The Journal of Nutrition Vol. 128 No. 12 December 1998, pp. 2512-2519

Dietary Fish Oil Affects Monoaminergic Neurotransmission and Behavior in Rats^1,2

Sylvie Chalon^{*, 3}, Sylvie Delion-Vancassel^*, Catherine Belzung, Denis Guilloteau, Anne-Marie Leguisquet, Jean-Claude Besnard^*, and Georges Durand^**

^* INSERM U316, Laboratoire de Biophysique Médicale et Pharmaceutique, 37200 Tours, France,  Laboratoire d'Ethologie et de Pharmacologie du Comportement, 37200 Tours, France and ^** INRA, Laboratoire de Nutrition et Sécurité Alimentaire, 78352 Jouy-en-Josas Cedex, France

	ABSTRACT

Abstract Introduction Methods Results Discussion References

We studied the effects of a fish oil enriched diet on fatty acid composition of cerebral membranes and on several neurochemical and behavioral variables of monoaminergic function in rats. The frontal cortex, striatum, hippocampus and cerebellum were studied in rats fed fish oil (FPO, 50% salmon oil + 50% palm oil), which provided an (n-6)/(n-3) polyunsaturated fatty acid (PUFA) ratio of 0.14 versus 6.19 in controls fed a diet containing a mixture of African peanut oil and rapeseed oil. In the FPO group compared to the control group, the major modifications in fatty acid composition of cerebral membranes included the following: higher levels in 22:6(n-3), lower levels in 20:4(n-6) and a significantly greater proportion of phosphatidylserine. Dopamine levels were 40% greater in the frontal cortex of rats fed FPO than from those fed the control diet. In this cerebral region there was also a reduction in monoamine oxidase B (MAO-B) activity and greater binding to dopamine D₂ receptors. By contrast, a lower binding to dopamine D₂ receptors (7%) was observed in the striatum. Ambulatory activity was also reduced in FPO-fed rats, possibly related to observed changes in striatal dopaminergic receptors. This suggested that the level of (n-6) PUFA, which was considerably lower in the FPO diet than in the control diet, could act on locomotion through an effect on striatal dopaminergic function, whereas the high level of (n-3) PUFA could act on cortical dopaminergic function.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Cerebral membranes contain high amounts of polyunsaturated fatty acids (PUFA)⁴ which cannot be synthesized and must therefore be obtained from the diet. It has been demonstrated by several authors that a chronic dietary deficiency in the precursor of (n-3) PUFA, -linolenic acid, acts on the lipid composition of brain membranes, inducing low levels of (n-3) PUFA compensated for by high amounts of (n-6) PUFA, especially 22:5(n-6), and also alters the learning ability in rodents (Bourre et al. 1984 and 1989, Enslen et al. 1991, Lamptey and Walker 1978, Yamamoto et al. 1987 and 1988). However, fewer data are available concerning the effects of high dietary intake of (n-3) PUFA. Recent studies showed that high dietary fish oil, providing high levels of ecosapentaenoic acid [EPA or 20:5(n-3)] and docosahexaenoic acid [DHA or 22:6(n-3)], induced changes in brain PUFA composition characterized by high levels of EPA and DHA compensated for by low amounts of 20:4(n-6) (arachidonic acid), 22:4(n-6) and 22:5(n-6) (Bourre et al. 1988 and 1990). These modifications seemed to be greater using fish oil as the source of (n-3) PUFA instead of the precursor compound, -linolenic acid (Alsted and Hoy 1992). Moreover, providing high dietary fish oil during the fetal period in rats modified brain fatty acid (FA) composition and also improved learning ability (Yonekubo et al. 1993 and 1994). Improvement of learning ability by dietary (n-3) supplementation has recently been confirmed using Morris' water maze (Jensen et al. 1996). All these reports on the effects of dietary (n-3) PUFA on rat behavior are in accordance with an important role of this PUFA in cerebral function.

In addition, relationships between essential fatty acid (EFA) status and several human psychiatric diseases have recently been observed. In particular, several studies have shown that in some schizophrenics, especially those with negative symptoms, red cell membranes were depleted in arachidonic acid, the major PUFA from the (n-6) family, and also in DHA, the major PUFA from the (n-3) family (Kaiya et al. 1991, Peet et al. 1996).

We recently hypothesized that dietary (n-3) PUFA intake could act on behavior through effects on specific neurotransmission systems. Accordingly, we showed that a chronic -linolenic acid diet deficiency in rats induced changes in dopaminergic and serotoninergic function in several cerebral regions (Delion et al. 1994 and 1996). Major neurochemical changes occurred in the frontal cortex, and this finding was compatible with the type of behavioral disturbances already described with this deficiency.

In this study, we therefore examined the effects of a fish oil dietary supply, rich in DHA (5.3%) and EPA (9.8%), and poor in 18:2(n-6) (1.7%) on lipid FA composition of brain membranes, monoaminergic neurotransmission and behavior in 2-mo-old male rats. The fish oil diet was provided by a mixture of salmon oil and hydrogenated palm oil. Animals under this diet were compared to control rats receiving an adequate level of (n-3) PUFA (160 mg per 100 g of diet) and (n-6)/(n-3) ratio (Bourre et al. 1989). We studied the lipid composition of specific cerebral regions, i.e., striatum (STR), frontal cortex (FCX), hippocampus (HPC) and cerebellum (CB), and several neurochemical and behavioral parameters of monoaminergic neurotransmission. Specific neurochemical variables such as endogenous dopamine (DA), noradrenaline (NA) and serotonin (5-HT) levels, monoamine oxidase activity and several DA and 5-HT binding sites were measured in cerebral areas under monoaminergic regulation such as the STR, FCX and HPC and in the CB as a reference region poor in monoaminergic innervation. Moreover, these measurements were associated with several behavioral tests which involve these cerebral areas, such as locomotor activity and anxiety.

View larger version (29K): [in this window] [in a new window]   Fig 1. Composition of total phospholiipids in 16:0 (A), 18:0 (B) and 18:1(n-9) (C) fatty acids in the frontal cortex (FCX), striatum (STR), hippocampus (HPC) and cerebellum (CB) of 2-mo-old rats fed a control diet or a fish oil-enriched diet (FPO, fish oil + palm oil) from weaning. Data are mean ± SD, n = 6. *Significantly different from control, P < 0.05 (ANOVA).

View larger version (31K): [in this window] [in a new window]   Fig 2. Composition of total phospholipids in 22:6(n-3) (A), 20:4(n-6) (B) and sum of (n-6) and (n-3) PUFA (C) in the frontal cortex (FCX), striatum (STR), hippocampus (HPC) and cerebellum (CB) of 2-mo-old rats fed a control diet or a fish oil-enriched diet (FPO, fish oil + palm oil) from weaning. Data are mean ± SD, n = 6. *Significantly different from control, P < 0.05 (ANOVA).

View larger version (40K): [in this window] [in a new window]   Fig 3. Proportion of phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin (SM) in the frontal cortex (FCX), striatum (STR), hippocampus (HPC) and cerebellum (CB) of 2-mo-old rats fed a control diet or a fish oil-enriched diet (FPO, fish oil + palm oil) from weaning. Data are mean ± SD, n = 6. *Significantly different from control, P < 0.05 (ANOVA).

View larger version (24K): [in this window] [in a new window]   Fig 4. Endogenous monoamine concentrations in the frontal cortex (FCX), striatum (STR), hippocampus (HPC) and cerebellum (CB) of 2-mo-old rats fed a control diet or a fish oil-enriched diet (FPO, fish oil + palm oil) from weaning. Data are mean ± SD, n = 6. *Significantly different from control, P < 0.05 (ANOVA).

View larger version (34K): [in this window] [in a new window]   Fig 5. Monoamine oxidase A (A) and B (B) activities in the frontal cortex (FCX), striatum (STR), hippocampus (HPC) and cerebellum (CB) of 2-mo-old rats fed a control diet or a fish oil-enriched diet (FPO, fish oil + palm oil) for weaning. Data are mean ± SD, n = 6. *Significantly different from control, P < 0.05 (ANOVA).

	MATERIAL AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diets.  Two generations of female Wistar rats originating from our laboratory were given a diet containing 6 g/100 g of total lipids in the form of a mixture of 60.5% African peanut oil and 39.5% rapeseed oil. This control diet provided ~1200 mg of linoleic acid and 160 mg of -linolenic acid per 100 g of diet with an (n-6)/(n-3) ratio equal to 6. Two weeks before mating, 20 female rats originating from the second generation were divided into two groups. The first group of 10 females received the above diet, i.e., the control diet, the second group of 10 females received a diet in which the peanut + rapeseed oil mixture was replaced by a fish oil + palm oil mixture (50% salmon oil-50% hydrogenated palm oil; FPO diet) providing ~1000 mg of (n-3) PUFA and 140 mg of (n-6) PUFA. The (n-6)/(n-3) ratio was equal to 0.1 in the FPO diet (Tables 1 and 2). At weaning, the male progeny (third generation) of both groups were given free access to the same diet as their respective dams and water (10 litters/dietary group). Animals were housed two per cage under standard controlled conditions of temperature, humidity and dark-light cycles (08.00 h to 20.00 h). They were studied at 2 mo of age. The experimental protocol was in compliance with appropriate guidelines from "Ministère de l'Agriculture, France."

Lipid analysis of cerebral regions.  Six rats per group were used for lipid analyses. They were killed by decapitation and four cerebral areas, the frontal cortex (FCX), the striatum (STR), the hippocampus (HPC) and the cerebellum (CB) were removed on ice. Tissues were homogenized using a Polytron Kinematica PT 1200 (Bioblock Scientific, Strasbourg, France) in 5 mL of a chloroform/methanol solution (2/1; v/v) in the presence of butylhydroxytoluene (0.02 g/L). Total lipids were extracted according to the procedure of Folch et al. (1957). The phospholipids (PL) were then separated from total lipids on a silica gel column (Supelclean tube LC-SI, St Germain-en-Laye, France). An aliquot of the PL (total PL) was used for FA composition analysis. FA methyl esters were separated using a gas chromatograph (FID 80, Fisons Instrument GC 8000, Arcueil, France), equipped with an on-column injector and capillary column (CP wax 52 CB; 50 m × 0.32 mm; Chrompack, Les Ulis, France). Components were identified by their equivalent chain lengths in comparison with standards, and the peaks were integrated by Nelson 600 and the results transferred onto Lotus 123 (SRA, Marcy l'Etoile, France). Another aliquot of PL was used for separation of PL classes by HPLC (Gold System model 126, Beckman Instruments, Gagny, France) equipped with a light-scattering detector (Cunow DDL 11, Cunow Cergy Pontoise, France). Aditionally, a fraction of total PL was separated by HPLC and each class (phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC) and sphingomyelin (SM)) was related to the total amount of PL. Significant differences between dietary groups were tested using one-way ANOVA and were considered significantly different when P < 0.05.

Determination of endogenous monoamine concentrations.  Rats (eight per group) were killed by decapitation, brains were rapidly dissected on ice, and the FCX, STR, HPC and CB were removed. Tissues were weighed, homogenized with an Ultra Turax T25 (Bioblock Scientific, France) in 0.5 mL perchloric acid (0.2 mol/L), EDTA (1 g/L) and Na₂S₂O₅ (1 g/L), and then centrifuged at 7310 × g (J2-21 M/E centrifuge and JA 21 rotor, Beckman Instrument, Gagny, France) for 10 min at 4°C. Supernatants were kept at 80°C until use. The serotonin (5-HT), dopamine (DA) and noradrenaline (NA) levels were determined by HPLC with electrochemical detection (electrodetector model 460, Waters Millipore, Milford, MA) according to a previously described procedure (Delion et al. 1994). The results were compared between the different dietary groups by one-way ANOVA, and values were considered significantly different when P < 0.05.

Autoradiographic studies.  S()-sulpiride, (+)-butaclamol hydrochloride and desipramine hydrochloride were purchased from RBI (Natick, MA), ketanserin tartrate was purchased from Janssen Research Products (Geel, Belgium), pargyline was obtained from Sigma (Saint-Quentin Fallavier, France) and mazindol was a gift from Sandoz (Rueil-Malmaison, France). [³H]-SCH-23390 (specific radioactivity, 2630.7 GBq/mmol), [³H]-YM-09151-2 (specific radioactivity, 3011.8 GBq/mmol), [³H]-spiperone (specific radioactivity, 799.2 GBq/mmol) and [³H]-mazindol (specific radioactivity, 629 GBq/mmol) were obtained from NEN (Paris, France).

Eight animals were used per dietary group. They were killed by decapitation and brains were removed and frozen; 20 µm-thick coronal sections were cut at 20°C using a cryostat (Jung Cryocut 1800, Leica, Rueil-Malmaison, France) and mounted onto clean gelatined microscope slides.

Binding to dopamine D₁ receptors, dopamine D₂ receptors, dopamine transporters and serotonin 5-HT₂ receptors was measured with specific tritiated ligands for each binding site, i.e., [³H]-SCH-23390, [³H]-YM-09151-2, [³H]-mazindol and [³H]-spiperone, respectively, using previously described procedures (Delion et al. 1994). In all experiments, sections were exposed to tritium-sensitive films (³H-Hyperfilms, Amersham, Les Ulis, France) with calibrated ³H-radioactive standards (Microscales, Amersham) for appropriate periods, i.e., 9 d for D₁ receptors, 5 wk for D₂ receptors, 5 wk at 4 °C for dopamine uptake sites and 3 mo at 4°C for 5-HT₂ receptors. The intensity of receptor binding was assessed using a computer-imaging system (IMSTAR, Paris, France). Specific binding was the difference between total binding in four serial sections minus nonspecific binding in four adjacent sections. Means obtained from the two group sections exposed on the same sensitive film were compared using Student`s t test for paired values and were considered significantly different when P < 0.05.

Monoamine oxidase assays.  2,5-Diphenyl-oxazole (PPO) was purchased from Sigma. 5-[¹⁴C]-hydroxytryptamine creatinine sulfate ([¹⁴C]-5-HT, specific radioactivity, 2.04 GBq/mmol) was obtained from Amersham, and [¹⁴C]-phenylethylamine hydrochloride ([¹⁴C]-PEA, specific radioactivity, 1.55 GBq/mmol) was obtained from NEN.

Eight animals were used per dietary group. They were killed by decapitation, the brains were quickly removed, and FCX, STR, HPC and CB were dissected on ice. The separated regions were homogenized with an Ultra Turax model T25 (Bioblock Scientific) in 10 volumes (wt/v) of ice cold 0.1 mol/L phosphate buffer (pH 7.4), and the resulting homogenates were used as enzyme sources to assay MAO activity.

MAO [EC 1.4.3.4., amine: oxygen oxydoreductase (deaminating) (flavine containing)] activity was radiochemically assayed by a conventional method (high substrate concentration method) using a previously described procedure (Delion et al. 1994). Statistical analyses were performed on the experimental data between the different dietary groups using one-way ANOVA. Differences were considered significantly different when P < 0.05.

Behavior studies.  Eight to 11 rats were studied in each behavior test.

Ambulatory activity test.  Animals were placed singly in a novel environment consisting of a circular corridor (10 cm wide and with walls 70 cm high, inner diameter 55 cm) made of steel with infrared photocells placed 2.5 cm above the grid floor at each cardinal point. A beam interruption of 0.25 s was required to activate the counters, and rapid movements were thus not registered. The activity count therefore represented basically ambulatory activity. The number of photocell interruptions was registered over 60 min following introduction of the animal in the apparatus.

Elevated plus maze.  The elevated plus-maze consisted of two opposing 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 (total entries). The proportion of entries into the open arms (open arm/total entries) was then calculated.

Statistics.  Comparisons between groups were done using the Mann-Whitney test.

	RESULTS

Abstract Introduction Methods Results Discussion References

Body weights.  The fish oil diet had no effect on the body weight of the rats. At the time of study, the FPO group weighed 250 ± 18 g versus 251 ± 30 g for the control group.

Lipid analysis of cerebral regions. 

FA composition of total PL.  Levels of saturated fatty acids (SFA) were modified in the FPO group compared to controls with a lesser amount of 16:0 in the STR and CB (Fig. 1A) and a greater amount of 18:0 in the FCX, STR and CB (Fig. 1B). The levels of the major monounsaturated fatty acid (MUFA), 18:1(n-9), were significantly greater in the FPO group than in controls in all the cerebral structures studied (Fig. 1C).

As expected, differences in FA composition between groups principally involved PUFA. The level of the major (n-3) PUFA, 22:6 (n-3), was greater in the STR and HPC of rats fed FPO (Fig. 2A). By contrast, the major (n-6) PUFA, 20:4 (n-6), was 30-50% lower in all four cerebral structures studied of rats fed FPO (Fig. 2B). The (n-6)/(n-3) ratio was 50% lower in the FPO group in all cerebral regions (about 0.5% vs 1% in the control group).

These differences in PUFA levels resulted in a lower total (n-6) + (n-3) PUFA level in the four areas studied in the FPO group than in controls (Fig. 2C).

Proportions of PL classes.  Proportions of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylinositol (PI) were not modified or were only slightly modified in the four cerebral regions of the FPO group compared to controls (Fig. 3). The proportion of sphingomyelin (SM) in the FPO group was not affected in the CB but was significantly lower in the other cerebral regions. By contrast, the proportion of phosphatidylserine (PS) was 30-100% greater in the four cerebral regions of the rats fed FPO than in controls (P < 0.05).

Endogenous monoamine concentrations.  Concentrations of monoamines (NA, DA and 5-HT) in the FCX, STR, HPC and CB are shown in Fig. 4, except for DA which was almost undetectable in the HPC and CB with our method.

NA levels were identical for the FPO and control groups in the four cerebral regions studied. 5-HT levels were similar in both groups in the STR, HPC and CB. However, a 20% higher level was observed in the FCX in the FPO group compared to the control group. In the FCX, DA levels were 40% greater than in the FPO group, while in the STR, DA levels tended to be lower in the FPO group (0.05 < P < 0.1).

Autoradiographic studies.  High intensity of binding to dopamine D₁ receptors was determined with [³H]-SCH-23390 in the STR (135 pmol/g wet tissue). There was no difference between groups in the specific binding of the radiolabeled ligand (Table 3).

High numbers of dopamine D₂ receptors were labeled in the STR but represented ~50% of the density of D₁ receptors. [³H]-YM-09151-2 also bound to D₂ receptors in the FCX (~10 pmol/g wet tissue). In the STR the FPO diet induced 7% lower specific binding (P < 0.05) compared to the control diet. In the FCX, the FPO diet induced 10% higher specific binding (P < 0.05) compared to the control diet.

A high density of dopamine uptake sites was observed using [³H]-mazindol in the STR, but the signal measured in the FCX was too low to be detected with the method used. There was no difference in the binding of [³H]-mazindol between the two dietary groups. The density of serotoninergic 5-HT₂ sites in the FCX did not differ between groups.

MAO activity assay.  A predominance of MAO-A over MAO-B activity (~60% versus 40%) was observed in all four cerebral regions (Fig. 5). Diet treatment did not affect MAO-A activity, but MAO-B activity in the FCX was 25% lower in rats fed the FPO diet than in thoses fed the control diet (P < 0.05).

Behavioral studies. 

Ambulatory activity test.  Ambulatory activity was 25% lower in the FPO group than in controls (P = 0.03). In FPO-fed rats the number of photocell crossings per h was 335 ± 18 versus 404 ± 24 in controls.

Elevated plus maze.  There was no significant effect of diet on the proportion of total entries or open arm/total entries (Table 4, P < 0.05).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

In this study fish oil-enriched diet modified the FA composition of the rat brain and also acted on several neurochemical and behavioral aspects of monoaminergic functions. We used a diet containing a mixture of fish oil and hydrogenated palm oil (FPO diet) to enhance dietary (n-3) PUFA supply. However, this diet also contained a 88% lower level of (n-6) PUFA than the control diet, consistent with the diet used by Yonekubo et al. (1994). With this diet, the supply of (n-3) PUFA was 15 times greater than the minimal requirement for adequate (n-3) PUFA levels in cerebral membranes (Bourre et al. 1993). The major change in the composition of dietary lipids in the FPO diet was a sixfold increase in total (n-3) PUFA, compensated for by a dramatic reduction in total (n-6) PUFA. However, the FPO diet also differed from the control diet in other FA families with reduced levels of MUFA and enhanced levels in SFA. The FPO diet induced modifications in FA composition of the brain regions studied, i.e., the striatum, frontal cortex, hippocampus and cerebellum. Little modification was observed in the levels of major SFA, 16:0 and 18:0, in brain membranes. By contrast, generally greater levels of the major MUFA, 18:1(n-9), was observed in all the cerebral regions studied. It seemed, therefore, that MUFA levels in brain membranes were not directly linked to MUFA levels in the diet, as has already been observed in whole brain and in liver from rats receiving a fish oil enriched diet (Bourre et al. 1990). Several differences in FA composition of brain membranes occurred for (n-3) PUFA and, more importantly, for (n-6) PUFA. The fish oil-enriched diet induced a rise in the major (n-3) PUFA, DHA, in the striatum and hippocampus, whereas no change occurred in the frontal cortex and cerebellum. By contrast, a dramatic reduction in the levels of the major (n-6) PUFA, 20:4(n-6), was observed in all four cerebral regions. This greater level of (n-3) PUFA, compensated for by lower levels of (n-6) PUFA, has already been described (Bourre et al. 1988 and 1990). However, it must be emphasized that there was not a complete balance between the variations in (n-6) and (n-3) PUFA families. This was expressed in the evolution of the total (n-6) + (n-3) PUFA levels which were reduced in the four cerebral regions studied. In addition, it appears from the present work that the different cerebral regions studied have specific sensitivity to (n-3) availability from the diet. This type of specific response has already been observed in chronic -linolenic acid deficiency, which induces reduction in DHA levels which are less pronounced in the frontal cortex than in the striatum and cerebellum (Delion et al. 1994).

The present study also showed that the fish oil-enriched diet affected the proportions of specific phospholipid classes, i.e., SM and especially PS, whereas other classes, i.e., PE, PC and PI were scarcely modified, if at all. In contrast, no differences in these proportions were observed in rats fed an -linolenic acid-deficient diet (Delion et al. 1994). Very little information is available on the proportion of different PL classes in brain membranes and on the effects of these proportions on cerebral functions. However, the composition of PL classes of cerebral membranes might change during aging (Delion et al. 1996 and 1997, Gaiti et al. 1982) and also in subjects suffering from Alzheimer's disease (Södeberg et al. 1992), but the meaning of these modifications remained unclear. Moreover, PS could have specific physiological and pharmacological effects on cerebral functions. Indeed, several studies have shown that endogenous PS can activate protein kinase C, Na⁺/K⁺-dependent ATPase, tyrosine hydroxylase and calcium uptake, and that exogenous PS can improve cognitive deficits associated with age (reviewed by Pepeu et al. 1996). It can therefore be assumed that the rise in cerebral proportions of PS induced by the dietary fish oil enrichment might have contributed to the changes that we observed in specific neurotransmission systems.

Because dietary fish oil had effects on lipid composition of several brain regions, it was valuable to relate these changes to neurochemical and behavioral parameters associated with these regions. We observed a reduction in locomotor activity in the (n-3) PUFA supplemented group, in agreement with previous results (Mills et al. 1988, Nakashima et al. 1993). However, our results did not distinguish between a reduced locomotion and a more rapid habituation to the experimental apparatus. This finding can be related to the tendency for reduction in DA levels and to the lower binding to dopamine D₂ receptors observed in the striatum in the FPO group, since decreased activity of the nigro-striatal dopaminergic pathway induces impairment in locomotor activity. The reduction of binding to dopamine receptors could be related to changes in the density and/or in the affinity of binding sites. We have previously observed that the -linolenic acid diet deficiency did not alter endogenous DA levels nor dopamine D₂ receptors in the striatum (Delion et al. 1994 and 1996), in agreement with the normal locomotor activity described in this deficiency (Yehuda et al. 1986).

Dietary (n-3) PUFA supplementation greatly increased the endogenous DA levels in the frontal cortex. This could be related to the significant reduction in cortical MAO-B activity measured in this dietary group since MAO-B is involved in the degradation of monoamines (Tipton et al. 1976). In agreement with this hypothesis, increases in DA release have also been observed after administration of MAO inhibitors in the rat (Butcher et al. 1990). In addition to the rise in DA levels in the frontal cortex in our study, a slightly higher binding to dopamine D₂ receptors was measured in the FPO group compared to controls. This must be related to the significant reduction in dopamine levels and the binding to D₂ receptors previously observed in dietary -linolenic acid-deficient rats (Delion et al. 1994). It seems, therefore, that dietary (n-3) PUFA might specifically act on the dopaminergic cortical function. These findings have recently been taken as the basis for a dopamine hypothesis linking (n-3) PUFA and cerebral function (Reisbick and Neuringer 1997). We demonstrated recently that the -linolenic acid deficiency could act on dopaminergic neurotransmission through effects on presynaptic dopamine storage processes (Zimmer et al. 1998).

In the present study, we found no effect of diet in the elevated plus maze, an animal model of anxiety (Pellow et al. 1985); this confirmed results already obtained by Nakashima et al. (1993). In addition, a slight rise in 5-HT levels was observed in the frontal cortex which remains unexplained.

In conclusion, we have shown that a fish oil diet affected several neurochemical and behavioral features of monoaminergic function. Few data have to date indicated the effects of the main PUFA of the (n-6) and (n-3) families, arachidonic acid and docosahexaenoic acid, on dopaminergic (L'Hirondel et al. 1995) and GABAergic (Hamano et al. 1996, Witt et al. 1994 and 1996) neurotransmissions, but there have been only in vitro experiments. We observed a reduction in locomotor activity accompanied by a decrease in binding to dopamine D₂ receptors in the striatum, but it must be emphasized that major neurochemical changes appeared in the frontal cortex. In this specific cerebral region a rise in endogenous DA levels occurred accompanied by a reduction in MAO-B activity and a slight increase in binding to dopamine D₂ receptors. These changes suggest a rise in cortical dopaminergic function compared to controls; moreover, they are the opposite of those observed in -linolenic acid-deficient rats (Delion et al. 1994 and 1996, Zimmer et al. 1998). We hypothesize that these modifications of cortical dopaminergic neurotransmission are related to learning ability, which is affected by -linolenic acid diet deficiency (Bourre et al. 1984 and 1989, Enslen et al. 1991, Lamptey and Walker 1978, Yamamoto et al. 1987 and 1988), and improved after (n-3) PUFA dietary supplementation (Jensen et al. 1996, Yonekubo et al. 1993 and 1994).

These results suggest that the level of (n-6) PUFA, which was low in the FPO diet, could affect locomotion, whereas the level of (n-3) PUFA could act on cognitive processes through effects on dopaminergic cortical function.

	FOOTNOTES

Manuscript received 22 April 1998. Initial reviews completed 15 June 1998. Revision accepted 31 August 1998.

	ACKNOWLEDGMENTS

We thank Jean-Paul Macaire, Alain Limard, Serge Barreau and Sylvie Bodard for their excellent technical assistance, and Doreen Raine for editorial assistance.

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