The Journal of Nutrition Vol. 128 No. 9 September 1998, pp. 1537-1542

-Linolenic Acid Deficiency Modifies Distractibility but Not Anxiety and Locomotion in Rats during Aging¹

Catherine Belzung², Anne-Marie Leguisquet, Serge Barreau, Sylvie Delion-Vancassel, Sylvie Chalon^*, and Georges Durand

Laboratoire d'Ethologie et de Pharmacologie du Comportement, F-37200 Tours, France; ^* INSERM 316, Laboratoire de Biophysique Médicale et Pharmaceutique, F-37200 Tours Cedex, France; and  INRA, Laboratoire de Nutrition et Sécurité Alimentaire, F-78352 Jouy en Josas Cedex, France

	ABSTRACT

Abstract Introduction Methods Results & Discussion References

In rodents, chronic dietary -linolenic acid deficiency decreases learning and memory and alters dopaminergic and serotoninergic neurotransmission. However, these two neurotransmitter systems are related mainly to attention, emotion and locomotion. Therefore, we decided to investigate the effects of dietary -linolenic acid deficiency in rats tested with animal models of distractibility (the distractometer procedure), anxiety (the elevated plus maze) and ambulatory activity (a circular corridor). Moreover, because these neurochemical modifications persist during aging, we decided to study the effects of aging on these behaviors by using rats aged 2, 6, 12 and 24 mo. An age-related decline in distractibility was observed that was accelerated by linolenic acid deficiency. Indeed, an age-related reduction in distractibility was found in so far as distraction time was reduced at the age of 12 mo in controls and at the age of 24 mo in deficient groups compared with 2-mo-old rats. Moreover, distraction time was significantly lower in 6- and 24-mo-old rats fed a deficient diet compared with age-matched controls. Anxiety was not modified by diet or age. Finally, a parallel decrease in locomotion was exhibited by rats fed both diets between 6 and 12 mo of age. Locomotion was not modified by diet. These results show that dietary -linolenic deficiency alters behavior in a very specific way; distractibility is modified by diet, whereas anxiety and locomotion are not, suggesting that particular brain areas may be altered.

	INTRODUCTION

Abstract Introduction Methods Results & Discussion References

Lipid concentration is very high in the central nervous system. Indeed, compared with other parts of the body, the brain lipid concentration ranks second, immediately after adipose tissue. These brain lipids contain a very high amount of polyunsaturated fatty acids (PUFA)³ derived from the essential fatty acid (FA) precursors linoleic [18:2(n-6)] and -linolenic acid [18:3(n-3)]. These two precursors must be obtained from the diet. The brain PUFA are actively accumulated during pre- and postnatal development and are essential components of the structural membrane lipids. More precisely, a diet deficient in [18:3(n-3)] modifies the FA neuronal membranes. It induces a dramatic loss of (n-3) PUFA, in particular docosahexaenoic acid (DHA) [22:6(n-3)], and a concomitant rise in the (n-6) PUFA, [22:5(n-6)] (Bourre et al. 1989, Yamamoto et al. 1988). These modifications sustain alterations of cerebral membrane function, architecture and fluidity. Moreover, they may modulate the binding of neurotransmitters to membrane receptors (Murphy 1990).

In recent studies (Delion et al. 1994 and 1996), we have shown that a chronic dietary -linolenic acid deficiency alters dopaminergic and serotoninergic neurotransmission in rats. In particular, we have observed a significantly lower concentration of endogenous dopamine, a lower density of dopaminergic D2 receptors, and a higher density of serotoninergic 5-hydroxytryptamine 2 (5-HT2) receptors in the frontal cortex of deficient rats.

Behavioral studies have demonstrated that chronic dietary -linolenic acid deficiency alters performance in learning paradigms, whereas supplementation induces the opposite effects. For example, deficiency impairs spatial learning in the Morris water maze (Francès et al. 1996, Nakashima et al. 1993), whereas supplementation improves it (Coscina et al. 1986). Moreover, -linolenic acid deficiency altered discriminative learning (Lamptey and Walker 1978, Morgan et al. 1981, Yamamoto et al. 1988) and emotional learning in the active avoidance test (Bourre et al. 1989). This last effect could be related to a modification of emotion level (Beuzen and Belzung 1995). However, this seems not to be the case because -linolenic acid deficiency does not modify anxiety (Francès et al 1995).

The next step would be to establish a relationship between the neurochemical (dopamine and serotonin function in the frontal cortex) and the behavioral modifications observed in animals deficient in -linolenic acid. As can be seen, behavioral studies that have been undertaken describe mainly modifications of learning or memory, whereas other functions such as attention or emotions have rarely been investigated (see Wainwright et al. 1994, Reisbick and Neuringer 1997).

High distractibility has been observed in human subjects with cortical lesions of the dorsolateral prefrontal cortex as well as in schizophrenia (Braff 1993, Crawford et al. 1995, Franke et al. 1994, Harvey and Peddley 1989), a disease characterized by hypodopaminergia in the prefrontal cortex and hyperdopaminergia in mesolimbic subcortical regions such as the ventral striatum (see Davis et al. 1991 for a review). Schizophrenia is usually treated with dopamine D2 receptor antagonists. Moreover the red cells of schizophrenic patients exhibit a depletion in docosahexaenoic acid, whereas (n-3) PUFA supplementation in the diet induces an improvement of schizophrenic symptoms (Mellor et al. 1996, Peet et al. 1996). An animal model of distractibility has recently been proposed in which stimulation of dopaminergic function increased distractibility, an effect blocked by D2 antagonists (Ågmo et al. 1997). Because dopaminergic function was decreased in rats fed a diet deficient in -linolenic acid, we decided to study the effects of such a diet by using this behavioral paradigm (Experiment 1).

Prefrontal cortex 5-HT2 serotoninergic receptors are involved in anxiety because postsynaptic stimulation of these receptors reduces exploration of the open arms in rats confronted with an elevated plus maze (Gibson et al. 1994, Stutzmann et al. 1991). Because frontal 5-HT2 receptor density was increased in deficient rats, we decided to study the effects of -linolenic deficiency using this experimental design (Experiment 2).

Behavioral modifications can be the result of changes in activity level; thus it was necessary to verify that the diet did not induce modifications of locomotion. We therefore investigated the effect of -linolenic acid deficiency on ambulatory activity in these animals (Experiment 3).

The decrease in 22:6(n-3) induced by dietary -linolenic acid deficiency partially attenuates during aging, even if this recovery is lower in the frontal cortex than in other brain areas (Delion et al. 1996). Indeed, the frontal cortex is more vulnerable to the effects of aging then most other brain regions in human neuroimaging studies (Raz 1996, Raz et al. 1997, Waldemar 1995). However, the neurochemical modifications observed in deficient subjects persist at all ages because the modifications observed during aging in controls parallel those observed in deficient animals (Delion et al. 1996). For these reasons, we examined the behavioral modifications during aging, using rats aged 2, 6, 12 and 24 mo.

	MATERIALS AND METHODS

Abstract Introduction Methods Results & Discussion References

Animals and diets.  Two generations of female Wistar rats, originating from our laboratory, were fed a diet containing 6% fat as African peanut oil (Lesieur-Alimentaire, Coudekerque, France) specifically deficient in -linolenic acid. This deficient diet provided ~1200 mg of linoleic acid but <6 mg of -linolenic acid/100 g of diet. Two weeks before mating, female rats originating from the second generation of the -linolenic acid-deficient rats were divided in two groups. The first group received the deficient diet, and the second group received a diet in which peanut oil was replaced by a mixture of peanut oil and rapeseed oil (60 and 40%, respectively). This diet (control) provided the same amount of linoleic acid as the deficient diet and also ~200 mg of -linolenic acid/100 g of diet [(n-6)/(n-3) = 6]. Diets were consumed ad libitum by both groups. At weaning, the male progeny of these two groups of female rats received the same diet as their respective dams. A two-generation model was used so that the offspring could not derive (n-3) PUFA from maternal stores during gestation and lactation. Furthermore, when the female rats were fed a control diet 2 wk before mating, their offspring exhibited a normal level of DHA in the phospholipids of their central nervous system at birth (Guesnet et al. 1997) and at weaning (Bourre et al. 1993). The composition of the diets and their fatty acid composition are reported in Tables 1 and 2. The experimental protocol was in compliance with applicable guidelines from the Ministère de l'Agriculture, France. Rats were tested at the ages of 2, 6, 12 and 24 mo. They arrived in the behavioral laboratory 2 wk before testing and were exposed to a reversed 12-h light:dark cycle, with lights off at 0800 h to allow observation during their high activity period, that is, when lights were off. Each rat was tested only once in a given procedure and was naive to the apparatus.

Distractibility. 

Apparatus.  A start box (20 × 20 × 30 cm high) was connected to a straight runway (200 × 10 × 10 cm high) ending in a goal box (20 × 20 × 30 cm high). In the middle of the goal box, a drinking dish was fixed to the floor. The apparatus was made of plywood with covers of opaque Plexiglas, making it possible to visually locate the rat inside the apparatus. Both the start and the goal box could be closed by manually operated sliding doors. At the middle of the runway, another runway could be perpendicularly connected (100 × 10 × 10 cm high). This additional runway ended in an empty box (30 × 30 × 30 cm). The dimensions of the empty box were different from the ones of the goal box, to assure that the subjects could distinguish both boxes (see Ågmo et al. 1997a and 1997b for more details).

Procedure.  Before the experiments began, the rats were allowed to drink an 180 g/L sucrose solution in their home cage for 48 h. This solution was later used as reinforcement in the distraction procedure. In addition to the ordinary water bottle, another bottle with 200 mL of the sucrose solution was freely available. After the home cage exposure to the sucrose solution, the rats were habituated to the apparatus during two sessions of 1 h each, separated by at least 24 h. During habituation, 5 mL of the sucrose solution was available in the goal box.

Acquisition consisted of three daily trials. On each trial, the rat was placed in the start box for 1 min. The door was then opened, and the rat was allowed a maximum of 5 min to enter the runway. Immediately after entry, the door was closed to prevent reentry. In the runway, the rat was allowed 5 min to reach the goalbox. Once inside the goalbox, the door was closed. One minute later, the rat was removed. On every trial, 0.5 mL of sucrose solution was available in the goal box. The following measures were recorded: running time (time from entering the runway, with 4 paws, until the animal was inside the goal box, with 4 paws,); lick latency (time from entry into the goal box, with 4 paws, until the beginning of licking the sucrose solution). Any rat that exceeded the maximum times allowed for entering the runway or the goal box or that did not drink at any of the trials was eliminated from the experiment. This procedure assured that only rats with reliable runway behavior were included in the test.

At the test, the additional arm was connected to the runway. One trial was performed. In addition to measures taken during acquisition, the distraction time was recorded. This is the time that the rat spent exploring the additional runway (4 paws inside). The running time recorded at the test does not include the distraction time (i.e., the distraction time was subtracted from the total running time).

Elevated plus maze. 

Apparatus.  The elevated plus-maze consisted of two opposing open arms (50 × 10 cm) and two enclosed arms (50 × 10 × 40 cm high), connected by a central platform (10 × 10 cm). The apparatus was elevated to a height of 50 cm above floor level. At the beginning of the 5-min test, the rat was placed on the central platform with the head facing an open arm. Behavior was videotaped, and later an observer recorded the number of entries into both arms. The proportion of entries into the open arms and the total number of entries was then calculated.

Ambulatory activity test.  Rats were individually placed in a novel environment consisting of a circular corridor (10 cm wide, with walls 70 cm high, inner diameter 55 cm) made of steel, with four infrared photocells placed 2.5 cm above the grid floor at equidistant locations within the circumference. A beam interruption of 250 ms was required to activate the counters. In that way, rapid movements were not registered. The activity count represents, therefore, basically ambulatory activity. The number of photocell interruptions was registered during each 60-min period after introduction of the rat to the apparatus.

Statistics.  Data were analyzed by two-factor ANOVA. In the event of a diet × age interaction, further simple effects ANOVA or t test where appropriate was conducted. A posteriori comparisons were made with Tukey's procedure.

	RESULTS AND DISCUSSION

Abstract Introduction Methods Results & Discussion References

Experiment 1: Distractibility.  The interaction age × diet was significant for distraction time (P < 0.05) and for running time (P < 0.01) but not for lick latency (P < 0.12).

No effect of diet was observed for lick latency [F(71,1) = 1.48, P < 0.22]. For distraction time and running time, no effect of diet appeared at 2 mo. At 6 mo, distraction time was lower in rats fed the deficient diet compared with age-matched controls. At 12 mo, running time was greater in the deficient group. Finally, at 24 mo, both distraction time and running time were lower in the deficient rats.

A significant effect of age appeared for lick latency [F(71,3) = 4.93, P < 0.004]. This was due to an increase in lick latency in control rats aged 2 or 6 mo, compared with control rats aged 12 or 24 mo.

In control groups, a significant effect of age for distraction time (P < 0.0001) but not for running time existed. This was due to an increase in this variable in 2-mo-old rats compared with 12-mo-old rats and in 6-mo-old rats compared with 12- or 24-mo-old rats (Fig. 1).

View larger version (15K): [in this window] [in a new window]   Fig 1. Effects of dietary -linolenic acid deficiency on distractibility in 2-, 6-, 12- and 24-mo-old male rats as assessed by running time (panel A), lick latency (panel B) and distraction time (panel C). Values are means ± SEM [n = 12, 9, 11 and 10 (controls) and 9, 8, 13 and 7 (deficient) at 2, 6, 12 and 24 mo, respectively]. °Indicates a significant difference between control and deficient diets (age-matched rats). Values assigned a different superscript in the control group (a-b) or in deficient group (x-y) were significantly different (P < 0.05).

In deficient rats, the effect of age appeared for distraction time and running time (P < 0.005 and P < 0.003, respectively). A posteriori comparisons revealed that 24-mo-old rats exhibited lower distraction time then 2- or 6-mo-old rats, whereas the effect on running time was due to a difference between 24-mo-old rats and 6- or 12-mo-old rats, with the latter two exhibiting longer running times.

Thus, rats fed a deficient diet exhibited lower distractibility than controls at the ages of 6 and 24 mo, whereas a decrease in distractibility occurred with age. Indeed, in controls, a reduction of distraction time was obvious at the age of 12 mo and persisted at 24 mo (compared with 2-mo-old rats). In deficient groups, a reduction in distraction time occurred only in the oldest rats, that is, at the age of 24 mo. This shift of age-related deficits between the two diet groups may explain the fact that there was no difference in distraction time between controls and deficient-diet groups at the age of 12 mo.

Diet could affect any of the determinants of runway behavior, including locomotor effects and changes in motivation or reward value of sucrose; thus the effects observed would be nonspecific (Ågmo et al. 1997a and 1997b). However, such an interpretation can be excluded in this experiment. Indeed, modifications of such determinants may not underlie the behavioral differences observed between rats fed the two diets because the modifications of running time are not concomitant with the changes in distractibility.

The reduction of distractibility observed in rats deficient in -linolenic acid can be explained by modification of the dopaminergic function. Indeed, an increase in distractibility is usually observed when the subcortical dopaminergic system is stimulated, for example, by pharmacologic agents such as amphetamine (Ågmo et al. 1997a and 1997b), so that the decrease of distractibility observed in deficient animals can be related to the reduction of the dopaminergic function exhibited by these animals (Delion et al. 1994 and 1996). However, schizophrenic subjects have a marked depletion in (n-3) PUFA levels in red blood cells (Horrobin et al. 1989 and 1994, Peet et al. 1996) so that one may expect that deficient animals would show increased distractibility. It should be noted that schizophrenic subjects exhibit a decrease in both (n-3) and in (n-6) PUFA series. Therefore, their (n-3)/(n-6) ratio may not be altered. For this reason, rats that are fed an -linolenic-deficient diet and exhibit a decrease in brain (n-3) balanced by an increase in (n-6) FA, i.e., an increase of the (n-6)/(n-3) ratio (Delion et al. 1994 and 1996), may not be an appropriate animal model of the FA modifications observed in schizophrenic patients.

It is tempting to speculate that the shift in age-related modifications of distractibility observed in deficient groups may be explained by the free-radical theory of aging (Harman 1956, 1968 amd 1994 Harman et al. 1976). PUFA are powerful oxidative substrates converted by free radical metabolism into lipid peroxides. The latter induce the production of additional free radicals, which cause oxidative alteration of the brain. The (n-3) PUFA are more autooxidizable than other PUFA having the same carbon chain length because of a higher unsaturation number (Cho et al. 1987, Cosgrove et al. 1987, Howard and Ingold 1967), so that long-term dietary deficiency in (n-3) PUFA would be expected to induce less susceptibility to lipid peroxidation and, according to the "free radical theory of aging," would lead to a shift in senescence-induced behavioral deficits. This is indeed what we observed because the changes in distractibility exhibited in rats during aging appeared earlier in control than in deficient rats. This result contradicts those of a study by Yamamoto et al. (1991), showing that rats fed an -linolenic-rich diet exhibited an increased learning ability in senescence by using a brightness-discrimination paradigm. Differences used in diet composition (-linolenic deficient diet vs. -linolenic-enriched diet) and in behavioral tests (an animal model of attentional processes vs. an animal model of learning and memory) may explain this discrepancy.

Elderly monkeys exhibit deficits in performance of the delayed-response task, an effect interpreted as an increase in the susceptibility to interference from irrelevant stimuli, that is, distractibility (Arnsten and Contant 1992, Coull 1994). This finding contrasts with the results of this study in which distractibility seems to decrease during aging. Concerning this discrepancy, it must be emphasized that, unlike delayed-response tasks, our procedure does not depend upon learning or the memorizing ability of the subjects (Ågmo et al. 1997a and 1997b). The occurrence of working memory deficits as well as cognitive deterioration have been repeatedly described during aging (Craik et al. 1989, Ingram et al. 1994, Schurman et al. 1986, Wiegersma and Meerstse 1990); thus it is difficult to accept an increase in distractibility with delayed-response tasks. However, the decrease in time spent in the additional arm that we observed in the oldest animals can also be interpreted by using frames other than distractibility. For example, alternative explanations could be a decrease in exploration drive or an increase in rigidity or behavioral perseveration because such modifications have been repeatedly reported to occur during normal aging (for a review see Lalonde and Badescu 1995).

Experiment 2: Elevated plus maze.  Two-factor ANOVA did not reveal age × diet interactions for the percentage of entries in the open arm (P < 0.72) and total entries (P < 0.09) (Fig. 2). No effect of diet appeared for the percentage of entries into the open arms [F(82,1) = 0.05, P < 0.81] or for total entries [F(82,1) = 0.24, P < 0.62]. An effect of age was observed for total entries [F(82,3) = 6.47, P < 0.001] but not for percentage of entries into the open arms [F(82,3) = 2.21, P < 0.09). Tukey's test revealed that this effect wass due to a decrease of total entries in aged rats (significant differences were observed between 2- and 24-mo-old rats in controls and between 2- and 12- or 24-mo as well as between 6- and 12-mo-old deficient rats).

View larger version (18K): [in this window] [in a new window]   Fig 2. Effects of dietary -linolenic acid deficiency on anxiety in the elevated plus maze test in 2-, 6-, 12- and 24-mo-old male rats as assessed by percentage of entries in the open arms (panel A) and the number of entries in the open and closed arms (panel B). Values are means ± SEM [n = 12, 11, 11 and 12 (controls) and 12, 12, 12 and 8 (deficient) at 2, 6, 12 and 24 mo, respectively]. Values assigned a different superscript in the control group (a-b) or in deficient group (x-z) were significantly different (P < 0.05).

The failure of dietary -linolenic acid deficiency to modify the percentage of entries into the open arms of the elevated plus maze shows that the diet does not modify anxiety. This result confirms the data of another study that reported absence of anxiety modification in rats that were deficient in (n-3) PUFA and confronted with various rodent models of anxiety such as the light/dark choice paradigm or the elevated plus maze test (Francès et al. 1995). Concerning the absence of age-related modifications in anxiety level, conflicting results have been reported in the literature. Indeed, by using the same experimental procedure as ours, Miyamoto et al. (1992) found an age-related decrease in anxiety, whereas Frussa-Filho et al. (1991) reported an increase in anxiety in old vs. young rats. The decrease in total entries exhibited by the oldest rats in our study can be related to the well-documented age-related decline of horizontal locomotion (see Lalonde and Badescu 1995 for a recent review).

Experiment 3: Ambulatory activity,  There was no interaction age × diet (P < 0.36) and no difference between diets was observed, whatever the age considered. In fact, an age-induced decrease in locomotion was observed in all rats [F(82,3) = 31.72, P < 0.0001]. In both diet groups, this effect was significant between 6 and 12 mo of age (Fig. 3). This age-induced decline in ambulatory activity parallels the reduction of entries into the arms of the elevated plus maze observed in the oldest rats.

View larger version (13K): [in this window] [in a new window]   Fig 3. Effects of dietary -linolenic acid deficiency on ambulatory activity in 2-, 6-, 12- and 24-mo-old male rats as assessed by number of beam interruptions. Values are means ± SEM [n = 12, 12, 11 and 12 (controls) and 12, 12, 11 and 8 (deficient) at 2, 6, 12 and 24 mo, respectively]. Values assigned a different superscript in the control group (a-b) or in deficient group (x-z) were significantly different (P < 0.05).

Previous studies dealing with the behavioral effects of PUFA deficiency have shown that dietary -linolenic deficiency induced learning and memory impairments (short-term memory in the Morris water maze, in discriminative learning procedures or in passive avoidance tests). Present data extend the deficits produced by (n-3)PUFA deficiency to another behavioral process, one linked to distractibility. Furthermore, this dietary deficiency does not affect the behavior of rodents in a nonspecific way. Indeed, distractibility is reduced in rats fed a deficient diet, whereas anxiety and locomotion are not. This may suggest that behaviors related to particular brain areas or neurotransmitter systems such as the dopaminergic function within the prefrontal cortex may be altered, whereas others, related to other brain structures such as the striatum, are not.

	ACKNOWLDGEMENT

Raymond Jegat built all of the behavioral testing equipment.

	FOOTNOTES

Manuscript received 17 December 1997. Initial reviews completed 20 March 1998. Revision accepted 18 May 1998.

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