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DHA Restriction Leads to Distortions in Visual Pathways in Early Development

The development of visual pathways from the retina to the visual cortex in the brain involves the proper arrangement of retinal neuronal axons within two brain structures along the route. These structures, the superior colliculus and the lateral geniculate nucleus, require precise alignment (topography) of the neural connections from the retina and other sensory tissues so that coordinated responses are possible (Figure). For example, connections with motor neurons permit eye movement; those with auditory neurons enable visual responses to sound. Coordinated sensory responses permit three-dimensional vision and the recognition of objects by touch.

During development, axonal projections of the retinal neurons converge with their target cells in the superior colliculus in specific patterns described as retinocollicular topography. The final maturation of this circuitry in rodents occurs in the first 2 to 3 weeks of postnatal development. This time frame constitutes a critical period during which disturbances from arachidonic acid blockade, sensory deprivation or nutritional restriction may impair the development of the retinocollicular topography and axonal plasticity. Such impairment may not be restored by repletion if restoration occurs past the critical period.

The primary period for the development of the human visual system is between 20 weeks’ gestational age and 2 to 3 years of life. The most critical processes in retinal development occur between 24 weeks’ gestation and 3 to 4 months of age. The synaptic circuitry of the retina is refined during postnatal development. Adverse events occurring between 24 and 40 weeks’ gestation, such as nutritional deprivation or injury, usually do not alter the structure of the eye, except in retinopathy of prematurity, but do affect visual function. It has been reported that disturbances in long-chain (LC) PUFA metabolism in early postnatal development disrupt the orderly arrangement of axons in the lateral geniculate nucleus and the superior colliculus. Blocking the activity of phospholipase A2 or 5-lipoxygenase during the critical postnatal period of visual maturation induced the sprouting of uncrossed axons in the collicular visual layers.

Extending their earlier studies on nutrition and visual function, Patricia Coelho de Velasco and colleagues in the laboratory of Claudio Serfaty at the Universidade Federal Fluminense, Brazil, examined the effects of pre- and postnatal n-3 LC-PUFA deprivation on the axonal topography of the retinocollicular and retinogeniculate structures in developing rats. The investigators initiated the DHA restriction prior to mating and continued it through 13, 28 or 42 postnatal days. The researchers fed the control animals a diet containing 5% fat from soybean oil, which contains linoleic and alpha-linolenic acids. Rats can convert alpha-linolenic acid to long-chain n-3 PUFAs. Restricted diets contained 5% coconut oil as the fat source, which has no omega-3 (n-3) PUFAs and only small amounts of linoleic acid. Animals consumed the diets from 5 weeks prior to mating through delivery and lactation until postnatal day 13, 28 or 42. One group of n-3 PUFA-restricted animals was repleted with oral fish oil providing 120 mg of DHA and 180 mg EPA per day from postnatal day 7 to 28. Another group of animals from each treatment protocol was given a retinal lesion at postnatal day 21 and continued on the same diets for 7 days until sacrifice at postnatal day 28.

The investigators measured the fatty acid profile of the superior colliculi at postnatal day 28. Axons and axon terminals in the tissue were labeled with horseradish peroxidase and quantified using imaging software. Optical densities of the axon terminals were assessed in the upper layers of the superior colliculus along the ventral border of the stratum griseum superficial (SGS). Axon terminals from the retina normally form clusters of uncrossed axons in this region, mainly along the dorsal-lateral and dorso-medial aspects. Similar densities were measured in the central region of the lateral geniculate nucleus.

Fatty acid analysis of the superior colliculus at postnatal day 28 showed that the n-3 PUFA-restricted animals had significantly lower DHA compared with the controls (Table). Significant increases in palmitic, stearic and oleic acids were observed, without changes in the levels of arachidonic acid. The effect of n-3 LC-PUFA restriction on the retinocollicular topography was examined in the second postnatal week, day 13, which is during the critical developmental period. Control animals exhibited a normal distribution of terminal labeling in clusters, while in the restricted animals, labeling was expanded and fused along the ventral border of the SGS. Axons were also scattered outside the main axon terminal zones.

The appearance of the terminal clusters observed in the dark field photomicrographs of the superior colliculus was confirmed by measurements of the optical densities in the terminal fields. Optical densities in the SGS of the n-3 LC-PUFA-restricted animals at days 13, 28 and 42 were significantly increased compared with the unrestricted animals, reflecting the fusion of axon terminals and expansion of the clusters. Interestingly, when the restricted animals were repleted with fish oil from postnatal day 7 to 28, optical density values were restored to those comparable to the unrestricted controls.

In addition, the researchers assessed the critical period window in both treatment groups in animals given a retinal lesion in the left eye at postnatal day 21. Previous studies showed that at this stage, a retinal lesion does not lead to axonal sprouting from uncrossed axons in the intact eye within one week of the lesion. This observation is consistent with the end of the critical developmental period. Axonal sprouting is the attempt by a neuron to form new connections after neuronal damage.

In this study, the investigators assessed axonal sprouting by measuring the optical density of the subpial collicular layers in the intact eye 7 days after the lesion, i.e., at postnatal day 28. Control animals exhibited little terminal labeling in the lateral superior colliculus one week after the lesion. In contrast, the n-3 LC-PUFA-restricted animals showed a pronounced sprouting response, extending to the lateral aspect of the superior colliculus. Axonal sprouting was reflected in the optical density measurements and the photomicrographs of the superior colliculus. In control animals, optical densities between the lateral and medial aspects of the superior colliculus did not differ significantly. In contrast, the n-3 LC-PUFA-restricted animals had approximately twice as much labeling in the lateral subpial aspect of the superior colliculus compared with the medial aspect. These observations, taken one week after the usual closure of the critical developmental period, suggest that the retinal lesion in the n-3 LC-PUFA-restricted animals induced an extension of synaptic plasticity in the uninjured eye.

Evidence that the axonal sprouting was related to the lesion and not the n-3 LC-PUFA restriction itself was shown in the optical densities the medial and lateral aspects of the superior colliculus in the control and restricted animals. In both groups without lesions, optical densities were lower in the lateral than the medial aspects, but did not differ significantly between the two locations. Only in the injured animals on the n-3 LC-PUFA-restricted diet were the optical densities significantly higher in the lateral than the medial aspects (15.3 ± 1.5 vs 10.2 ± 1.3 absorbance units, P < 0.05).

The key findings from this study showed that n-3 LC-PUFA restriction during the critical period of visual development, resulted in significantly reduced levels of DHA in the superior colliculus and the lateral geniculate nucleus, two brain structures essential for proper visual function. Reduced DHA concentrations in the superior colliculus have been reported in baboons that were not restricted in n-3 LC-PUFAs during fetal development, but were fed infant formula lacking LC-PUFAs. The n-3 LC-PUFA restriction in this study led to disturbances in the topographical maps of these structures, resulting in the expansion and fusion of the axon terminals from the retina to these sites. The provision of fish oil from postnatal day 7 to 28 reversed the effects of n-3 LC-PUFA dietary restriction. The n-3 LC-PUFA-restricted animals exhibited a prolonged period of synaptic plasticity, as observed in the spread of axonal sprouting in the lateral region of the superior colliculus at postnatal day 28, suggesting a delayed closure of the critical period.

In their discussion of these observations, the authors commented that their observations could best be explained by DHA deprivation delaying axonal pruning and the closure of the critical period of synaptic plasticity. Other research has shown that DHA affects gene expression in the hippocampus and improves synaptic plasticity and axonal growth. The investigators further pointed out that normally the development of sensory connections in these structures is achieved through the selective elimination of misplaced axons and the stabilization of correct placements with their synapses. These studies increase our understanding of how DHA contributes to the proper development of the visual system in early life.

de Velasco PC, Mendonça HR, Borba JM, Andrade da Costa BL, Guedes RC, Navarro DM, Santos GK, Faria-Melibeu AD, Campello Costa P, Serfaty CA. Nutritional restriction of omega-3 fatty acids alters topographical fine tuning and leads to a delay in the critical period in the rodent visual system. Exp Neurol 2012;234:220-229. PubMed

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