Dietary DHA during development affects depression-like behaviors and biomarkers that emerge after puberty in adolescent rats
2014; Elsevier BV; Volume: 56; Issue: 1 Linguagem: Inglês
10.1194/jlr.m055558
ISSN1539-7262
AutoresMichael Weiser, Kelly Wynalda, Norman Salem, Christopher M. Butt,
Tópico(s)Early Childhood Education and Development
ResumoDHA is an important omega-3 PUFA that confers neurodevelopmental benefits. Sufficient omega-3 PUFA intake has been associated with improved mood-associated measures in adult humans and rodents, but it is unknown whether DHA specifically influences these benefits. Furthermore, the extent to which development and puberty interact with the maternal diet and the offspring diet to affect mood-related behaviors in adolescence is poorly understood. We sought to address these questions by 1) feeding pregnant rats with diets sufficient or deficient in DHA during gestation and lactation; 2) weaning their male offspring to diets that were sufficient or deficient in DHA; and 3) assessing depression-related behaviors (forced swim test), plasma biomarkers [brain-derived neurotrophic factor (BDNF), serotonin, and melatonin], and brain biomarkers (BDNF) in the offspring before and after puberty. No dietary effects were detected when the offspring were evaluated before puberty. In contrast, after puberty depressive-like behavior and its associated biomarkers were worse in DHA-deficient offspring compared with animals with sufficient levels of DHA. The findings reported here suggest that maintaining sufficient DHA levels throughout development (both pre- and postweaning) may increase resiliency to emotional stressors and decrease susceptibility to mood disorders that commonly arise during adolescence. DHA is an important omega-3 PUFA that confers neurodevelopmental benefits. Sufficient omega-3 PUFA intake has been associated with improved mood-associated measures in adult humans and rodents, but it is unknown whether DHA specifically influences these benefits. Furthermore, the extent to which development and puberty interact with the maternal diet and the offspring diet to affect mood-related behaviors in adolescence is poorly understood. We sought to address these questions by 1) feeding pregnant rats with diets sufficient or deficient in DHA during gestation and lactation; 2) weaning their male offspring to diets that were sufficient or deficient in DHA; and 3) assessing depression-related behaviors (forced swim test), plasma biomarkers [brain-derived neurotrophic factor (BDNF), serotonin, and melatonin], and brain biomarkers (BDNF) in the offspring before and after puberty. No dietary effects were detected when the offspring were evaluated before puberty. In contrast, after puberty depressive-like behavior and its associated biomarkers were worse in DHA-deficient offspring compared with animals with sufficient levels of DHA. The findings reported here suggest that maintaining sufficient DHA levels throughout development (both pre- and postweaning) may increase resiliency to emotional stressors and decrease susceptibility to mood disorders that commonly arise during adolescence. 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The work reported here sought to examine whether DHA sufficiency throughout development (gestation, preweaning, prepubescence, and adolescence) in rats could positively affect behavioral measures (forced swim test [FST]) and biomarkers (serotonin, melatonin, and brain-derived neurotrophic factor [BDNF]) associated with mood before or after puberty. The periods of dietary DHA supplementation were designed to include the majority of neural development as well as the transition from adolescence to adulthood in the rat (8–9 weeks). The study design also included the investigation of potential effects of DHA removal at weaning to model the postweaning drop in DHA intake that occurs in some infants. Furthermore, we wanted to determine whether feeding a postweaning diet rich in DHA to offspring weaned from DHA-deficient dams could affect these measures. Overall, the results described here suggest that DHA supplementation is required throughout development (pre- and postweaning) to positively affect mood-related behavioral measures and biomarkers after puberty in adolescent rats. Sprague-Dawley rats were housed individually (dams during gestation and during lactation with offspring) or in pairs (males after weaning) in polycarbonate cages in a temperature and humidity controlled environment, on a 12 h:12 h light:dark cycle, with chow and water ad libitum. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Colorado (Boulder, CO) and were performed according to the Guide for the Care and Use of Laboratory Animals (8th edition, National Research Council). The DHA-deficient and DHA-sufficient diet were prepared by Dyets Inc. (Bethlehem, PA) and were based on the AIN-93G formulation (31Reeves P.G. Nielsen F.H. Fahey Jr, G.C. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet.J. Nutr. 1993; 123: 1939-1951Crossref PubMed Scopus (6798) Google Scholar), with 7% total fat derived from a custom fat blend containing olive, hydrogenated coconut, safflower, soybean, and docosahexaenoic acid-rich single-cell oils. Composition of the two diets is shown in Table 1. Each diet contained, as a percentage of total FAs, ∼11% linoleic acid (18:2n6) and 0.5% α-linolenic acid (18:3n3) but differed in amount of DHA (22:6n3; 0% for deficient vs. 0.89% for sufficient) and therefore total n3 FAs (0.61% vs. 1.50%) and n6:n3 ratio (17.89% vs. 7.55%).TABLE 1.Compositions of diets used (g/100 g diet)IngredientDHA DeficientDHA SufficientCasein20.0020.00l-Cystine0.300.30Sucrose10.0010.00Cornstarch39.7539.75DyetroseaTrademark Dyets, Inc. Carbohydrate composition (%): monosaccharides, 1; disaccharides, 4; trisaccharides, 5; tetrasaccharides and higher, 90.13.2013.20Cellulose5.005.00Mineral Mix 210025bAIN-93G mineral mix (mg/100 g diet): calcium, 500; phosphorus, 156.1; potassium, 360; sodium, 101.9; chloride, 157.1; sulfur, 30; magnesium, 50.7; iron, 3.5; copper, 0.6; manganese, 1; chromium, 0.1; iodine, 0.02; selenium, 0.02; fluoride, 0.1; boron, 0.05; molybdenum, 0.02; silicon, 0.5; nickel, 0.05; lithium, 0.01; vanadium, 0.01.3.503.50Vitamin Mix 310025cAIN-93VX vitamin mix (U/100 g diet): thiamin, 0.6 mg; riboflavin, 0.6 mg; pyridoxine, 0.7 mg; niacin, 3 mg; pantothenate, 1.6 mg; folate, 0.2 mg; biotin, 0.02 mg; cyanocobalamin, 2.5 mg; vitamin A, 400 IU; Vitamin E, 7.5 IU; Vitamin D3, 100 IU; Vitamin K1, 0.08 mg.1.001.00Choline0.250.25Fat composition:7.007.00Olive oil1.681.53Coconut oil2.242.14Safflower oil2.812.87Soybean oildTert-butylhydroquinone free.0.270.28DHA-S oileProvided by DSM Nutritional Products.—0.19FA composition:(% Total FAs)18:1n945.4845.1118:2n610.8810.9618:3n30.520.5020:4n60.010.0222:6n3—0.89ΣSat41.6040.75ΣMono46.8846.42ΣPUFA11.5212.83Σn30.611.50Σn610.9111.33n6:n317.897.5518:1n9, oleic acid; 18:2n6, linoleic acid; 18:3n3, α-linolenic acid; 20:4n6, arachidonic acid (ARA); 22:6n3, DHA; ΣMono, sum of monounsaturated fatty acids; Σn3, sum of omega-3 fatty acids; Σn6, sum of omega-6 fatty acids; ΣPUFA, sum of polyunsaturated fatty acids; ΣSat, sum of saturated fatty acids; n6:n3, ratio of omega-6 to omega-3 fatty acids.a Trademark Dyets, Inc. Carbohydrate composition (%): monosaccharides, 1; disaccharides, 4; trisaccharides, 5; tetrasaccharides and higher, 90.b AIN-93G mineral mix (mg/100 g diet): calcium, 500; phosphorus, 156.1; potassium, 360; sodium, 101.9; chloride, 157.1; sulfur, 30; magnesium, 50.7; iron, 3.5; copper, 0.6; manganese, 1; chromium, 0.1; iodine, 0.02; selenium, 0.02; fluoride, 0.1; boron, 0.05; molybdenum, 0.02; silicon, 0.5; nickel, 0.05; lithium, 0.01; vanadium, 0.01.c AIN-93VX vitamin mix (U/100 g diet): thiamin, 0.6 mg; riboflavin, 0.6 mg; pyridoxine, 0.7 mg; niacin, 3 mg; pantothenate, 1.6 mg; folate, 0.2 mg; biotin, 0.02 mg; cyanocobalamin, 2.5 mg; vitamin A, 400 IU; Vitamin E, 7.5 IU; Vitamin D3, 100 IU; Vitamin K1, 0.08 mg.d Tert-butylhydroquinone free.e Provided by DSM Nutritional Products. Open table in a new tab 18:1n9, oleic acid; 18:2n6, linoleic acid; 18:3n3, α-linolenic acid; 20:4n6, arachidonic acid (ARA); 22:6n3, DHA; ΣMono, sum of monounsaturated fatty acids; Σn3, sum of omega-3 fatty acids; Σn6, sum of omega-6 fatty acids; ΣPUFA, sum of polyunsaturated fatty acids; ΣSat, sum of saturated fatty acids; n6:n3, ratio of omega-6 to omega-3 fatty acids. The experimental design of the study is illustrated in Fig. 1. Timed-pregnant Sprague-Dawley rats were obtained from Harlan Laboratories (Indianapolis, IN) at embryonic day 4 and fed either a DHA-deficient or a DHA-sufficient diet. Shortly after parturition, on postnatal day (P) 1, pups were sexed, culled to liters of 10, and matched for sex and cross-fostered equally among the dams fed similar diets. At P16, male offspring were weaned from their mothers and fed either a DHA-deficient or a DHA-sufficient diet. We chose to examine only the male offspring in this study because including female rats would require more than twice the number of animals placed on study in order to provide sufficient statistical power given that the day of estrous for each female would be an additional cofactor. This design resulted in four groups that will be referred to as follows: 1) deficient (DHA-deficient maternal and postweaning diets), 2) preweaning sufficient (DHA-sufficient maternal diet and DHA-deficient postweaning diet), 3) postweaning sufficient (DHA-deficient maternal diet and DHA-sufficient postweaning diet), and 4) sufficient (DHA-sufficient maternal and postweaning diets). Offspring were tested in the FST in two separate cohorts at either P39–P40 or P59–P60, but not at both time points. Thirty minutes following the final swim test, each animal was euthanized via decapitation. Whole brain was extracted and frozen in cold isopentane (approximately −30°C) and stored at −80°C (brains were not perfused with saline or buffer, but rather frozen directly after decapitation and extraction). Trunk blood was collected into K2-EDTA-coated vacutainers (BD Biosciences), inverted to mix, and centrifuged at 1,000 g for 15 min, and the resulting plasma aliquots were stored at −80°C until assayed. The cell pellet was then mixed with a 5-fold volume of saline, centrifuged at 1,000 g for 5 min (wash repeated twice), and the resulting pellet containing the red blood cell (RBC) fraction was stored at –80°C until assayed. After necropsy, brains were thawed on ice and bisected sagittally down the longitudinal cerebral fissure and cerebellar vermis; one hemisection was used for FA analysis, and one hemisection was regionally dissected and stored at –80°C until further processing. Tissues (RBCs, plasma, and brain) from the current study were analyzed for FA composition by gas chromatography. Sample preparation was optimized for each tissue matrix. Briefly, the plasma was aliquoted and dried under evaporative nitrogen; brain tissues were lyophilized, homogenized, and weighed; and RBCs were vortexed, then aliquoted directly for assays. Internal standard (trinonadecanoic acid or pentadecanoic acid in toluene) was then added to each sample, and direct transesterification was accomplished by the addition of 1.5 N methanolic hydrochloric acid. Samples were heated to 100°C for 2 h. Following methylation, saturated sodium chloride was added, and the lipids were extracted into toluene for direct injection. Calibration curves were generated using GLC-502B (Nu-Chek Prep, Elysian, MN) for FA reference standards, with trinonadecanoic acid or pentadecanoic acid for the internal standard. Samples were analyzed on an Agilent 6890 gas chromatograph (split injection) equipped with a flame ionization detector. A 30 m × 0.32 mm × 0.2 µm SP-2380 fused silica capillary column (Supelco, Bellafonte, PA) was used with hydrogen as the carrier gas. The oven was temperature programmed from 140°C to 190°C at 5°C/min and held for 1 min at 190°C, then increased to 260°C at a rate of 17°C/min and held for 3 min for a total run time of 18.12 min. The flame ionization detector was set at 285°C. FA data are expressed as a wt percentage of total FAs. Animals were acclimated to the test room overnight prior to testing. The test was performed over two consecutive days. On day 1, the animals were acclimated to the test (0800 h to 1300 h) by placing them in a Plexiglas cylindrical container (45 cm × 20 cm; Stoelting Co., Wood Dale, IL) filled with 30 cm of fresh water (25°C) for 15 min, after which they were toweled dried and returned to their home cage. On day 2 (24 h later), the test was performed for a total swim time of 5 min, after which the rats were toweled dried and returned to their home cage. Both trials were recorded by a digital video camera secured to the ceiling above the cylinders. Total time swimming, immobile, and climbing, and number of dives were measured post hoc by an experimenter blind to the group assignments. Total time immobile was measured in real-time by behavioral software (ANY-maze, Stoelting Co.), and confirmed by the post hoc analysis. Swimming was defined as movement of the forelimbs and hind limbs that did not break the surface of the water. Immobility was defined as absence of any movement except for slight movements necessary for the animal to keep its head above water. Climbing was defined as rapid movement of the forelimbs that did not break the surface of the water. Dives were counted when the animal submerged its head in an effort to find an escape below the surface of the water. Plasma testosterone concentration was determined via a competitive ELISA (catalog number EIA-1559; DRG International Inc., Mountainside, NJ) according to the manufacturer's specifications. Plasma samples were run neat in triplicate. All samples were analyzed in one assay. The intra-assay variance was 7.5%. The limit of detection for this assay is 0.083 ng/ml. Plasma serotonin concentration was assayed via a competitive ELISA (catalog number RE59121; IBL International Inc., Hamburg, Germany). Plasma samples were centrifuged for 2 min at 10,000 g to ensure a platelet-free sample, and a 100 µl aliquot of the supernatant was taken for the assay. The manufacturer's "Sample B" protocol (for platelet-free plasma) was followed as specified. All samples were analyzed in one assay. The intra-assay variance was 9.6%. The limit of detection for this assay is 0.014 ng/ml. Plasma melatonin concentration was analyzed via a competitive ELISA (catalog number RE54021; IBL International Inc.) according to the manufacturer's specifications. All samples were analyzed in one assay. The intra-assay variance was 8.2%. The limit of detection for this assay is 1.6 pg/ml. Plasma and brain BDNF was determined via a two-site sandwich ELISA (catalog number TB257; Promega Corp., Madison, WI) according to the manufacturer's specifications with modifications as determined via empirical testing on similar samples. Plasma samples were centrifuged for 2 min at 10,000 g to ensure a platelet-free sample, and a 100 µl aliquot of the supernatant was taken for the assay. Brain tissue samples were homogenized in buffer [100 mM Tris-HCl, 400 mM NaCl, 4 mM EDTA, 0.05% sodium azide, 0.2% Triton-X, 2% BSA (fraction V), protease inhibitor cocktail (Roche Cat# 539137; 1:100 dilution), and 0.1 mM PMSF]. Buffers containing BSA have been shown to improve BDNF recovery from brain tissue samples (32Szapacs M.E. Mathews T.A. Tessarollo L. Ernest Lyons W. Mamounas L.A. Andrews A.M. Exploring the relationship between serotonin and brain-derived neurotrophic factor: analysis of BDNF protein and extraneuronal 5-HT in mice with reduced serotonin transporter or BDNF expression.J. Neurosci. Methods. 2004; 140: 81-92Crossref PubMed Scopus (125) Google Scholar). Homogenization buffer (10× volume by weight of tissue) was added to each sample, and tissue was homogenized with an ultrasonic tissue disruptor (Misonix XL2000) on setting 4 for 30 s. Homogenates were cleared at 16,000 g for 30 min at 4°C, and 100 µl aliquots of the supernatant were stored at −80°C until assayed. Plasma and brain samples were treated with 4 µl of 1.0 M HCl for 15 min, neutralized with 4 µl of 1.0 M NaOH, and diluted with 392 µl sample buffer (1:5) on day 1 of the assay. Changes to the manufacturer's protocol included the following: BDNF standard curve was serially diluted from provided stock standard prior to addition to plate (rather than in plate), initial sample incubation with anti-BDNF coated plate was performed at 4°C for 24 h with no shaking, and incubation with secondary antibody was done at 4°C for 20 h with no shaking. All other portions of the protocol were completed according to the manufacturer's recommendations. Brain BDNF content was normalized to wet weight of each tissue sample because the specific homogenization buffer used precluded the ability to measure protein levels in the homogenates due to interference by BSA with standard protein assays. The intra-assay variance was 6.5%, and the interassay variance was 9.7%. The limit of detection for this assay is 15.6 pg/ml. Data were analyzed with SPSS, version 16.0 (IBM, Armonk, NY) and visualized with Prism version 5.4 (GraphPad Inc., La Jolla, CA). Main effects were detected via one-way ANOVA or multivariate ANOVA where α (P) levels less than 0.05 were considered statistically different. In the case of a main effect, ANOVA analysis was followed by Tukey's post hoc tests for pairwise comparisons to determine significant differences between groups. Data from the dams (two groups) were analyzed by the Student's t-test with Welch's correction in cases where Levene's test for equality of variances was significant. Correlations were detected via two-tailed Pearson correlation calculations where P levels less than 0.05 were considered statistically significant. Data sets exhibiting skewed distribution frequencies were transformed with log10 or square-root calculations to improve their frequency distributions prior to analysis. Outlier detection was conducted using Grubb's test prior to any other analyses. All data are expressed as the group mean ± SEM. Mean plasma testosterone concentration was lower in animals at P40 (0.67 ± 0.06 ng/ml) than those at P60 (2.66 ± 0.16 ng/ml) as determined by an unpaired one-tailed t-test with Welch's correction (t(85) = 11.34, P < 0.0001). There were
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