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Dietary n-3 fatty acid restriction during gestation in rats: neuronal cell body and growth-cone fatty acids^1,2,3

¹ From the Department of Paediatrics, University of British Columbia, Vancouver, Canada.

² Supported by a grant from the Medical Research Council, Canada.

³ Reprints not available. Address correspondence to SM Innis, University of British Columbia, Department of Paediatrics, BC Research Institute for Child and Family Health, 950 West 28th Avenue, Vancouver, V5Z 4H4, Canada. E-mail: sinnis{at}unixg.ubc.ca.

	ABSTRACT

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Growth cones are membrane-rich structures found at the distal end of growing axons and are the predecessors of the synaptic membranes of nerve endings. This study examined whether n-3 fatty acid restriction during gestation in rats alters the composition of growth cone and neuronal cell body membrane fatty acids in newborns. Female rats were fed a standard control diet containing soy oil (8% of fatty acids as 18:3n-3 by wt) or a semisynthetic n-3 fatty acid–deficient diet with safflower oil (0.3% of fatty acids as 18:3n-3 by wt) throughout normal pregnancy. Experiments were conducted on postnatal day 2 to minimize the potential for contamination from synaptic membranes and glial cells. Dietary n-3 fatty acid restriction resulted in lower docosahexaenoic acid (DHA) concentrations and a corresponding higher docosapentaenoic acid concentration in neuronal growth cones, but had no effects on neuronal cell body fatty acid concentrations. These studies suggest that accretion of DHA in growth cones, but not neuronal cell bodies, is affected by n-3 fatty acid restriction during gestation. Differences in other fatty acids or components between the semisynthetic and the standard diet, however, could have been involved in the effects on growth-cone DHA content. The results also provide evidence to suggest that the addition of new membrane fatty acids to neurons during development occurs along the shaft of the axon or at the growth cone, rather than originating at the cell body.

Key Words: Brain • growth cones • docosahexaenoic acid • 22:6–3 • arachidonic acid • 20:4–6 • rats • n–3 fatty acids

	INTRODUCTION

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The structural membrane lipids of the central nervous system and retina are characterized by the presence of large amounts of docosahexaenoic acid (DHA; 22:6n-3) which is accumulated largely during growth and development. The accretion of DHA in synaptic endings of the brain and in the retina is reduced when the dietary intake of n-3 fatty acids, including DHA and -linolenic acid (ALA; 18:3n-3), is restricted during gestation and lactation (1, 2). The decrease in DHA due to dietary n-3 fatty acid deficiency is accompanied by a compensatory increase in the amounts of the corresponding n-6 fatty acid, docosapentaenoic acid (DPA; 22:5n-6).

The formation of synaptic membranes occurs late in neuronal maturation (3). As neuronal proliferation nears completion, the differentiation process begins with axon sprouting followed by the formation of growth cones, axon elongation, pathfinding, target selection, and a final transformation of growth cones to mature synaptic endings. The maturation of growth cones to synapses is accompanied by changes in fatty acid composition (4). Growth cones are 75% lipid, are morphologically and compositionally distinct from synapses (5), and are thought to play a role in guiding axons to their often distant targets.

Time-lapse cinematography of axon extension in cell culture has shown growth cones appearing as large, flattened, veil-like, membrane-rich surfaces with considerable motility (6). The addition of lipid and protein to the growing axon may result from the addition of new membrane at the growing tip, as well as lateral diffusion of membrane components from the cell body (6, 7). The present study examined the extent to which dietary restriction of n-3 fatty acids caused by feeding a diet very low in ALA during gestation alters the fatty acid composition of growth cones and neuronal cell bodies in newborn rats.

	MATERIALS AND METHODS

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Animals and diets
Wistar Rats (Animal Care, University of British Columbia, Vancouver, Canada) were housed and bred with free access to food and water. Female rats were assigned to receive either a standard control diet containing soybean oil with 8% of fatty acids as ALA (Purina Rodent Laboratory Chow #5001; Purina Mills, St Louis) (n = 5) or a low-ALA semipurified diet with safflower oil (0.3% of fatty acids as ALA) (n = 4) from 3 d before mating. The semipurified diet contained, per 100 g diet food, 20 g safflower oil, 20 g vitamin-free casein (US Biochemical, Cleveland), 20 g sucrose, 28.75 g starch, 5 g nonnutritive cellulose (Teklad Test Diets, Madison, WI), 1 g vitamin mix [(AOAC no. 40055; Teklad Test Diets, Madison, WI) which provided, per kg of each diet (1087 kJ): 20000 IU vitamin A, 2000 IU vitamin D, 100 mg vitamin E acetate, 5 mg menadione, 5 mg thiamine-HCl, 8 mg riboflavin, 40 mg pyridoxine-HCl, 40 mg niacin, 40 mg pantothenic acid, 2000 mg choline, 100 mg myoinositol, 100 mg p-aminobenzoic acid, 0.4 mg biotin, 2 mg folic acid, and 30 mg vitamin B-12], and 5 g mineral mix [(Bernhart-Tomarelli salt mix; General Biochemicals, Chagrin Falls, OH) which provided, per kg of complete diet: 1.1 g calcium carbonate, 36.8 g calcium phosphate, 0.1 g citric acid, 23 mg cupric citrate.1/2 H₂0, 4.0 g potassium diphosphate, 294 mg ferric citrate.5 H₂0, 1.3 g MgO, 418 mg manganese citrate, 0.5 mg K₂O, 3.4 g potassium sulfate, 1.5 g NaCl, 1.1 g sodium phosphate, 66.5 mg zinc citrate.2 H₂0 with an additional 78 mg Mn²⁺, 60 µg Se²⁺, 1.0 g choline chloride, and 3.0 g L-methionine]. Thus, differences other than ALA were present between the control and n-3 fatty acid–deficient semisynthetic diet. Newborn pups were studied on postnatal day 2 because at that time neurogenesis is nearly complete, axon formation predominates over synaptogenesis, and glial proliferation is not yet prevalent (4). The contamination of growth cones by synaptic and glial membranes was, therefore, minimized. All procedures were reviewed and approved by the University of British Columbia Animal Care Committee and conformed to the guidelines of the Canadian Council on Animal Care.

Tissue preparation and analysis
On postnatal day 2, whole brains were removed and pooled within each litter. The method for isolation of the neuronal cell fractions was adapted from previous reports (5, 8, 9). Briefly, the tissue was homogenized in 8 volumes of 1 mmol N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid containing 0.32 mol sucrose/L and 1.0 mmol MgCl₂/L, pH 7.3, with 5 strokes in a polytetrafluoroethylene-glass homogenizer. The homogenate was centrifuged at 1660 x g for 15 min at 4°C in a Beckman J6B low-speed centrifuge (Beckman, Mississauga, Canada). A portion was frozen at -70°C and the remainder was used to prepare the neuronal fractions. Growth-cone particles were isolated from the low-speed supernate (5) and washed (8), and the neuronal cell body particles were isolated from the low-speed pellet (9). A portion of the isolated particles was frozen at -70°C and the remainder was used to prepare the growth-cone and neuronal cell body membranes (8). Total lipids were extracted and fatty acid methyl esters were prepared, separated, identified, and quantified by gas-liquid chromatography exactly as described previously (10). All results are presented as means ± SEMs. Differences between means were compared using unpaired t tests.

	RESULTS

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Growth cones prepared from pups born to rats fed the n-3 fatty acid–deficient diet had 50% lower concentrations of DHA, corresponding higher concentrations of DPA, and a 7-fold higher ratio of DPA to DHA than the neuronal growth cones of pups born to rats fed the control diet (Table 1). The concentrations of 22-carbon fatty acids (n-6 plus n-3) in the neuronal growth cones, however, were not significantly different between the pups from the n-3 fatty acid–deficient and the control groups. The analysis of the cell body fatty acids, in contrast to the neuronal growth cones, however, found no significant differences in the concentrations of DHA and eicosapentaenoic acid (22:5n-3) between pups born to rats fed the n-3 fatty acid–deficient diet and those born to rats fed the control diet.

	DISCUSSION

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Alternatively, the differences between the concentrations of DHA and DPA in the growth cone and cell body after dietary n-3 fatty acid restriction may be interpreted as evidence that the neuronal cell body takes precedence over growth-cone membranes for DHA when the availability of DHA is limited. However, it is also plausible that the maternal n-3 fatty acid stores available in gestation provided a sufficient amount of n-3 fatty acids for the synthesis of cell body membranes during the proliferative phase of neurogenesis, but became depleted with the onset of membrane-rich growth-cone formation. Although dietary n-3 fatty restriction was imposed throughout pregnancy, n-3 fatty acids from maternal adipose tissue would be available for placental transfer during the proliferative phase of neurogenesis, which is predominantly an in utero event. Furthermore, the demand for n-3 fatty acids would be expected to substantially increase with growth-cone membrane formation and axon extension in the brain.

The difference in the response to DHA and DPA in the neuronal cell bodies compared with growth cones in the n-3 fatty acid–deficient newborns suggests that the addition of new membrane lipid to axons occurs along the shaft of the axon, at the distal growth cone, or both, rather than originating in the cell body with transfer along the axon shaft. Similar suggestions have been made by others (7, 11). Further studies are required to determine whether the reduced growth-cone concentrations of DHA and corresponding increases in DPA affect axon elongation, pathfinding, target selection, or synaptogenesis.

	REFERENCES

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