS, Souza DO, et al. Increased locomotor response to amphetamine, but not other psychostimulants, in adult mice submitted to a low-protein diet. Physiol Behav 2004;83(1):129 – 33.
[27] Ogura T, Ogata M, Akita H, Jitsuki S, Akiba L, Noda K, et al. Impaired acquisition of skilled behavior in rotarod task by moderate depletion of striatal dopamine in a pre-symptomatic stage model of Parkinson’s disease. Neurosci Res 2005;51(3):299 – 308. [28] Bengelloun WA, Nelson DJ, Zent HM, Beatty WW. Behavior of male and female rats with septal lesions: influence of prior gonadectomy. Physiol Behav 1976;16(3):317 – 30. [29] El Ayadi A, Errami M. Interactions between neomycin and cerebral dopaminergic and serotoninergic transmission in rats. Therapie 1999;54:595 – 9. [30] Louilot A. Effet de divers traitements pharmacologiques sur le me´tabolisme de la dopamine dans le noyau accumbens et le tubercule olfactif, utilisation de la voltametrie differentielle in vivo. Unpublished thesis, Universite´ de Bordeau II; 1983. [31] Hernandes ADS, Franc¸olin-Silva AL, Valadares CT, Fukuda MTH, Almeida SS. Effects of different malnutrition techniques on the behavior of rats tested in the elevated T-maze. Behav Brain Res 2005;162(2):240 – 5. [32] Almeida SS, Tonkiss J, Galler JR. Prenatal protein malnutrition affects the social interactions of juvenile rats. Physiol Behav 1996;60(1): 197 – 201. [33] Smart JL, Whatson TS, Dobbing J. Thresholds of response to electric shock in previously undernourished rats. Br J Nutr 1975;34(3):511 – 6. [34] Trzcinska M, Tonkiss J, Galler JR. Influence of prenatal protein malnutrition on behavioral reactivity to stress in adult rats. Stress 1999;3(1):71 – 83. [35] Tonkiss J, Almeida SS, Galler JR. Prenatally malnourished female but not male rats show increased sensitivity to MK-801 in a differential reinforcement of low rates task. Behav Pharmacol 1998;9(1):49 – 60. [36] Tonkiss J, Trzcinska M, Shultz P, Vincitore M, Galler JR. Prenatally protein malnourished rats are less sensitive to the amnestic effects of medial septal infusions of chlordiazepoxide. Behav Pharm 2000; 11(6):437 – 46.