Dietary DHA during development affects depression-like behaviors and biomarkers that emerge after puberty in adolescent rats

Abstract

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.

The prevalence of depressive disorders increases dramatically after the rapid hormonal, physical, and neural changes of puberty (1, 2) from 1% of the population under 12 years old to ∼25% of the population by the end of adolescence (3), and is more commonly diagnosed in girls than boys (2). Nearly half of depressed adolescents are treatment resistant, and selective serotonin reuptake inhibitors (SSRIs) that are typically effective for treating depressive disorders in adults have been shown to exacerbate suicidal behavior in some teens (4). These issues highlight the need for alternative approaches to juvenile depression, of which diet may be one (5, 6).

Mood is likely regulated by interactions between limbic and cortical brain regions (7). The volume of gray matter in the frontal and parietal lobes first increases during early development and then declines after puberty, possibly due to synaptic pruning (8). Thus, as the brain matures, these regions may be impacted by environmental factors such as stress, diet, and exercise. One particularly important dietary factor for optimal brain development is the omega-3 PUFA DHA (9). Interestingly, dietary consumption of DHA in the United States has dropped significantly during the 20th century coinciding with a significant rise in the prevalence of depressive disorders (10, 11), and it has been suggested that these two phenomena are linked (12).

DHA is the principal omega-3 PUFA in mammalian and human tissues (13, 14). It is particularly abundant in the central nervous system where it is enriched in synaptic membranes, astrocytes, growth cones, and mitochondria (15). Brain tissue is ∼60% lipid (dry wt.), and DHA represents 5% to 10% of those lipids, including nearly half of the FAs comprising membrane aminophospholipids (16, 17). Unfortunately, conversion to DHA from dietary precursors is very limited in humans [<0.1% (18, 19)], and thus preformed DHA consumption is needed for optimal brain content (20). Sources of preformed DHA include foods such as fatty fish, organ meats, and eggs, or supplementation with oils derived from fish or microalgae.

DHA is acquired during gestation via maternal stores by way of placental transfer, and it is acquired after birth from mother’s milk or formula. As DHA is acquired primarily through the diet after birth, a decline in preformed DHA intake typically occurs immediately following weaning. This circumstance creates some developmental risk because weaning is a critical period for synaptogenesis and myelination. Thus, suboptimal DHA intake may influence synapse pruning, myelination, inhibitory synaptogenesis, and so forth, during this time. Indeed, DHA deficiency throughout development leads to decreased DHA levels in neural tissue and is associated with deficits in psychomotor development (21), problem solving (22), reading skills (23), visual acuity (24), and attention (25). Furthermore, emerging clinical data support a link between sufficient DHA intake with a reduction in the incidence and/or symptomatic relief of adolescent depression (5, 26–29), and there exists an abundance of clinical evidence supporting a positive role for the omega-3 PUFA EPA in the treatment of adult depression (30). However, the studies examining the importance of DHA, in particular, are limited in scope and interpretation, and no longitudinal studies have been reported to date. It is therefore unknown whether DHA sufficiency during development can help to establish optimal emotional resiliency to depressive mood states.

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.

MATERIALS AND METHODS

Animals

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).

Diet composition

The DHA-deficient and DHA-sufficient diet were prepared by Dyets Inc. (Bethlehem, PA) and were based on the AIN-93G formulation (31), 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)

Ingredient	DHA Deficient	DHA Sufficient
Casein	20.00	20.00
l-Cystine	0.30	0.30
Sucrose	10.00	10.00
Cornstarch	39.75	39.75
Dyetrosea	13.20	13.20
Cellulose	5.00	5.00
Mineral Mix 210025b	3.50	3.50
Vitamin Mix 310025c	1.00	1.00
Choline	0.25	0.25
Fat composition:	7.00	7.00
Olive oil	1.68	1.53
Coconut oil	2.24	2.14
Safflower oil	2.81	2.87
Soybean oild	0.27	0.28
DHA-S oile	—	0.19
FA composition:	(% Total FAs)
18:1n9	45.48	45.11
18:2n6	10.88	10.96
18:3n3	0.52	0.50
20:4n6	0.01	0.02
22:6n3	—	0.89
ΣSat	41.60	40.75
ΣMono	46.88	46.42
ΣPUFA	11.52	12.83
Σn3	0.61	1.50
Σn6	10.91	11.33
n6:n3	17.89	7.55

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.

^aTrademark Dyets, Inc. Carbohydrate composition (%): mono­saccharides, 1; disaccha­rides, 4; trisaccharides, 5; tetrasaccharides and higher, 90.

^bAIN-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.

^cAIN-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.

^dTert-butylhydroquinone free.

^eProvided by DSM Nutritional Products.

Experimental design

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.

Fig. 1.

Experimental design of the study. Timed pregnant females (embryonic day 4) were provided either a base diet containing no DHA or an equivalent diet containing DHA as ∼1% of the total FAs throughout the gestation and lactation periods. At weaning, male offspring were either provided the base diet or the DHA-containing diet. Behavior was assessed in the FST before and after puberty (P40 and P60, respectively) in separate cohorts (each animal was only tested once). Tissue was taken at both time points for analysis of biomarkers for mood.

FA analysis

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.

FST

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.

Biomarker analysis

Testosterone.

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.

Serotonin.

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.

Melatonin.

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.

BDNF.

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 (32). 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.

Statistical analysis

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.

RESULTS

Plasma testosterone

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 no significant group differences at either P40 or P60 (data not shown). Therefore, P40 and P60 were likely within the pre-/peripubertal and postpubertal time periods, respectively.

Body and brain weight

There were no differences in body weight between dams on either the DHA-sufficient or DHA-deficient diet after parturition or after weaning, and no differences were detected in whole brain weight at the time of euthanization following weaning.

Among the offspring, there were no group differences in body weight at either P40 or P60, or brain mass at P40. However, analysis by one-way ANOVA detected a significant main effect of group on brain weight at P60 [F_(3,61) = 6.16, P = <0.001]. Tukey’s post hoc test indicated that preweaning sufficient animals had significantly lower whole brain weight (1.982 ± 0.012 g) than either sufficient (2.088 ± 0.025 g, P = 0.021) or deficient (2.114 ± 0.024 g, P < 0.0001) animals, but not postweaning sufficient animals (2.072 ± 0.026 g, P = 0.080).

FA analysis

Diet affected the DHA status of peripheral and central tissues in the dam at the time of euthanization (P21; 39 days on diet). Sufficient dams had higher DHA content in RBCs (4.39 ± 0.09% of total FAs, N = 8) than deficient dams (1.96 ± 0.11%, N = 9; t₍₁₅₎ = 16.80, P < 0.0001), and higher DHA content in brain (13.50 ± 0.20%, N = 8) than deficient dams (12.59 ± 0.17%, N = 9; t₍₁₅₎ = 3.50, P = 0.0016) as determined by an unpaired one-tailed t-test. In the offspring, supplementation of the maternal or postweaning diets with DHA had profound effects on the FA composition of central and peripheral tissues both before and after puberty. The FA composition of brain, RBCs, plasma, and their statistical analyses are detailed in Tables 2, 3, and 4, respectively. In addition, the data for the primary PUFAs of interest within brain tissue [DHA, 22:6n3; arachidonic acid (ARA, 20:4n6); and docosapentaenoic acid (DPAn6; 22:5n6)] are graphed in Fig. 2. As found in previous studies, dietary supplementation of DHA during any developmental time period (maternal, postweaning, or throughout) significantly increased brain DHA content both before and after puberty relative to deficient controls. Furthermore, at P40 sufficient animals had significantly higher brain DHA content (14.80% of total FAs) than preweaning (12.96%) and postweaning (13.56%) sufficient cohorts, both of which had higher brain DHA content than the deficient animals (9.50%). At P60, brain DHA content was similar in the sufficient and postweaning sufficient groups (14.01% and 13.51%, respectively), and both groups had significantly greater brain DHA content than both the preweaning sufficient and deficient groups (11.89% and 9.71%, respectively).

TABLE 2.

Dietary DHA affects brain FA profiles

FA	Age (P)	Deficient	Prewean Sufficient	Postwean Sufficient	Sufficient	F	P	Power
16:0	40	18.32 ± 0.07a	18.16 ± 0.10ab	18.20 ± 0.08a	17.87 ± 0.07b	5.68	0.002	0.94
	60	18.64 ± 0.14	18.60 ± 0.17	18.39 ± 0.15	18.57 ± 0.15		NS
18:1n9	40	16.87 ± 0.09a	17.42 ± 0.14bc	17.13 ± 0.12ab	17.73 ± 0.09c	11.02	<0.001	1.00
	60	17.37 ± 0.13a	17.63 ± 0.14a	17.84 ± 0.15a	17.87 ± 0.13a	2.97	0.038	0.68
18:2n6	40	0.66 ± 0.01a	0.65 ± 0.01a	0.68 ± 0.01ab	0.71 ± 0.01b	7.02	<0.001	0.97
	60	0.46 ± 0.01a	0.44 ± 0.01a	0.52 ± 0.01b	0.49 ± 0.01b	19.67	<0.001	1.00
18:3n3	40	0.10 ± 0.00	0.09 ± 0.00	0.09 ± 0.00	0.09 ± 0.00		NS
	60	0.09 ± 0.00	0.09 ± 0.00	0.08 ± 0.00	0.08 ± 0.00		NS
20:4n6	40	10.57 ± 0.05a	10.30 ± 0.04b	9.98 ± 0.07c	9.81 ± 0.05c	42.69	<0.001	1.00
	60	9.62 ± 0.06a	9.45 ± 0.08a	8.99 ± 0.07b	9.12 ± 0.08b	16.72	<0.001	1.00
20:5n3	40	n.d.	n.d.	n.d.	n.d.		NS
	60	0.06 ± 0.00	0.06 ± 0.00	0.06 ± 0.00	0.07 ± 0.00		NS
22:5n6	40	5.47 ± 0.17a	2.32 ± 0.07b	2.67 ± 0.19b	1.22 ± 0.03c	181.22	<0.001	1.00
	60	4.32 ± 0.16a	2.35 ± 0.15b	1.32 ± 0.03c	0.86 ± 0.03d	172.92	<0.001	1.00
22:5n3	40	0.13 ± 0.00a	0.11 ± 0.00a	0.13 ± 0.00a	0.17 ± 0.01b	11.00	<0.001	1.00
	60	0.15 ± 0.01a	0.15 ± 0.01a	0.19 ± 0.01b	0.20 ± 0.01b	10.85	<0.001	1.00
22:6n3	40	9.50 ± 0.24a	12.96 ± 0.08b	13.56 ± 0.26b	14.80 ± 0.08c	151.10	<0.001	1.00
	60	9.71 ± 0.20a	11.89 ± 0.18b	13.51 ± 0.15c	14.01 ± 0.13c	128.70	<0.001	1.00
ΣSat	40	44.51 ± 0.08a	44.35 ± 0.08ab	44.43 ± 0.09ab	44.17 ± 0.09b	2.94	0.039	0.673
	60	44.68 ± 0.10	44.58 ± 0.14	44.53 ± 0.12	44.50 ± 0.12		NS
ΣMono	40	27.95 ± 0.16ab	28.07 ± 0.15a	27.40 ± 0.19b	27.90 ± 0.20ab	2.83	0.045	0.655
	60	29.96 ± 0.21	30.02 ± 0.30	29.82 ± 0.29	29.68 ± 0.28		NS
ΣPUFA	40	27.53 ± 0.15a	27.58 ± 0.13a	28.17 ± 0.16b	27.93 ± 0.16ab	4.13	0.009	0.83
	60	25.37 ± 0.21	25.40 ± 0.19	25.65 ± 0.22	25.82 ± 0.20		NS
Σn3	40	10.29 ± 0.28a	13.77 ± 0.14b	14.31 ± 0.26b	15.62 ± 0.17c	106.13	<0.001	1.00
	60	10.28 ± 0.19a	12.46 ± 0.18b	14.10 ± 0.15c	14.63 ± 0.13c	137.25	<0.001	1.00
Σn6	40	17.05 ± 0.20a	13.63 ± 0.11b	13.66 ± 0.21b	12.12 ± 0.05c	175.99	<0.001	1.00
	60	14.92 ± 0.18a	12.78 ± 0.19b	11.40 ± 0.10c	11.05 ± 0.09c	135.88	<0.001	1.00
n6:n3	40	1.68 ± 0.5a	0.99 ± 0.02b	0.96 ± 0.04b	0.78 ± 0.05c	144.56	<0.001	1.00
	60	1.46 ± 0.04a	1.03 ± 0.02b	0.81 ± 0.01c	0.76 ± 0.01c	194.48	<0.001	1.00
ARA:DHA	40	1.12 ± 0.02a	0.80 ± 0.00b	0.74 ± 0.02b	0.66 ± 0.00c	152.72	<0.001	1.00
	60	1.00 ± 0.02a	0.80 ± 0.01b	0.67 ± 0.01c	0.65 ± 0.01c	182.42	<0.001	1.00

16:0, palmitic acid; 18:1n9, oleic acid; 20:5n3, EPA; 22:5, docosapentaenoic acid (DPAn3 and DPAn6); n.d., not detectible; NS, not significant in multifactorial ANOVA. Data are presented as the mean percent of total FAs ± the SEM. The degrees of freedom and their errors for the analysis of each FA at age P40 were 3 and 68, and at age P60 were 3 and 67, respectively. Significant differences (P < 0.05) between groups were determined with Tukey’s post hoc test and are designated by a lack of common superscripts across each row.

Fig. 2.

Dietary DHA supplementation during development affected the concentration of FAs in brain tissue. A: Before puberty, dietary DHA at any time point resulted in higher brain DHA concentration relative to controls on a DHA-deficient diet, and DHA throughout development increased brain DHA content to a greater extent than DHA during only the preweaning or postweaning period. B: After puberty, dietary DHA at any time point resulted in higher brain DHA concentration relative to deficient controls, and DHA throughout development or after weaning increased brain DHA content higher than DHA during only the preweaning period. C: Before puberty, dietary DHA at any time point resulted in lower brain ARA concentration relative to deficient controls, and DHA throughout development or after weaning decreased brain ARA content to a greater extent than DHA during only the preweaning period. D: After puberty, dietary DHA postweaning, regardless of maternal diet, resulted in lower brain ARA concentration relative to deficient controls, yet DHA supplementation just during the preweaning period did not affect brain ARA content. E: Before puberty, dietary DHA at any time point resulted in lower brain DPA(n6) concentration relative to deficient controls, and DHA throughout development decreased brain DPA(n6) content to a greater extent than DHA during only the preweaning or postweaning period. F: After puberty, dietary DHA at any time point resulted in lower brain DPA(n6) content relative to deficient controls, DHA throughout development decreased brain DPA(n6) content lower than DHA during only the preweaning or postweaning period, and DHA supplementation only after weaning decreased brain DPA(n6) concentration lower than DHA during just the preweaning period. Data are presented as averages ± the SEM. Significant differences (P < 0.05) between groups were determined by one-way ANOVA followed by Tukey’s post hoc tests and are indicated by an absence of shared superscripts.

With the other brain PUFAs of interest, again similar to previous studies, there were differences in ARA and DPAn6 brain content that tracked inversely to the change in brain DHA levels (Table 2). For example, ARA and DPAn6 were highest in the brains from animals that were not supplemented with DHA during development, and lowest in those supplemented throughout development. DPAn6 brain content was quicker to change in response to postweaning diet than was DHA, and ARA levels responded at rates somewhat in-between those of DHA and DPAn6. As expected, these changes led to a corresponding decrease in the omega-6 to omega-3 (n6:n3) ratio with improved brain DHA status, while total PUFA content of brain was largely unaffected.

Dietary supplementation with DHA also changed the FA profiles of RBCs (Table 3) and plasma (Table 4) in a fashion similar to changes seen in brain tissue both before and after puberty. However, changes in FA composition following postweaning dietary change (deficient to sufficient, or vice versa) were more rapid in both tissues than in brain. For example, the 24 day period from weaning (P16) to prepuberty (P40) was long enough to allow the DHA levels in RBCs from preweaning deficient (postweaning sufficient) offspring to reach those of animals maintained on pre- and postweaning DHA-supplemented diets (5.04% vs. 5.38% of total FAs, respectively). EPA was found in very low levels in brain (n.d. to 0.07% total FAs), RBCs (0.06 to 0.21% total FAs), and plasma (0.09 to 0.30% total FAs). However, dietary DHA did cause a small but significant increase in EPA levels in RBCs (Table 3) and plasma (Table 4), and this may in part reflect retroconversion from DHA.

TABLE 3.

Dietary DHA affects RBC FA profiles

FA	Age (P)	Deficient	Prewean Sufficient	Postwean Sufficient	Sufficient	F	P	Power
16:0	40	27.16 ± 0.33	26.16 ± 0.76	27.74 ± 0.52	27.92 ± 0.75		NS
	60	25.04 ± 0.66	25.77 ± 0.22	26.52 ± 0.12	26.03 ± 0.14		NS
18:1n9	40	11.63 ± 0.21	11.52 ± 0.34	11.03 ± 0.20	11.42 ± 0.30		NS
	60	10.47 ± 0.26	10.22 ± 0.16	10.11 ± 0.21	10.20 ± 0.24		NS
18:2n6	40	5.12 ± 0.08	5.08 ± 0.10	5.36 ± 0.10	5.29 ± 0.12		NS
	60	4.96 ± 0.10a	4.83 ± 0.10a	5.54 ± 0.10b	5.04 ± 0.09a	8.97	<0.001	0.994
18:3n3	40	0.04 ± 0.01	0.04 ± 0.01	0.04 ± 0.01	0.04 ± 0.01		NS
	60	0.05 ± 0.01	0.04 ± 0.01	0.04 ± 0.01	0.04 ± 0.01		NS
20:4n6	40	25.67 ± 0.53a	26.16 ± 0.23a	23.16 ± 0.66b	22.68 ± 0.93b	7.60	<0.001	0.98
	60	27.32 ± 0.29a	26.97 ± 0.37a	24.45 ± 0.29b	24.60 ± 0.34b	22.30	<0.001	1.00
20:5n3	40	0.09 ± 0.01a	0.10 ± 0.01a	0.21 ± 0.02b	0.20 ± 0.01b	18.76	<0.001	1.00
	60	0.06 ± 0.01a	0.07 ± 0.01a	0.18 ± 0.01b	0.15 ± 0.01b	40.14	<0.001	1.00
22:5n6	40	2.32 ± 0.08a	1.94 ± 0.06b	1.75 ± 0.07b	1.48 ± 0.08c	24.03	<0.001	1.00
	60	2.24 ± 0.06a	2.19 ± 0.15a	1.33 ± 0.03b	1.31 ± 0.04b	38.69	<0.001	1.00
22:5n3	40	0.64 ± 0.01	0.65 ± 0.02	0.65 ± 0.02	0.65 ± 0.03		NS
	60	0.49 ± 0.01a	0.48 ± 0.01a	0.60 ± 0.02b	0.56 ± 0.01b	13.16	<0.001	1.00
22:6n3	40	1.51 ± 0.04a	2.54 ± 0.04b	5.04 ± 0.21c	5.38 ± 0.28c	110.52	<0.001	1.00
	60	1.59 ± 0.03a	2.28 ± 0.30b	5.34 ± 0.11c	5.34 ± 0.10c	149.66	<0.001	1.00
ΣSat	40	44.01 ± 0.57	43.61 ± 0.61	44.87 ± 0.71	45.10 ± 1.09		NS
	60	44.67 ± 0.45	45.29 ± 0.15	45.28 ± 0.25	45.11 ± 0.19		NS
ΣMono	40	18.48 ± 0.27	18.19 ± 0.44	17.52 ± 0.28	17.79 ± 0.35		NS
	60	17.08 ± 0.37	16.46 ± 0.19	16.20 ± 0.25	16.43 ± 0.31		NS
ΣPUFA	40	37.52 ± 0.61	38.20 ± 0.31	37.61 ± 0.94	37.12 ± 1.36		NS
	60	38.25 ± 0.33	38.25 ± 0.25	38.52 ± 0.41	38.45 ± 0.29		NS
Σn3	40	2.43 ± 0.06a	3.47 ± 0.07b	6.08 ± 0.23c	6.41 ± 0.29c	107.66	<0.001	1.00
	60	2.32 ± 0.05a	2.86 ± 0.32b	6.28 ± 0.13c	6.19 ± 0.11c	140.87	<0.001	1.00
Σn6	40	34.32 ± 0.58a	34.37 ± 0.27a	31.13 ± 0.72b	30.27 ± 1.07b	8.72	<0.001	1.00
	60	35.63 ± 0.32a	34.99 ± 0.42a	32.04 ± 0.31b	31.73 ± 0.31b	33.91	<0.001	1.00
n6:n3	40	14.21 ± 0.28a	9.96 ± 0.17b	5.21 ± 0.14c	4.80 ± 0.11c	558.72	<0.001	1.00
	60	15.46 ± 0.27a	13.07 ± 0.79b	5.12 ± 0.08c	5.16 ± 0.11c	167.87	<0.001	1.00
ARA:DHA	40	17.06 ± 0.34a	10.33 ± 0.16b	4.71 ± 0.16c	4.35 ± 0.16c	743.19	<0.001	1.00
	60	17.22 ± 0.31a	13.68 ± 0.89b	4.60 ± 0.07c	4.64 ± 0.12c	189.82	<0.001	1.00

Data are presented as the mean percent of total FAs ± the SEM. The degrees of freedom and their errors for the analysis of each FA at age P40 were 3 and 68, and at age P60 were 3 and 66, respectively. Significant differences (P < 0.05) between groups were determined with Tukey’s post hoc test and are designated by a lack of common superscripts across each row.

TABLE 4.

Dietary DHA affects plasma FA profiles

FA	Age (P)	Deficient	Prewean Sufficient	Postwean Sufficient	Sufficient	F	P	Power
16:0	40	20.51 ± 0.29	20.43 ± 0.35	20.48 ± 0.34	21.16 ± 0.33		NS
	60	21.75 ± 0.44	21.30 ± 0.25	22.12 ± 0.42	21.50 ± 0.34		NS
18:1n9	40	34.45 ± 0.51a	34.57 ± 0.69a	31.92 ± 0.95b	33.17 ± 0.35ab	3.57	0.019	0.77
	60	33.69 ± 0.74a	32.59 ± 0.64ab	31.38 ± 0.48ab	30.37 ± 0.62b	5.18	0.003	0.91
18:2n6	40	9.67 ± 0.27ab	9.53 ± 0.24a	10.63 ± 0.33b	9.93 ± 0.21ab	3.40	0.023	0.74
	60	8.20 ± 0.19a	8.45 ± 0.22ab	9.50 ± 0.27c	9.13 ± 0.24bc	6.87	<0.001	0.97
18:3n3	40	0.22 ± 0.01	0.22 ± 0.02	0.26 ± 0.03	0.24 ± 0.01		NS
	60	0.15 ± 0.01	0.15 ± 0.02	0.17 ± 0.01	0.16 ± 0.01		NS
20:4n6	40	8.42 ± 0.44ab	9.52 ± 0.48a	8.66 ± 0.57ab	7.50 ± 0.37b	3.12	0.032	0.70
	60	9.27 ± 0.48ab	10.22 ± 0.55a	8.12 ± 0.44b	10.06 ± 0.58ab	3.08	0.033	0.70
20:5n3	40	0.15 ± 0.01a	0.14 ± 0.02a	0.30 ± 0.03b	0.24 ± 0.02b	16.04	<0.001	1.00
	60	0.09 ± 0.01a	0.11 ± 0.01a	0.19 ± 0.01b	0.19 ± 0.01b	27.33	<0.001	1.00
22:5n6	40	0.76 ± 0.05a	0.75 ± 0.04a	0.59 ± 0.02b	0.53 ± 0.2b	9.04	<0.001	0.99
	60	0.97 ± 0.06a	1.09 ± 0.10a	0.60 ± 0.02b	0.69 ± 0.03b	13.18	<0.001	1.00
22:5n3	40	0.04 ± 0.01a	0.04 ± 0.01a	0.10 ± 0.03a	0.05 ± 0.03a	2.80	0.047	0.65
	60	0.06 ± 0.00a	0.07 ± 0.01ab	0.08 ± 0.01ab	0.09 ± 0.01b	4.25	0.008	0.84
22:6n3	40	0.59 ± 0.03a	0.74 ± 0.03a	2.91 ± 0.14b	2.47 ± 0.08c	230.78	<0.001	1.00
	60	0.73 ± 0.03a	1.11 ± 0.21a	2.78 ± 0.12b	3.21 ± 0.10b	89.94	<0.001	1.00
ΣSat	40	35.37 ± 0.21	35.10 ± 0.33	35.53 ± 0.38	35.93 ± 0.33		NS
	60	35.18 ± 0.50	35.36 ± 0.30	35.86 ± 0.49	35.08 ± 0.37		NS
ΣMono	40	43.04 ± 0.68a	42.31 ± 0.71a	39.31 ± 1.02b	41.49 ± 0.47ab	4.63	0.005	0.87
	60	44.10 ± 0.75a	42.17 ± 0.86ab	41.46 ± 0.68ab	40.09 ± 0.83b	4.70	0.005	0.88
ΣPUFA	40	21.59 ± 0.75a	22.59 ± 0.68ab	25.16 ± 0.95b	22.58 ± 0.57ab	4.12	0.010	0.83
	60	20.71 ± 0.69a	22.47 ± 0.83ab	22.68 ± 0.77ab	24.83 ± 0.85b	4.74	0.010	0.88
Σn3	40	1.14 ± 0.04a	1.29 ± 0.05a	3.73 ± 0.22b	3.16 ± 0.09c	115.49	<0.001	1.00
	60	1.11 ± 0.04a	1.54 ± 0.21a	3.29 ± 0.14b	3.73 ± 0.11b	88.90	<0.001	1.00
Σn6	40	19.74 ± 0.68	20.55 ± 0.61	20.67 ± 0.72	18.75 ± 0.47		NS
	60	19.19 ± 0.61	20.52 ± 0.69	19.04 ± 0.64	20.72 ± 0.74		NS
n6:n3	40	17.38 ± 0.22a	16.14 ± 0.36b	5.66 ± 0.16c	5.96 ± 0.09c	731.00	<0.001	1.00
	60	17.33 ± 0.31a	15.24 ± 0.87b	5.85 ± 0.16c	5.55 ± 0.09c	164.45	<0.001	1.00
ARA:DHA	40	14.19 ± 0.33a	12.73 ± 0.23b	2.94 ± 0.09c	3.02 ± 0.09c	783.17	<0.001	1.00
	60	12.61 ± 0.26a	11.35 ± 0.75a	2.94 ± 0.10b	3.10 ± 0.10b	157.53	<0.001	1.00

Data are presented as the mean percent of total FAs ± the SEM. The degrees of freedom and their errors for the analysis of each FA at age P40 were 3 and 68, and at age P60 were 3 and 66, respectively. Significant differences (P < 0.05) between groups were determined with Tukey’s post hoc test and are designated by a lack of common superscripts across each row.

Depression-like behavior

Before puberty (P40), there were no differences among the four groups in measures of passive behaviors (time immobile; Fig. 3A) or active behaviors (time climbing and dives; Fig. 3C and Fig. 3E). Interestingly, after puberty there was a shift toward more average time spent immobile (Fig. 3B; 175.5 min vs. 144.2 min prepuberty), less average time spent climbing (Fig. 3D; 12.4 min vs. 46.8 min prepuberty), and a lower mean number of dives (Fig. 3F; 0.44 vs. 1.25 prepuberty) in the animals deficient in DHA throughout development. At P60, one-way ANOVA detected a main effect of group on time spent immobile (F_(3,50) = 6.73, P < 0.001), time spent climbing (F_(3,54) = 6.93, P < 0.0001), and number of dives (F_(3,61) = 3.40, P = 0.023). Tukey’s post hoc pairwise analysis indicated that completely sufficient animals spent significantly less time immobile (P = 0.026) and more time climbing (P < 0.001) and performed more dives (P = 0.045) than the animals deficient in dietary DHA throughout development. In addition, preweaning sufficient animals spent significantly more time climbing (P = 0.007) than deficient cohorts, yet were no different from deficient animals in measures of immobility or dives. Postweaning sufficient (preweaning deficient) offspring exhibited behavior similar to the completely deficient controls. At P60, brain DHA level was negatively correlated to immobility time (−0.293, P = 0.016) and positively correlated to climbing time (0.376, P = 0.002) and number of dives (0.224, P = 0.036). No correlations were seen at P40.

Fig. 3.

Dietary DHA inhibited postpuberty increases in depressive-like behaviors in the FST. Diet did not affect immobility (a passive behavior) before puberty at P40 (A), and only dietary DHA throughout development reduced immobility after puberty at P60 (B). Similarly, diet did not affect climbing or diving (active behaviors) before puberty (C and E, respectively). After puberty, dietary DHA during the gestation and lactation (preweaning) period, regardless of postweaning diet, increased climbing (D). However, only dietary DHA throughout development increased diving (F). Data are presented as averages ± the SEM. Significant differences (P < 0.05) between groups were determined by one-way ANOVA followed by Tukey’s post hoc tests and are indicated by an absence of shared superscripts.

Plasma biomarkers

For plasma BDNF, one-way ANOVA did not identify a main effect of group at P40 (Fig. 4A) but did at P60 (Fig. 4B; F_(3,59) = 2.93, P = 0.041). Tukey’s post hoc analysis determined that only animals receiving DHA supplementation throughout development had significantly higher plasma BDNF concentrations after puberty than the deficient animals (P = 0.039). Both pre- and postweaning DHA supplementation was required to positively affect plasma BDNF concentration. Additionally, plasma BDNF concentration was positively correlated to plasma, but not brain, DHA levels (0.211, P = 0.049) at P60. No correlation was seen at P40.

Fig. 4.

Dietary DHA mitigated postpuberty decreases in plasma biomarkers associated with mood. Diet did not affect plasma levels of BDNF or serotonin before puberty (A and C, respectively), and only DHA supplementation throughout development increased plasma BDNF and serotonin after puberty (B and D, respectively). Before puberty, only DHA supplementation throughout development increased plasma melatonin (E). After puberty, dietary DHA during the postweaning period, regardless of maternal diet, increased plasma melatonin concentration (F). Data are presented as averages ± the SEM. Significant differences (P < 0.05) between groups were determined by one-way ANOVA followed by Tukey’s post hoc tests and are indicated by an absence of shared superscripts.

With plasma serotonin, one-way ANOVA did not detect a main effect of group at P40 (Fig. 4C) but did at P60 (Fig. 4D; F_(3,64) = 7.55, P < 0.0001). Tukey’s post hoc analysis indicated that compared with deficient control animals, only animals receiving DHA supplementation throughout development had significantly higher plasma serotonin concentrations after puberty (P = 0.002). DHA supplementation was required during gestation, lactation, and postweaning periods to positively affect plasma serotonin concentration. Plasma serotonin was positively correlated to brain DHA levels at P60 (0.346, P = 0.002), but not at P40.

For plasma melatonin, one-way ANOVA identified a main effect of group before puberty at P40 (Fig. 4E; F_(3,56) = 4.54, P = 0.006) and after puberty at P60 (Fig. 4F; F_(3,58) = 11.77, P < 0.0001). Before puberty, Tukey’s post hoc analy­sis showed that only offspring that received DHA throughout development had significantly increased plasma concentrations of melatonin (P = 0.015). Preweaning sufficient animals had melatonin concentrations that were approaching those of sufficient animals, but it appeared that DHA deficiency during the 24 day window between weaning and P40 was long enough to impair the effect of preweaning dietary DHA in these animals as there was only a trend for significance as compared with deficient controls (P = 0.088). After puberty, both groups that received postweaning dietary DHA, regardless of maternal diet, exhibited significantly increased plasma melatonin concentrations as compared with deficient controls (sufficient, P < 0.0001; postweaning sufficient, P = 0.001). Dietary supplementation with DHA after weaning was necessary to enhance plasma melatonin concentrations after puberty, and DHA supplementation during only gestation and lactation was ineffective in modulating plasma melatonin after puberty. Plasma melatonin was positively correlated to brain DHA levels at P40 (0.418, P < 0.001) and at P60 (0.509, P < 0.001).

Brain BDNF

One-way ANOVA identified a significant main effect of group on BDNF content in hippocampus (Fig. 5A; F_(3,43) = 4.23, P = 0.010) and hypothalamus (Fig. 5B; F_(3,61) = 3.99, P = 0.012) at P60. Tukey’s post hoc analysis indicated that sufficient animals had significantly higher BDNF content in both the hippocampus (P = 0.015) and hypothalamus (P = 0.017) as compared with deficient controls. These effects were absent in animals from DHA-sufficient mothers that lacked DHA supplementation after weaning (preweaning sufficient), although there was a slight trend toward increased hippocampal BDNF in this cohort (P = 0.194). DHA supplementation after weaning in offspring from DHA-deficient mothers (postweaning sufficient) was able to increase hippocampal BDNF levels (P = 0.030), but not hypothalamic BDNF content. Brain DHA levels were positively correlated to both hippocampal BDNF content (0.454, P < 0.001) and BDNF content in the hypothalamus (0.259, P = 0.019).

Fig. 5.

Dietary DHA throughout development increased BNDF in the hippocampus (A) and hypothalamus (B) when measured at P60, an effect that was absent in animals where dietary DHA was removed at weaning. Dietary DHA supplementation in deficient offspring increased hippocampal, but not hypothalamic, BDNF at P60. Data are presented as averages ± the SEM. Significant differences (P < 0.05) between groups were determined by one-way ANOVA followed by Tukey’s post hoc tests and are indicated by an absence of shared superscripts.

DISCUSSION

This study demonstrated that DHA supplementation provided to rats during early and late development increased brain DHA levels considerably and affected depressive-like behaviors and mood-associated biomarkers that emerged following puberty. Removal of DHA supplementation after weaning (late development) negated nearly all of these behavioral and biochemical effects, whereas starting dietary DHA supplementation after weaning was too late to affect most measures. The circulating biomarkers chosen in this study are clinically reported to be affected by at least one or more successful therapies aimed at treating depression (antidepressant drugs, cognitive behavioral therapy, electroconvulsive treatment, etc.). Overall, we observed an interesting pubertal shift in all of the peripheral biomarkers to lower plasma concentrations postpuberty as compared with prepuberty. This decrease coincided with the overall increase in depressive-like behaviors seen postpuberty in the DHA-deficient offspring.

Maternal and postweaning dietary supplementation with DHA had an effect on overall FA composition of tissues that depended greatly on the timing and duration of supplementation, the location of the tissue, and the specific FA of interest. Addition of DHA to the maternal diet successfully increased brain DHA content in the offspring that was further enhanced or diminished depending on whether DHA was provided in the postweaning diet. Deficient offspring had ∼30% less brain DHA than sufficient offspring both before and after puberty, or a difference of about 4% to 5% of total FAs. This spread is similar to the difference (−22% on average) that has been noted in postmortem tissue from depressed patients as compared with their healthy counterparts (33). In fact, DHA was the only FA found to be significantly altered in these postmortem brains. Another interesting phenomenon observed in this study was the effect of diet change at the time of weaning on brain DHA content before and after puberty. Switching preweaning sufficient animals to a DHA-deficient diet at weaning caused a slow decline in brain DHA content (−15% relative to sufficient animals) that did not reach the levels observed in deficient controls by the end of puberty (44 days). This rate was clearly slower than the rate of increase seen the preweaning deficient animals (+39% relative to deficient animals) indicating that the developing brain was able to retain DHA to some extent in the face of dietary deficiency. Indeed, previous studies show that accretion is relatively faster than depletion of DHA in neural tissue (34). However, infants are typically weaned to rather DHA-poor foods and children’s diets are usually no better than the usual Western adult diet, provid­ing plenty of time throughout childhood and adolescence for depletion of DHA from neural tissue. Unfortunately, longitudinal data on DHA status in children from birth to adolescence are lacking, but in one particular study, healthy term infants who were weaned at 4–6 months of age to formula without DHA had significant losses in RBC DHA content at 1 year of age (−50%) as compared with levels measured at the time of weaning. Conversely, infants fed formula containing DHA not only maintained tissue levels of DHA but had increases in RBC DHA content (24%) relative to baseline levels at weaning and improved measures of visual system development as compared with infants fed DHA-deficient formula (35). Thus, the depletion of brain DHA content after weaning in this study may be comparable to what is observed in humans following the postweaning drop in DHA consumption usually seen in infants and continued throughout development into puberty. These data indicate that the time between weaning and puberty was too short to overcome the deficiency induced by the maternal diet, but long enough to cause a substantial loss of brain DHA with a deficient postweaning diet.

In this study, maternal and postweaning dietary DHA supplementation reduced postpubertal measures of depression-like behaviors that increased in deficient animals after puberty. We used the modified FST to measure depression-like behaviors before and after puberty (36). Typical antidepressants limit the amount of passive coping (immobility) and promote the amount of active coping (swimming/climbing) in adult animals (37, 38). In juvenile animals, immobility is particularly sensitive to SSRIs and not tricyclic antidepressants and thereby effectively models the clinical evidence observed in cases of adolescent depression (39). The data from this study indicated that subsequent to puberty there was a shift in deficient animals toward a depression-like behavioral phenotype and biomarker profile. This shift may be akin to the increased incidence of depression following puberty in humans. Because the FST requires two tests separated by 24 h, it is entirely possible that the stress of the first test differentially sensitized the animals to the second test, subsequently influencing the behavioral responses seen. Accordingly, prior stressors have been shown to affect behavior in a synergistic fashion with omega-3 deficiency (40, 41). Animals provided preweaning dietary supplementation with DHA displayed a more active postpubertal coping style than deficient controls. However, improvements in all active behaviors (climbing and diving) and in passive behavior (immobility) were only observed in sufficient animals. This indicated that these effects were dependent on early and late organizational (developmental) effects of DHA supplementation, as switching to a DHA-rich postweaning diet in preweaning deficient offspring was unable to affect these measures. However, climbing behaviors were exclusively dependent on an early organizational effect of DHA, as increased time climbing was seen only in preweaning sufficient and entirely sufficient offspring, but not postweaning sufficient or deficient offspring. These data show that both maternal and postweaning dietary DHA supplementation were required to effectively buffer the postpubertal rise in passive behaviors and fall in active behaviors seen in the deficient controls. Conversely, postweaning dietary DHA was too late, and maternal-only DHA supplementation was insufficient, to overcome all aspects of this shift in behavior.

In line with the decrease in time immobile, there was a higher plasma concentration of serotonin detected in sufficient animals from this study relative to their deficient counterparts. This was only observed in the sufficient group, and not maintained in preweaning sufficient or rescued in postweaning sufficient animals, suggesting that the effect of dietary DHA supplementation on this measure was dependent on its actions in early and late development. Previous reports suggest that early, but not late, omega-3 PUFA supplementation can restore brain serotonin and dopamine levels in omega-3 PUFA-deficient rats. Taken with our results, it may be that peripheral serotonin levels are more dependent upon current omega-3 PUFA status, whereas brain serotonin levels are more sensitive to early developmental omega-3 PUFA intake (42). Interestingly, in our study there was an apparent drop in plasma serotonin levels after puberty in all animals, a phenomenon that has been reported previously in boys (43, 44). Importantly, concentrations of circulating serotonin and its precursor tryptophan have been reported to be lower in depressed adults relative to healthy controls (45, 46). Brain serotonin content is well known to be dependent on plasma tryptophan concentration (47), and the circulating serotonin level may also be an indicator of brain serotonin concentration (48). In a recent study, rats supplemented with high-DHA fish oil from gestation to adulthood exhibited decreased immobility in the FST and had increased levels of serotonin in the hippocampus and cortex, effects that were inhibited by acute treatment with a 5-HT_1A serotonin receptor antagonist (49). This suggests that optimal DHA status may enhance serotonergic neurotransmission, thereby improving depressive-like behaviors. Our data extend this previous study to suggest that optimal DHA status during both early and late development is important for measures of juvenile mood and peripheral indicators of serotonin function following puberty. Interestingly, 5-HT_1A receptors are highly expressed at birth in humans but decline rapidly in expression through adolescence (50, 51). Thus, it is intriguing to speculate that this decline is somehow buffered by enhanced levels of DHA in brain, perhaps by facilitating 5-HT_1A receptor activity.

Addition of DHA to the postweaning diet, regardless of maternal diet, also increased plasma levels of melatonin relative to deficient offspring, an effect absent in preweaning sufficient animals. This indicates that plasma melatonin levels may be more acutely dependent on DHA tissue content than on organizational effects of DHA. Melatonin has an important role in the entrainment of many biological systems (including sleep/wake cycles) to the central circadian clockmaker, the suprachiasmatic nucleus. Mood disorders have been associated with disruptions in circadian rhythms that govern essential function such as sleep, eating, and neuroendocrine function. Depressive symptoms are typically highest in morning, suggesting a phase advance of circadian rhythm, and depressed adolescents have been shown to have altered rest-activity rhythms (52). Interestingly, we observed a substantial drop in plasma melatonin concentration after puberty, an effect that has been previously reported in humans (53, 54). Circulating melatonin has been shown to be low in depressed patients, and positively affected by SSRI treatment (55, 56). Corroborating our effects reported here, a diet low in omega-3 PUFAs has been shown to decrease plasma melatonin in hamsters (57), and exogenous melatonin and melatonin receptor agonists administered to rats and mice exert antidepressant-like effects in the FST (58, 59) possibly through a central serotonin-dependent mechanism (60). Both serotonin and melatonin receptor systems have been linked to the expression of BDNF in the brain (61, 62), an important neural growth factor implicated in the neurotrophin theory of depression (63).

In accordance with the increases in plasma melatonin and serotonin, there was a higher plasma concentration of BDNF detected in sufficient animals from this study relative to their deficient counterparts. As with serotonin, but not melatonin, this increase was only observed in the sufficient group, and not maintained in preweaning sufficient or rescued in postweaning sufficient animals. This suggests that the effect of dietary DHA supplementation on plasma BDNF was dependent on its actions in early and late development. Furthermore, in alignment with melatonin and serotonin, plasma BDNF concentrations were lower after puberty, a phenomenon also reported in humans (64). Circulating levels of BDNF correlate well with the expression of BDNF in brain, at least in rodents (65), thus providing a convenient indicator of central BDNF status. It also appears that there is evidence, albeit controversial, that BDNF can cross the blood-brain barrier (66, 67). Interestingly, peripheral administration of BDNF in mice reportedly decreased immobility in the FST after 2 weeks of treatment (68) suggesting that circulating BNDF may have a functional effect on behavioral measures of mood. BDNF has a prominent role in shaping neurotransmission and plasticity in the brain by aiding in the growth, maintenance, and survival of neurons (69, 70), including the normal development and function of serotonin neurons (71). Deficits of neural plasticity appear to be associated with depression (72). Accordingly, peripheral BDNF has been shown to be lower in depressed patients as compared with healthy counterparts, and these levels rise with antidepressant treatment (73, 74). Furthermore, increased hippocampal BDNF immunoreactivity was detected in postmortem samples from patients who were treated with antidepressants (75). BDNF protein levels are highest in the hippocampus and hypothalamus, two brain regions that exhibit a high degree of plasticity and are essential elements of the neuroendocrine response to stress (76, 77). The hypothalamus in particular also plays a critical role in the onset, duration, and completion of puberty (78, 79). Thus, increases in the levels of hypothalamic and hippocampal BDNF content may provide an adolescent enhanced resiliency to stressors and negative mood states during a time of great physical, mental, and hormonal change.

In this study, dietary DHA supplementation throughout early and late development increased postpubertal hippocampal and hypothalamic BDNF protein levels relative to deficient offspring. Loss of dietary DHA after weaning resulted in BDNF protein levels no different than deficient animals; however, preweaning deficient animals placed on a postweaning DHA-supplemented diet had increased BDNF protein levels in the hippocampus but not the hypothalamus. The effects seen in hypothalamus are similar to the observations of plasma BDNF concentrations in this study. This suggests that BDNF protein expression in the hypothalamus is dependent upon effects of DHA during both early and late development, but that hippocampal BDNF is sensitive to current DHA tissue status and/or late developmental effects of DHA. Other studies have also reported an effect of an omega-3 sufficient diet during development on brain levels of BDNF protein. For example, rats fed DHA-adequate diets from gestation to 18 weeks old had elevated BDNF protein in the hippocampus and hypothalamus (80), and those fed an α-linolenic acid sufficient diet from weaning to 18 weeks old had elevated BDNF protein expression in the frontal cortex (81) compared with animals on deficient diets. In addition, fish oil supplementation during gestation and lactation increased hippocampal and cortical BDNF protein levels in 21-day-old and 90-day-old rats (49). Our data are in line with these reports but suggest that the effects of dietary DHA during development on brain BDNF protein levels are likely dependent on the timing of DHA supplementation in a brain region-specific manner. These changes in BDNF protein expression may have contributed to the behavioral phenotype observed in the FST because acute infusion of BDNF into the midbrain or hippocampus has been shown to decrease depression-like behavior in the FST (82, 83).

Our study expands upon those described previously that have examined the effect of dietary omega-3 PUFA consumption, or lack thereof, during development on depression-like behaviors in rats. Both Naliwaiko et al. (84) and Vines et al. (49) reported that fish oil supplementation throughout gestation, lactation, and after weaning resulted in male offspring that exhibited reduced immobility in the FST compared with controls when tested as adults. In addition, studies by Ferraz et al. (85) and DeMar et al. (86) indicated that feeding male rats diets supplemented with fish oil or α-linolenic acid, respectively, after weaning (P21) into adulthood for 6 months decreased FST immobility compared with unsupplemented controls. On the other hand, McNamara et al. (87) reported that feeding omega-3 PUFA-sufficient diets from weaning to adulthood (P21–P90) in female rats had no effect on FST behavior compared with omega-3 PUFA-insufficient controls. This suggests that there may be a sex difference in the effect of postweaning omega-3 PUFA supplementation on depressive-like behaviors. However, similar to the females in McMamara et al. (87), we did not observe a benefit of postweaning DHA on time spent immobile in male offspring when examined at P60. Taken together with the previous studies, this suggests that perhaps postweaning omega-3 PUFA supplementation is most beneficial in males when provided past adolescence into adulthood or when behaviors are assessed in adulthood rather than late adolescence. What is unknown is whether pre- and postweaning omega-3 PUFA supplementation in female offspring has similar effects as those seen in our study with the male offspring. Regarding developmental timing of supplementation, Ferraz et al. (88) fed fish oil either during gestation and lactation or only after weaning and found that both groups of male rat offspring had reduced immobility in the FST at 90 days of age as compared with controls on base diet alone. However, the FA composition of the base diet and the tissue levels of omega-3 PUFAs were not reported, making comparisons with our study difficult. Furthermore, we examined changes in behavior across puberty, and thus our assessments were performed on adolescents rather than adults, and Ferraz et al. (88) did not have a group supplemented both before and after weaning. In particular, our study demonstrated that both pre- and postweaning DHA was required to positively affect all measures of depressive-like behavior when measured during postpubertal adolescence. Similar to Ferraz et al. (88), Chen and Su (89) provided fish oil either only to the dam or to the dam and male offspring after weaning. Unfortunately, they did not have a completely omega-3 PUFA-deficient group because offspring from dams fed deficient diets were placed on postweaning sufficient diets containing α-linolenic acid and DHA. They examined FST behaviors at P70, a time point closer to adolescence, and concluded that only preweaning fish oil was effective in reducing immobility in the FST at that age. However, offspring in the postweaning supplemented group were obtained from dams provided fish oil; thus, any separate effects of postweaning supplementation beyond those attributable to supplementation during the preweaning period are truly not inferable from data reported in Chen and Su (89).

Adolescent depression typically follows a recurrent episodic course (90). In this regard, perhaps DHA deficiency may be seen as a diathesis, or preexisting vulnerability (akin to a genetic predisposition), that is activated by developmental events such as puberty-dependent changes in brain connectivity and hormone secretion, or by intense psychological stressors (death of a loved one, emotional abuse, parental divorce, etc.). However, there are limited clinical studies that have examined the link between omega-3 PUFAs and juvenile depression. In a case-control study comparing RBC FA profiles in 150 depressed and 161 healthy adolescents, there was a higher percentage of DHA, but not EPA (20:5n3), present in the RBCs from healthy controls as compared with their depressed counterparts (28). Interestingly, there was an increase in RBC α-linolenic acid content in depressed adolescents suggesting that the conversion of α-linolenic acid to EPA, which is already inefficient in humans (∼0.1% conversion), may be impaired in these individuals. In a cross-sectional study, DHA content was lower (−16%) in the RBCs of depressed teenagers with eating disorders than those with eating disorders and no depressive symptoms (91). In a cross-sectional study of Japanese teenagers ages 12 to 15, there was an inverse correlation of EPA plus DHA intake with symptoms of depression in boys, but not girls (5). The lifetime major depressive disorder prevalence rates in Japan are some of the lowest in the world (92). However, to achieve these rates it may be necessary to attain the DHA concentrations found in the RBCs of Japanese adults. Such a feat likely requires a daily DHA intake of 400–700 mg by children and 700–1,000 mg by adults in the United States (93). Along these lines, a handful of intervention studies have been reported to date. Nemets et al. (27) observed improved scores in tests for symptoms of depression in depressed children ages 6 to 12 that were given 400 mg of EPA plus 200 mg of DHA daily for 1 month as compared with children who received placebo. Clayton et al. (26) provided 360 mg of EPA plus 1,560 mg of DHA as an adjunct to pharmacotherapy in 18 female bipolar depressed teens (average 16 years old) that had been stabilized for 6 weeks on lithium and valproate. Compared with within-individual baseline data, the supplementation caused a substantial elevation of RBC DHA and EPA content, and a significant decrease in clinical scores of depression. McNamara et al. (29) provided fish oil at doses of 2.4 g/day or 16.2 g/day in a small open-label trial for 10 weeks to adolescents with SSRI-resistant depression and observed symptom remission in 40% and 100% of cases, respectively. These studies provide an initial indication of the effect that optimal dietary DHA consumption might have on adolescent mood. It remains to be determined how maintenance of the optimal tissue levels of DHA throughout pregnancy, nursing, childhood, and adolescence affects the prevalence, duration, or severity of juvenile depression.

It should also be noted that several randomized, clinical trials have indicated that oral supplementation with fish oil, which contains substantial (but variable) amounts of the omega-3 PUFAs DHA and EPA, can improve clinical measures of depression in adults [for meta-analysis see (30)]. These effects have largely been attributed to EPA (94); however, only one direct side-by-side comparison between pure EPA and pure DHA has been reported. Mozaffari-Khosravi et al. (95) reported that EPA but not DHA was effective as an adjunct therapy to antidepressants in young adults with mild-to-moderate depression. Yet, in this study the effect of antidepressant cotherapy on the efficacy of either omega-3 PUFA is unknown, and the plasma FA profiles of the participants was not reported. The effect of EPA may be attributable to its anti-inflammatory actions in the periphery (96). However, in agreement with our FA data, only trace levels of EPA are found in the brain where upon entry it is rapidly β-oxidized and metabolized (97, 98). Furthermore, erythrocyte DHA, but not EPA, content was shown to be lower in SSRI-resistant depressed adolescents compared with healthy counterparts (29). Given that DHA, but not EPA, is highly enriched in neural tissue and is essential for brain development, it may play a critical role in the developmental factors that shape the etiology of adolescent depression, a supposition in alignment with the results of our study.

The data reported here present a totality of pubertal changes in depression-like behaviors and central and peripheral biomarkers of depression in male rats and strongly suggest that these changes can be positively modulated by DHA supplementation throughout development. These data highlight the particular importance of dietary supplementation with preformed DHA after weaning, a time when most infants are transitioned to foods containing low levels of omega-3 PUFAs, a trend that continues onward into adulthood. The clinical evidence regarding the role of DHA and omega-3 PUFAs on adolescent depression is emerging yet limited, but given the intervention and epidemiological data currently available and potential societal impact, further studies are certainly warranted.