Rebecca Sheaff Greiner

Rats with low levels of brain docosahexaenoic acid show impaired performance in olfactory-based and spatial learning tasks

Studies were carried out to determine if decreased levels of central nervous system docosahexaenoic acid (DHA), a result of consuming an n-3-deficient diet, had an effect on learning- and memory-related behaviors in adult male rats. Females were reared on an n-3-deficient or n-3-adequate diet beginning at 21 d of life. Their male pups, the F2 generation, were weaned to the diet of the dam and tested at 9–12 wk of age. An olfactory-based discrimination and Morris water maze task were used to assess performance. Whole brain was collected after the behavioral experiments and central nervous system fatty acid content was analyzed in olfactory bulb total lipid extracts. F2 generation male rats consuming the n-3-deficient diet had an 82% decrease in DHA compared to rats consuming the n-3-adequate diet. The n-3-deficient animals made significantly more total errors in a 7-problem, 2-odor discrimination task compared to the n-3-adequate group. Furthermore, the escape latency in the Morris water maze task was significantly longer for the n-3-deficient rats compared to the n-3-adequate rats. These results indicate that rats with decreased DHA levels in the central nervous system perform poorer in these tasks compared to rats with higher DHA levels and suggest the presence of learning deficits in these animals.

Cognitive deficits in docosahexaenoic acid-deficient rats.

This study investigated the influence of brain docosahexaenoic acid (DHA) deficiency on simple and complex olfactory-based learning and memory in 2nd generation (F2) adult male rats. Rats raised and maintained on either an n-3-adequate or an n-3-deficient diet were tested for acquisition of an olfactory learning set and an olfactory memory task, and for motivation to obtain a water reward. Despite a 76% decrease in brain DHA, n-3-deficient rats were able to acquire most simple 2-odor discrimination tasks but were deficient in the acquisition of a 20-problem olfactory learning set. This deficit could not be attributed to changes in sensory capacity but, instead, appeared to represent a deficit in higher order learning.

Nutritional Deprivation of α‐Linolenic Acid Decreases but Does Not Abolish Turnover and Availability of Unacylated Docosahexaenoic Acid and Docosahexaenoyl‐CoA in Rat Brain

Abstract: We applied our in vivo fatty acid method to examine concentrations, incorporation, and turnover rates of docosahexaenoic acid (22:6 n‐3) in brains of rats subject to a dietary deficiency of α‐linolenic acid (18:3 n‐3) for three generations. Adult deficient and adequate rats of the F3 generation were infused intravenously with [4,5‐3H]docosahexaenoic acid over 5 min, after which brain uptake and distribution of tracer were measured. Before infusion, the plasma 22:6 n‐3 level was 0.2 nmol ml‐1 in 18:3 n‐3‐deficient compared with 10.6 nmol ml‐1 in control rats. Brain unesterified 22:6 n‐3 was not detectable, whereas docosahexaenoyl‐CoA content was reduced by 95%, and 22:6 n‐3 content in different phospholipid classes was reduced by 83‐88% in deficient rats. Neither plasma or brain arachidonic acid (20:4 n‐6) level was significantly changed with diet. Docosapentaenoic acid (22:5 n‐6) reciprocally replaced 22:6 n‐3 in brain phospholipids. Calculations using operational equations from our model indicated that 22:6 n‐3 incorporation from plasma into brain was reduced 40‐fold by 18:3 n‐3 deficiency. Recycling of 22:6 n‐3 due to deacylation‐reacylation within phospholipids was reduced by 30‐70% with the deficient diet, but animals nevertheless continued to produce 22:6 n‐3 and docosahexaenoyl‐CoA for brain function. We propose that functional brain effects of n‐3 deficiency reflect altered ratios of n‐6 to n‐3 fatty acids.

n-3 Fatty acid deficiency decreases phosphatidylserine accumulation selectively in neuronal tissues.

We have previously shown that the docosahexaenoate (22∶6n−3) status in membrane phospholipids influences the biosynthesis and accumulation of phosphatidylserine (PS) in brain microsomes and C6 glioma cells. In the present study, we investigated whether the observed effect of membrane docosahexaenoic acid status on PS accumulation is universal or occurs specifically in neuronal tissues. We observed that rat brain cortex, brain mitochondria, and olfactory bulb, where 22∶6n−3 is highly concentrated, contain significantly higher levels of PS in comparison to liver and adrenal, where 22∶6n−3 is a rather minor component. Phospholipid molecular species analysis revealed that in brain cortex, mitochondria, and olfactory bulb 18∶0,22∶6n−3 was the most abundant species representing 45–65% of total PS. In nonneuronal tissues such as liver and adrenal, 18∶0,20∶4n−6 was the major PS species. Dietary depletion of n−3 fatty acids during prenatal and postnatal developmental periods decreased the brain 22∶6n−3 content by more than 80%, with a concomitant increase in 22∶5n−6 in all tissues. Under these conditions, an approximately 30–35% reduction in total PS in rat brain cortex, brain mitochondria, and olfactory bulb was observed, while PS levels in liver and adrenal were unchanged. The observed reduction of PS content in neuronal membranes appears to be due to a dramatic reduction of 18∶0,22∶6n−3-PS without complete replacement by 18∶0,22∶5n−6-PS. These results establish that variations in membrane 22∶6n−3 fatty acid composition have a profound influence on PS accumulation in neuronal tissues where 22∶6n−3 is abundant. These data have implications in neuronal signaling events where PS is believed to play an important role.

Alterations in brain function after loss of docosahexaenoate due to dietary restriction of n-3 fatty acids.

The concentration of the major polyunsaturated fatty acid (PUFA) in brain, docosahexaenoate, may be markedly reduced by two or more generations of dietary restriction of sources of n-3 fatty acids in the diet. Such a deficiency was induced through the feeding of safflower oil as the principal source of essential fatty acids. The reference point for this diet was an n-3 adequate diet to which alpha-linoleate and docosahexaenoate were added through the addition of a small quantity of flax seed or algael oils, respectively. The loss of brain DHA was associated with poorer performance in spatial tasks and an olfactory-cued reversal learning task. No difference could be observed in the hippocampal gross morphology. This study demonstrates the importance of providing a source of n-3 fatty acids during mammalian growth and development.

Olfactory discrimination deficits in n−3 fatty acid-deficient rats

Docosahexaenoic acid (DHA), a long chain n-3 fatty acid, is present in high concentrations in the central nervous system. Although the role that DHA may play in neural function is not well understood, infants fed formulas containing low levels of n-3 fatty acids have decreased visual acuity and neurodevelopmental test scores. The present experiment assessed whether dietary manipulations that decrease the concentration of DHA in the brain interfered with olfactory-based learning. We fed rats a diet that provided adequate n-3 fatty acids or a diet that was deficient in n-3 fatty acids for two generations. The second generation n-3-deficient group had 81% less brain DHA (82% less in olfactory bulb) compared to the n-3-adequate group and made significantly more errors in a series of olfactory-cued, 2-odor discrimination tasks compared to the adequate group. These results suggest that lower levels of central nervous system DHA lead to poorer performance in a series of simple odor discrimination tasks.

A decrease in cell size accompanies a loss of docosahexaenoate in the rat hippocampus.

Rats raised on n-3 essential fatty acid deficient diets demonstrate spatial memory deficits. To investigate neuroanatomical correlates of these deficits, morphological analysis of the hippocampus were carried out. Adult, female rats were raised for three generations on n-3 deficient or n-3 supplemented diets. Two n-3 deficient diets contained adequate linoleic acid (LA), or high linoleic acid (high LA), and two supplemented diets contained LA supplemented with alpha-linolenic acid (+LNA), or linoleic supplementation with alpha- linolenic and docosahexaenoic acids (+LNA/DHA). The total fatty acid composition of the hippocampus revealed a profound loss (90%) in docosahexaenoic acid (DHA) in the hippocampi of LA and high LA animals compared to those on +LNA and +LNA/DHA diets with a reciprocal increase in docosapentaenoic acid (DPAn-6) in all phospholipid species. The volume, density; total number, and cell body size of neurons in CA1-3, granular and hilar layers of the hippocampus were measured at septal and temporal locations using unbiased stereology. No differences were detected in any of these measures except for in cell body size; CA1 pyramidal neurons in the LA group were significantly (p < 0.04) smaller than neurons in the +LNA/DHA group at the septal location.

Docosapentaenoic acid does not completely replace DHA in n-3 FA-deficient rats during early development.

The reciprocal replacement of DHA by docosapentaenoic acid (DPAn−6) was studied in rats that consumed an n−3 FA-deficient or n−3 FA-adequate diet. Dams were fed the two experimental diets from weaning and throughout pregnancy and lactation. Their pups were then fed the respective diets after weaning. Cortex FA analysis was performed at various times (0, 5, 10, 20, 50, and 91 d) after birth to determine whether DPAn−6 completely replaced DHA in the n−3-deficient group. Cortical DHA levels were significantly lower (average 86%) in the n−3-deficient rats. DPAn−6 increased significantly in the n−3-deficient rats starting with a 6.5-fold increase at day 0 up to a 54-fold increase at day 91 compared with the n−3-adequate group. However, this significant increase did not completely replace the loss of DHA at postnatal days 5, 10, and 20 in which there was still an 11.5, 10.3, and 8.0% deficit in the sum of DHA and DPAn−6, respectively, in the n−3-deficient group. Once docosatetraenoic (DTA) and arachidonic acids (AA) were included in the sum (DHA+DPAn−6+DTA+AA), the levels between the two groups were similar, These results suggest that not only DPAn−6 but also other n−6 FA, including DTA and AA, replace DHA in n−3-deficient rats. The lack of total 22-carbon (22C) FA in the brain during the rapid membrane biogenesis that occurs during early development could be a factor in the nervous ystem functional deficits associated with n−3 FA deficiency.

Chronic nutritional deprivation of n-3 α-linolenic acid does not affect n-6 arachidonic acid recycling within brain phospholipids of awake rats

Using an in vivo fatty acid model and operational equations, we reported that esterified and unesterified concentrations of docosahexaenoic acid (DHA, 22 : 6 n‐3) were markedly reduced in brains of third‐generation (F3) rats nutritionally deprived of α‐linolenic acid (18 : 3 n‐3), and that DHA turnover within phospholipids was reduced as well. The concentration of docosapentaenoic acid (DPA, 22 : 5 n‐6), an arachidonic acid (AA, 20 : 4 n‐6) elongation/desaturation product, was barely detectable in control rats but was elevated in the deprived rats. In the present study, we used the same in vivo model, involving the intravenous infusion of radiolabeled AA to demonstrate that concentrations of unesterified and esterified AA, and turnover of AA within phospholipids, were not altered in brains of awake F3‐generation n‐3‐deficient rats, compared with control concentrations. Brain DPA‐CoA could be measured in the deprived but not control rats, and AA‐CoA was elevated in the deprived animals. These results indicated that AA and DHA are recycled within brain phospholipids independently of each other, suggesting that recycling is regulated independently by AA‐ and DHA‐selective enzymes, respectively. Competition among n‐3 and n‐6 fatty acids within brain probably does not occur at the level of recycling, but at levels of elongation and desaturation (hence greater production of DPA during n‐3 deprivation), or conversion to bioactive eicosanoids and other metabolites.

Impact of dietary n−3 FA deficiency on rat bone tissue FA composition

The effect of dietary n−3 FA deficiency on bone tissue FA composition was evaluated in growing rats. Two mixtures combining hydrogenated coconut oil with safflower oil served as the n−3-deficient dietary treatments and provided two levels of linoleic acid (LA). The n−3 treatments were formulated with added α-linolenic acid (LNA) from flaxseed oil (diet LNA) or LNA plus DHA, and both were balanced for LA. This study showed that bone is sensitive to changes in dietary n−3 FA and that DHA is more effective than LNA in maintaining DHA levels in these tissues.