Impact of maternal n-3 polyunsaturated fatty acid deficiency on dendritic arbor morphology and connectivity of developing Xenopus laevis central neurons in vivo

Abstract

Docosahexaenoic acid (DHA, 22:6n-3) is an essential component of the nervous system, and maternal n-3 polyunsaturated fatty acids (PUFAs) are an important source for brain development. Here, the impact of DHA on developing central neurons was examined using an accessible in vivo model. Xenopus laevis embryos from adult female frogs fed n-3 PUFA-adequate or deficient diets were analyzed every 10 weeks for up to 60 weeks, when frogs were then switched to a fish oil-supplemented diet. Lipid analysis showed that DHA was significantly reduced both in oocytes and tadpoles 40 weeks after deprivation, and brain DHA was reduced by 57% at 60 weeks. In vivo imaging of single optic tectal neurons coexpressing tdTomato and PSD-95-GFP revealed that neurons were morphologically simpler in tadpoles from frogs fed the deficient diet compared with the adequate diet. Tectal neurons had significantly fewer dendrite branches and shorter dendritic arbor over a 48 h imaging period. Postsynaptic cluster number and density were lower in neurons deprived of n-3 PUFA. Moreover, changes in neuronal morphology correlated with a 40% decrease in the levels of BDNF mRNA and mature protein in the brain, but not in TrkB. Importantly, switching to a fish oil-supplemented diet induced a recovery in DHA content in the frog embryos within 20 weeks and diminished the deprivation effects observed on tectal neurons of Stage 45 tadpoles. Consequently, our results indicate that DHA impacts dendrite maturation and synaptic connectivity in the developing brain, and it may be involved in neurotrophic support by BDNF.

Keywords: DHA; Xenopus laevis; dendrite; in vivo imaging; maternal diet; tectal neuron.

Figure 1.

Experimental plan. Detailed experimental plan is stated in Materials and Methods. Briefly, after 4 week acclimatization, frogs were divided into two groups, n-3 PUFA-adequate or n-3 PUFA-deficient diets were fed to frogs for 60 weeks, and then both groups were fed fish oil-supplemented n-3 PUFA-adequate diet for an additional 20 weeks up to week 80. Ovulation (Ovu.) was induced every 10 weeks after the first ovulation, and neuron imaging was performed at 40, 60, and 80 weeks.

Figure 2.

n-3 PUFA contents in oocytes from female frogs fed special diets over 80 weeks. DHA (A), ALA (B), and total n-3 PUFA (C) contents in oocytes from frogs fed special diets over the 80 week study. Data are mean ± SEM. *p < 0.05, compared with week 0 (before change in diet) (one-way ANOVA followed by Dunnett' multiple comparison test). ***p < 0.001, compared with week 0 (before change in diet) (one-way ANOVA followed by Dunnett' multiple comparison test). n = 4–6 samples per time point. Ade, Adequate diet; Def, deficient diet.

Figure 3.

Morphology of tectal neurons in tadpoles from frogs fed n-3 PUFA-adequate or n-3 PUFA-deficient diets. Representative tectal neurons expressing tdTomato (red) together with GFP-PSD-95 (green) in tadpoles from frogs fed n-3 PUFA-adequate and -deficient diets at (A) 20, (B) 40, and (C) 60 weeks. Neuron imaging was performed at 0 h (Stage 45), 24 and 48 h later as indicated. Scale bar, 20 μm.

Figure 4.

Tectal neurons have fewer branches in n-3 PUFA-deprived brain. A, Total branch number, (B) total dendritic length, and (C) average branch length were analyzed in tectal neurons in tadpoles from frogs fed the n-3 PUFA-adequate (Ade) or deficient (Def) diets for 60 weeks. Data are mean ± SEM. N = 24 neurons for the adequate, and n = 25 neurons for the deficient group, with one neuron analyzed per tadpole. Tadpoles analyzed were obtained from 6 and 5 frogs, respectively. *p < 0.05, **p < 0.01, ***p < 0.001; asterisks above gray bars indicate comparison with time-matched adequate group. (Tukey test).

Figure 5.

Dendritic arbors are morphologically simpler in n-3 PUFA-deprived brain. Branch order was analyzed in tectal neurons at (A) Stage 45, and (B) 24 h and (C) 48 h after the first imaging. D, DCI was calculated at Stage 45 as defined by the equation (Marshak et al., 2007). Data are mean ± SEM. N = 24 neurons for the adequate (Ade), and n = 25 neurons for the deficient (Def) group, with one neuron analyzed per tadpole. Tadpoles analyzed were obtained from 6 and 5 frogs, respectively. *p < 0.05, compared with the adequate group (unpaired t test). **p < 0.01, compared with the adequate group (unpaired t test). ***p < 0.001, compared with the adequate group (unpaired t test).

Figure 6.

New dendrite branch addition is altered in neurons from tadpoles with n-3 PUFA deficiency. The number of new branches and of stable branches was counted every 24 h to determine dendrite dynamics. The absolute number of (A) new branches added and (B) stable branches were reduced in the n-3 PUFA-deficient (Def) group. However, as percentage of total, the number of new branches (C) and stable branches (D) was not changed. Data are mean ± SEM. N = 24 for the adequate (Ade) and n = 25 for the deficient group. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7.

Postsynaptic site number and density are decreased in n-3 PUFA-deprived tectal neurons. A, The total number and (B) density of PSD95-GFP puncta were determined for tectal neurons in tadpoles at Stage 45 (0 h) and 48 h later (Stage 47; 48 h) from frogs fed the n-3 PUFA-adequate (Ade) or deficient (Def) diets for 60 weeks. B, Postsynaptic site density was calculated using total synapse number and total dendrite arbor length (Fig. 4A) for each individual neuron. Data are mean ± SEM. N = 9 for the adequate and deficient groups. *p < 0.05 with unpaired t test. **p < 0.01.

Figure 8.

Reduction of mature BDNF and PSD-95 protein levels in the tadpole brain. A, Representative immunoblots and (B) relative expression levels of pro-BDNF, mature-BDNF, TrkB, and PSD-95 and β-actin, and (C) relative total BDNF mRNA levels in tadpole brains from frogs fed n-3 PUFA-adequate (Ade) and deficient (Def) diets for 60 weeks. Tissues from Stage 45 tadpoles obtained from 6 frogs fed the adequate diet and 5 frogs fed the deficient diet were collected for Western blot and quantitative RT-PCR analyses. For Western blot, data were normalized to β-actin expression, which served as internal control for protein loading. Data are expressed as change in values relative to the adequate group. Data are mean ± SEM. *p < 0.05 (unpaired t test).

Figure 9.

Maternal DHA supplementation reverses morphological deficits in neurons and in BDNF expression caused by n-3 PUFA deprivation. Representative tectal neurons in Stage 45 tadpoles from frogs fed the (A) adequate and (B) deficient diet for 60 weeks and then switched to a fish oil-contained n-3 PUFA-adequate diet for 20 weeks. Sample neurons were from two different tadpoles, each obtained from oocytes from two different frogs in each group (A, B). C, Total branch numbers, (D) total dendrite arbor length, and (E) dendritic arbor complexity index (DCI) were measured. N = 19 neurons for the adequate group (Ade), and n = 13 neurons for the deficient group (Def) in tadpoles from 5 frogs per group. Relative expression levels of (F) pro-BDNF and mature-BDNF, and of (G) total BDNF mRNA, measured in brains of Stage 45 tadpoles from frogs at week 80 (n = 4 or 5). Data are mean ± SEM.

Figure 10.

Maternal n-3 PUFA condition does not influence retinal axonal projections to the brain. Representative coronal sections of Stage 45 tadpoles from frogs fed the (A–D) n-3 PUFA-adequate (Ade) or (A′–D′) n-3 PUFA-deficient (Def) diet for 60 weeks and immunostained with a neurofilament-associated protein antibody (green) illustrate the normal projection patterns of retinal axons (A, A′) out of the eye and along the optic nerve (on), and (B, B′) at the level of the optic chiasm (oc). Projection of axonal fibers along (C, C′) the optic tract (ot) and (D, D′) the rostral tectum (tect) were also similar in Stage 45 tadpoles from both groups. Counterstaining with DAPI (blue) reveals normal size and cellular organization in the (A, A′) retina, and (B–D, B′–D′) brains of tadpoles from frogs fed the adequate (A–D) and deficient (A′–D′) diets. rgc, Retinal ganglion cell layer; v, ventricle. Dorsal is up, ventral is down. Scale bar, 50 μm.