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Department of Paediatrics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4
2To whom correspondence should be addressed. E-mail: sinnis{at}interchange.ubc.ca.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • docosahexaenoic acid • brain • behavior • dopamine • piglets • dietary fatty acids
The nonmyelin membranes of the central nervous system contain high concentrations of the long-chain PUFA docosahexaenoic acid [22:6(n-3)], which is selectively enriched in synaptic plasma membrane phosphatidylethanolamine (PE)2 and phosphatidylserine (PS), as well as in the (n-6) fatty acid arachidonic acid [20:4(n-6)] (1). The amount and types of (n-6) and (n-3) fatty acids in the diet of young infants are of considerable interest because human milk contains both 22:6(n-3) and 20:4(n-6), whereas fats of vegetable origin, and until recently all infant formulas, contain the precursor essential fatty acids 
-linolenic acid [18:3n-3)] and linoleic acid [18:2(n-6)], respectively, but no 22:6(n-3) or 20:4(n-6) (2). It is well established that dietary deficiency of (n-3) fatty acids results in a decrease in 22:6(n-3), with a compensatory increase in C-22(n-6) fatty acids in the developing brain (1). In rodents, the severe depletion of brain 22:6(n-3) that results from feeding a diet restricted in (n-3) fatty acids through two or more generations is accompanied by decreased exploratory activity in novel environments and decreased performance in brightness discrimination, habituation and olfactory-based learning tasks (1,3–8). Changes in looking behavior in a novelty task, increased stereotyped behavior and polydypsia have also been reported in monkeys who were deprived of (n-3) fatty acids in gestation and postnatal life (9,10). More recent studies addressing the biological mechanisms that explain the relationship between 22:6(n-3) and behavior have shown that diet-induced depletion of 22:6(n-3) from the brain is accompanied by altered metabolism of dopamine, a key neurotransmitter that is involved in many of the cognitive advances of early childhood (11–14).
Studies of autopsy tissue have reported lower 22:6(n-3) and higher levels of 20:4(n-6), 22:4(n-6) and 22:5(n-6) in brain phospholipids of human infants who had been fed formulas containing 18:2(n-6) and 18:3(n-3) but no 20:4(n-6) or 22:6(n-3) than in infants who had been breast-fed (15). The functional importance of the differences in brain (n-3) and (n-6) fatty acids that result from a relatively short duration of postnatal feeding with diets of differing fatty acid composition in term gestation infants, however, is still unclear. Randomized trials with term infants fed formulas with 22:6(n-3) and 20:4(n-6) compared with formulas with no 22:6(n-3) or 20:4(n-6) have reported both beneficial effects and no effects on measures of neural development (16–21). One of the difficulties in studies with human infants is the inability to control genetic and environmental variables other than the formula diet that may also influence scores on tests of cognitive development. We have shown that brain and synaptic membrane 22:6(n-3) and dopamine are lower in piglets fed a milk-substitute diet low in (n-3) fatty acids for as little as 2–3 wk after birth than in piglets fed formulas resembling milk with 18:3(n-3) and 22:6(n-3) (11,22,23). Piglets can be raised in a controlled environment, and are readily bottle-fed from birth, thus enabling manipulation of dietary fatty acid intake without affecting other nutrients. In addition, the contribution of fat to the total energy in milk, and the composition of the milk fatty acids in pig milk is similar to that of humans (24). Therefore, we sought to establish a behavioral test for piglets that is responsive to diet-induced changes in brain 22:6(n-3) and to perturbation of brain dopamine. Such a model would enable further studies to delineate that effect of specific dietary (n-3) and (n-6) fatty acids, their balance and requirements in the early postnatal period on both the composition of the brain fatty acids and their effects at the behavioral level.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Yorkshire piglets (Kintail Meats, Langley, Canada) were fed from d 1of life with one of two diets that were identical in composition (25) except for the fat, n = 6/group. One of diets contained 1.2% energy 18:2(n-6) and 0.05% energy 18:3(n-3) as the only (n-6) and (n-3) fatty acids (low PUFA diet). The other diet contained 10.7% energy 18:2(n-6), 1.1% energy 18:3(n-3), 0.3% energy 20:4(n-6) and 0.3% energy 22:6(n-3) (high PUFA diet), (Table 1). The lower amount of PUFA in the low PUFA diet was compensated for by higher amounts of saturated fatty acids, particularly 12:0 and 14:0 compared with the high PUFA diet. The amounts of (n-6) and (n-3) fatty acids in the high PUFA diet were chosen to be within the range present in human milk (2,25,26). Arachidonic acid [20:4(n-6)] was included because this fatty acid is always present in human milk and pig’s milk and because studies with preterm infants have shown that supplementation with 22:6(n-3) without the addition of 20:4(n-6) can reduce blood lipid 20:4(n-6) and growth (27–29). The 20:4(n-6) and 22:6(n-3) were provided as single cell triglycerides, with 20:4(n-6) from the fungus Mortierella alpina and 22:6(n-3) from the algae Crypthecodinium cohnii (Martek Biosciences, Columbia, MD). The housing and care of the piglets were identical to that reported previously (11,25). The procedures were approved by the Animal Care Committee of the University of British Columbia and conformed to the guidelines of the Canadian Council on Animal Care.
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The elevated plus maze was based on that described by Andersen et al. (30), with modification appropriate for the size of 18- to 30-d-old piglets. The maze consisted of two opposing open arms and two opposing enclosed arms in the shape of a plus sign that was elevated 1 m above the ground. The two open arms were 1.25 m x 0.5 m with a 3-cm surrounding ledge to prevent the piglets from falling. The two enclosed arms measured 1.25 m x 0.5 m with 3 enclosed sides and 0.6-m high walls. The maze was located in a separate animal testing facility with plain walls and no visual cues. Piglets were tested individually. For testing, each piglet was placed in the center of the maze facing a closed arm; behavior was recorded for 5 min using a video camera equipped with a CCTV lens, aperture 3.5–8.0 mm F1.4, a time lapse video cassette recorder and a video monitor (National Electronics, Vancouver, Canada). Tapes were coded, then scored for the number of open arm entries, number of closed arm entries, time spent in the open arms and time spent in the closed arms. Each tape was scored by two independent observers. All of the piglets were tested between 0900 and 1000 h, based on preliminary studies with 24-h video-recording of natural behavior to assess peak activity in the usual diurnal cycle. Preliminary studies were also done to assess the duration of changes in behavior in the maze task after drug administration, with assessments of behavior at 30-min intervals up to 8 h, then at 24 h after oral dosing. No effects on behavior were evident at 8 or 24 h after drug administration. The reported half-life of an oral dose of sulperide is 6.3–8.4 h (31), and of L-dihydroxyphenylalanine (L-Dopa) with carbidopa is 2 h (32). Home cage behavior was assessed by video-recording piglets from 0900 to 1100 h in their home cage in groups of three per diet group at 26 d of age. The tapes were coded and scored for stereotyped behavior (snout-rubbing), biting, head thrusting and activity (upright/lying).
A randomized cross-over design was used to investigate behavior in the elevated plus maze and changes in behavior in response to pharmacologic manipulation of brain dopamine in piglets fed the low and high PUFA diets. The behavior of all piglets was tested on the elevated plus maze at 18 d of age. Then, at 20 d of age, three piglets in each diet group were given sulperide (20 mg/kg) (Sigma-Aldrich Canada, Oakville, Canada), a dopamine D2 receptor blocker, and tested 2 h later on the elevated plus maze. The remaining 3 piglets in each diet group were given the dopamine precursor L-dihydroxyphenylalanine (L-Dopa; 45 mg/kg), with Carbidopa (1 mg/kg) (Sigma-Aldrich Canada) (33,34) and tested 2 h later on the elevated plus maze. Carbidopa is an aromatic amino acid decarboxylase inhibitor that blocks peripheral dopamine degradation, thus allowing a greater proportion of the L-Dopa administered to reach the brain. At 22 d of age, the treatments were reversed and piglets retested.
Tissue collection.
At 30 d of age, piglets were anesthetized (ketamine hydrochloride, 37.5 mg/kg, MTC Pharmaceuticals, Cambridge, Canada; and xylazine hydrochloride, 3.75 mg/kg, Bayvet Division, Chenango, Etobicoke, Canada) then killed by intracardiac injection of 200 mg/kg pentobarbital. The brain was rapidly removed and weighed, and the frontal cortex was dissected and immediately frozen in liquid nitrogen and stored at -80°C until analysis.
Analytical methods.
Frontal cortex total lipids were extracted (34) and phosphatidylcholine (PC), PE, PS and phosphatidylinositol (PI) were separated using a HPLC [Waters 2690 Alliance HPLC (Milford, MA)], equipped with an autosampler and column heater with a Waters YMC-Pack Diol 120NP, 25 cm x 4.6 mm i.d., normal phase column, 5-µm particle size and 12-nm pore size column. The solvent system was a quarternary system of (by volume) hexane/petroleum ether, 97:3; methanol/triethylamine/acetic acid 765:15:13; acetone/triethylamine/acetic acid, 765:15:13; and isopropanol/acetic acid, 800:40 in a linear gradient with a flow rate of 2 mL/min (35). The column eluant was split 10:90 to an evaporative light scattering detector (Alltech, model 2000, Mandel Scientific, Guelph, Canada) and a fraction collector (Gilson FC204, Mandel Scientific). Fatty acid components in separated phospholipids were quantified as their respective FAME using a Varian 3400 GLC equipped with a flame ionization detector, Varian Star data system and a 30 m x 0.25 mm i.d. glass capillary SP 2330 column (11,25).
Statistical analyses.
Significant differences between the treatment groups for behavioral data were analyzed by two-way ANOVA. When significant effects were found, formal tests for significant difference due to diet or drug treatment were made using least significant difference, with preplanned comparisons to assess the effects of no drug or a specific drug within and between the diet groups. Differences were considered significant at P < 0.05. Significant differences between treatment groups for frontal cortex phospholipid fatty acids and home cage behavior testing were analyzed by independent t test. The assessment of home cage behavior considered each group of three piglets as n = 1, with n = 2/diet group. All analyses were done using SPSS version 10.0 (Chicago, IL).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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30, 22 and 40%, in frontal cortex PE, PS and PC compared with piglets fed the high PUFA diet (P < 0.01) (Table 2). In addition, piglets fed the low PUFA diet had significantly lower 20:5(n-3) and 22:5(n-3) and higher 20:4(n-6) and 22:4(n-6) in frontal cortex PE and PS, and lower 20:5(n-3) and higher 22:5(n-6) in frontal cortex PC than piglets fed the high PUFA diet to 30 d of age. Differences in the fatty acid composition of PI were small and involved lower 22:4(n-6) and higher 20:5(n-3), both of which represented 
1.0% frontal cortex fatty acids, in piglets fed the low compared with the high PUFA diet.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Many studies have shown that a diet deficient in (n-3) fatty acids results in reduced 22:6(n-3) in developing brain, with a compensatory increase in (n-6) fatty acids, particularly 22:5(n-6) (1,3–10,22,23,36,37). The decrease in brain 22:6(n-3) and increase in (n-6) fatty acids in rodents fed a (n-3) fatty acid-deficient diet through several generations is accompanied by changes in behavior (3–8), decreased endogenous dopamine and dopamine D2 receptors in frontal cortex, but not in the cerebellum or striatum (12,13), and a specific reduction in dopamine vesicles in presynaptic terminals of the frontal cortex (37). In addition, prolonged (n-3) fatty acid deficiency in rats has been shown to result in decreased extracellular dopamine in nucleus accumbens, decreased dopamine release in response to stimulation and reduced expression of the vesicular monoamine transporter2 in frontal cortex and nucleus accumbens (14,38,39) Our previous studies also found decreased endogenous dopamine in frontal cortex of piglets fed a diet deficient in (n-3) fatty acids for 18 d after birth (11). The available evidence thus suggests that brain 22:6(n-3) may be involved in regulation of dopamine synthesis, the dopamine vesicular pool, and potentially also D2 receptor binding, although the mechanisms of these effects remain to fully elucidated.
The importance of a dietary source of 22:6(n-3) compared with its precursor 18:3(n-3) and the effect of the dietary balance of 18:2(n-6) to 18:3(n-3) on the desaturation and elongation of 18:3(n-3) with respect to the provision of 22:6(n-3) for optimal growth and development of the brain and retina after term birth are still incompletely understood (15–21). The newborn piglet brain weighs 13 g at birth and increases threefold to 52 g at 30 d of age; body weight increases over fourfold from 1.5 kg at birth to 7.5 kg at 30 d in piglets raised in our facility. This rapid increase in brain and body weight during the first 4 wk after birth in piglets, together with the ease of bottle-feeding with a milk replacer of defined fat composition resembling human milk or milk substitutes, facilitates studies on the effects of dietary fat on the composition of newly formed membrane lipids. However, although sensitive tests of behavior have been used extensively in studies with rodents fed (n-3) fatty acid-deficient diets, behavioral measures to elucidate the physiologic significance of early differences in dietary lipid intake on the developing piglet brain are not well established.
Our studies clearly showed that diet-induced decreases in brain 22:6(n-3) resulting from differences in the composition of fat fed in the first 2–3 wk after birth are accompanied by altered behavior in young piglets. Because the diets fed also resulted in differences in frontal cortex (n-6) fatty acids, which included higher PE, PS and PC 22:5(n-6) and lower PE and PC 18:2(n-6) in piglets fed the low compared with the high PUFA diet, we cannot conclude a specific cause and effect between brain 22:6(n-3) and behavior. The interpretation of differences in behavior on the elevated plus maze, however, is complex and may involve independent or interactive effects on anxiety/fear, novelty seeking and exploratory behavior, and activity. The elevated plus maze is a commonly used test of fear in rodents, and when adapted for studies with piglets, pharmacologic agents that reduce anxiety resulted in decreased avoidance of entry into the open arms of the maze (36). The total number of arm entries and entries into the closed arms of the maze, on the other hand, appear to be a reflection of measures of activity, which allows the test to separate activity and fear of novelty (26,30,40). In our studies, piglets fed the low PUFA diet had a significantly lower number of total arm entries and entries into the open arms of the maze, and spent significantly longer in the closed, but not open arms, than piglets fed the high PUFA diet. Although this pattern suggests decreased activity, it is also consistent with increased fear/anxiety of residence in the open arms of the maze. The acute administration of L-Dopa with Carbidopa significantly increased the number of entries into the closed arms in piglets fed both the low and high PUFA diets. In addition, the decreased average amount of time spent per entry into the closed arms of the maze by piglets fed the low PUFA fatty acid diet when given L-Dopa with Carbidopa further suggests a relation between decreased brain 22:6(n-3), dopamine and decreased activity and/or increased fear/anxiety. Consistent with this suggestion, increases in extracellular dopamine in rodents have been shown to increase activity (41), whereas decreased levels of dopamine in the frontal cortex are associated with slow exploration and increased levels of fear (42). Takeuchi et al. (43) recently reported that second generation rats fed a (n-3) fatty acid-deficient diet had fewer entries into the open arms of an elevated plus maze and increased stress behaviors, which were reduced by supplementation with 22:6(n-3) (43). This further supports the possibility that decreased activity and increased time spent in the closed arms of the maze by piglets with low brain 22:6(n-3) may be explained by increased anxiety/fear rather than decreased activity. However, the dopaminergic pathway of nucleus accumbens, which is also affected by (n-3) fatty acid deficiency (14,38), is known to be involved in locomotor activity (44–46).
In the studies reported here, as in previous work (4–12,22,23,36,37), the decrease in frontal cortex 22:6(n-3) in piglets fed the low PUFA diet was accompanied by an increase in 22:5(n-6), 22:4(n-6) and 20:4(n-6) in specific phospholipids, even though this diet was relatively low in 18:2(n-6). This raises the possibility that biochemical or behavioral changes associated with reduced brain 22:6(n-3) may reflect a specific essential role of 22:6(n-3), or may be secondary to perturbation of normal brain (n-6) fatty acid metabolism. Recent studies have shown that 22:6(n-3) is involved in G-protein-coupled receptor activity (47). In addition, 22:6(n-3) inhibits both Ca2+-ATPase and Na,K-ATPase in cerebral cortex synaptosomal membranes (48). However, 20:4(n-6) also plays important roles in many aspects of signal transduction in neural tissues, which also include G-protein–coupled receptors, ion channel activities and dopamine release (49–51). Thus, although our studies showed that the diet-induced depletion of postnatal piglet frontal cortex 22:6(n-3) results in altered behavior on an elevated plus maze, which is modifiable by acute administration of L-Dopa, it is possible that the changes in behavior involve changes in dopamine metabolism in regions other than frontal cortex, and may be the result of the effects of the diet on brain (n-6) fatty acids rather than specific effects of reduced 22:6(n-3). Our studies also do not preclude the possibility that altered metabolism of other neurotransmitters, including serotonin (11,13), is involved in the altered behavioral response in animals fed (n-3) fatty acid–deficient diets.
In summary, we showed that measures of behavior on an elevated plus maze provide a simple functional assessment that is sensitive to depletion of brain 22:6(n-3) and the accompanying changes in (n-6) fatty acids in neonatal piglets fed a diet low in (n-3) fatty acids during early postnatal development. We also showed that treatment with L-Dopa decreases the time spent in the closed arms and increases activity on the maze test in piglets with diet-induced depletion of brain 22:6(n-3), suggesting that these parameters are influenced by increasing brain dopamine. Our work establishes the neonatal piglet as a model with which to study the behavioral effects of diet-induced changes in brain 22:6(n-3); this may useful in studies to elucidate the functional significance of differences in brain (n-3) and (n-6) fatty acids that result from differences in dietary intake and balance of these fatty acids.
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FOOTNOTES |
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3 Abbreviations used: L-Dopa, L-dihydroxyphenylalanine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine.
Manuscript received 10 April 2003. Initial review completed 11 June 2003. Revision accepted 3 July 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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different from piglets fed the same diet with no drug, P < 0.05.