Developmental effects of dietary n-3 fatty acids on activity and response to novelty

Abstract

Insufficient availability of n-3 polyunsaturated fatty acids (PUFA) during pre- and neonatal development decreases accretion of docosahexaenoic acid (DHA, 22:6n-3) in the developing brain. Low tissue levels of DHA are associated with neurodevelopmental disorders including attention deficit hyperactivity disorder (ADHD). In this study, 1st- and 2nd-litter male Long-Evans rats were raised from conception on a Control diet containing alpha-linolenic acid (4.20 g/kg diet), the dietarily essential fatty acid precursor of DHA, or a diet Deficient in alpha-linolenic acid (0.38 g/kg diet). The Deficient diet resulted in a decrease in brain phospholipid DHA of 48% in 1st-litter pups and 65% in 2nd-litter pups. Activity, habituation, and response to spatial change in a familiar environment were assessed in a single-session behavioral paradigm at postnatal days 28 and 70, inclusive. Activity and habituation varied by age with younger rats exhibiting higher activity, less habituation, and less stimulation of activity induced by spatial novelty. During the first and second exposures to the test chamber, 2nd-litter Deficient pups exhibited higher levels of activity than Control rats or 1st-litter Deficient pups, and less habituation during the first exposure, but were not more active after introduction of a novel spatial stimulus. The higher level of activity in a familiar environment, but not after introduction of a novel stimulus is consistent with clinical observations in ADHD. The observation of this effect only in 2nd-litter rats fed the Deficient diet suggests that brain DHA content, rather than dietary n-3 PUFA content, likely underlies these effects.

Fig. 1. Schematic of the experimental apparatus (A) and test protocol (B)

Fig. 2. Effects of the experimental treatments on brain phospholipid fatty acid composition

Data for docosahexaenoic acid (DHA, 22:6n-3), docosapentaenoic acid (DPA, 22:5n-6), and arachidonic acid (AA, 20:4n-6) are presented as the group means ± SEM (n = 5–6 per group selected at random from the total sample). The percentages of DHA and DPA in brain phospholipids were not different between 1^st- and 2^nd-litter Control pups. For all ages, brain DHA of 1^st- and 2^nd-litter Deficient pups was decreased compared to 1^st- and 2^nd-litter Controls (P<0.001). Brain DHA of 2^nd-litter Deficient pups was also lower than 1^st-litter Deficient pups (P<0.001). Brain DPA of 1^st- and 2^nd-litter Deficient pups was higher than their respective control groups at all ages (P<0.001). There was a main effect of litter on brain AA such that 1^st-litter pups had 8% higher AA than 2^nd-litter pups (P<0.001), but there was no effect of diet or age.

Fig. 3. Effects of experimental treatments on body weight

Data are presented as the group means ± SEM. Sample sizes for ages 28, 35, 42, 49, 56, and 70 days, respectively, were 1^st-litter Control: 13, 9, 9, 16, 17, and 14; 2^nd-litter Control: 11, 13, 14, 11, 15 and 11; 1^st-litter Deficient: 15, 17, 19, 20, 14, and 19; 2^nd-litter Deficient: 12, 9, 9, 9, 13, and 12. There was a significant effect of litter on weight, with 2^nd-litter pups weighing 4% less than 1^st-litter pups (P<0.001), but no main effect of treatment or interaction of litter and treatment.

Fig. 4. Activity of Control rats in the test paradigm across postnatal development

Activity was assessed on the basis of distance traveled. Data are presented across time blocks (A) and as the main effect for each test period (B). Data were collected in 5-min time blocks, were analyzed by repeated-measures ANOVA with factors of age and time block, and are presented as the group means ± SEM. Sample sizes for ages 28, 35, 42, 49, 56, and 70 days, respectively, were 24, 29, 33, 29, 32, and 31 reflecting the combined 1^st and 2^nd Control litters into a single Control group. In Panel A, there were significant main effects of age and time block, and interactions of age with time block during each of the test Periods (P<0.01). For clarity, differences between ages in each time block are not indicated. *P<0.05 v. P70 by ANOVA and Dunnett’s test.

Fig. 5. Habituation, recovery of activity, and novelty-induced stimulation of activity across postnatal development in Control rats

Data are presented as group means ± SEM for each of the three test Periods. Sample sizes for ages 28, 35, 42, 49, 56, and 70 days, respectively, were 24, 29, 33, 29, 32, and 31 reflecting the combined 1^st and 2^nd Control litters into a single Control group. Period 1 habituation was calculated as ratio of the distances traveled in time blocks 6 and 1. Period 2 recovery was the ratio of distances traveled in time blocks 9 and 6. Period 2 habituation was the ratio of distances traveled in time blocks 12 and 9. Period 3 stimulation was the ratio of distances traveled in time blocks 15 and 12. Period 3 habituation was the ratio of distances traveled in time blocks 18 and 15. *P<0.05 v. P70 by ANOVA and Dunnett’s test.

Fig. 6. Effects of dietary n-3 fatty acid content and litter on activity in the test paradigm across development

Activity was assessed on the basis of distance traveled. Data were collected in 5-min time blocks, were analyzed by repeated-measures ANOVA with factors of treatment, age and time block, and are presented as group means ± SEM. Sample sizes for ages 28, 35, 42, 49, 56, and 70 days, respectively, for Control rats were 24, 29, 33, 29, 32, and 31 reflecting the combined 1^st and 2^nd Control litters. Sample sizes for 1^st litter Deficient rats were 15, 17, 20, 19, 16, and 19; and 2^nd litter Deficient rats were 12, 12, 10, 9, 13, and 12. There was no significant interaction of treatment and age. The main effect of treatment on distance traveled is presented in Fig. 7.

Fig. 7. Main effects of dietary n-3 fatty acid content and litter on total distance traveled (A) and habituation, recovery of activity, and novelty-induced stimulation of activity (B)

Data are presented as group means ± SEM for each of the three test Periods. Sample sizes were 178 for Control, 106 for 1^st-litter Deficient, and 68 for 2^nd-litter Deficient. Habituation, recovery, and stimulation were calculated as described for Fig. 5. *P<0.05 v. Control, ^†P<0.05 v. 1^st-litter Deficient by ANOVA and Tukey’s test.