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Omega-3 PUFA Deficiency Abolishes Endocannabinoid-Linked Neuronal Functions

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The endocannabinoid signaling system is an important route through which long-chain polyunsaturated fatty acids (LC-PUFAs) affect behavior, mood, responses to stress, anxiety, memory, energy balance and other systems. Cannabinoid receptors and the substances that activate or antagonize them (endocannabinoids) are under intensive investigation for their involvement in substance abuse, addiction, impaired neurotransmission, memory, emotion, obesity and many other situations. Cannabinoid receptors are widely distributed in human tissues, but are especially concentrated in the brain. These receptors regulate synaptic plasticity, which is the ability of neurons to alter the strength of their response. Plasticity also results from changes in the number of receptors on a synapse.

The cannabinoid receptors CB1 and CB2 are coupled to G-proteins in the neuronal cell membrane through which they stimulate or inhibit other processes. In laboratory mice, stimulation of the endocannabinoid system increases feeding, enhances the reward aspects of eating and promotes lipogenesis. In contrast, a novel antagonist of CB1 was reported to reduce food intake and body weight. In obese men, higher levels of the endocannabinoid 2-arachidonoyl-glycerol (2-AG) are directly related to the level of intra-abdominal adiposity.

The two best known ligands for the cannabinoid receptors are derived from the omega-6 (n-6) LC-PUFA arachidonic acid (ARA). These are anandamide (N-arachidonoylethanolamine) and 2-AG. Endocannabinoid levels are affected by the relative amounts of ARA and DHA in the diet. Dietary supplementation with ARA and DHA led to significantly increased levels of anandamide and its DHA-form as well as higher levels of the ARA- and DHA-2-acylglycerols in several regions of the neonatal piglet brain. Piglets not consuming these LC-PUFAs had significantly lower concentrations of these substances compared with the supplemented and sow-milk-fed piglets.

Mice fed n-3 PUFA-deficient diets had greater amounts of 2-AG in brain than mice fed sufficient n-3 PUFAs. Feeding the animals a DHA-rich fish oil diet was accompanied by a decrease in brain 2-AG.

Others have reported that mice fed a DHA-enriched diet had significantly increased levels of docosahexaenoylethanolamine in the brain, with correspondingly lower levels of anandamide. Dietary DHA did not significantly affect the brain content of ARA or any of the 6 glycerol-ester endocannabinoids measured, except for eicosapentaenoylglycerol.

Several questions emerge from these and other studies. One addresses the functional links between dietary LC-PUFA consumption and the production of different endocannabinoid species. Another is the functional differences between the various endocannabinoids, especially the ethanolamine and glycerol types, which respond differently to dietary n-3 LC-PUFA changes. In this report, a French team of investigators addressed the functional effects of lifelong n-3 PUFA deficiency by examining synaptic plasticity in the prefrontal cortex of mice.

The researchers fed female mice throughout gestation and lactation 6%-fat diets based on African peanut oil, deficient in n-3 PUFAs and rich in linoleic acid. They used a rapeseed oil diet rich in alpha-linolenic acid as a control. The offspring consumed the same diets after weaning throughout life and were studied from ages 6 to 16 weeks. The investigators examined the fatty acids in the brain, endocannabinoid-dependent and independent synaptic plasticity, cannabinoid receptor coupling, circulating endocannabinoid levels and synaptic currents and behaviors related to the diet-induced changes in cannabinoid receptor function. They focused on the prefrontal cortex (Figure 1), the region associated with executive tasks and rewards, emotional behavior and depression. All evaluations were conducted in the n-3-PUFA-deficient offspring of the mothers fed the n-3-PUFA-deficient diet.

The investigators first established that the n-3-PUFA-deficient diet led to significant decreases in DHA, n-3 docosapentaenoic acid and eicosapentaenoic acid (EPA) in the whole brain and prefrontal cortex, which were accompanied by significant increases in n-6 docosapentaenoic acid and n-6 tetraenoic acid.

To assess synaptic plasticity, they applied tetanic stimulation at 10 Hz to slices of prefrontal cortex from animals fed the n-3 PUFA adequate and deficient diets. Tetanic stimulation is the delivery of very rapid electrical stimuli to prefrontal cortex synapses that induces synaptic responses. In this experiment, tetanic stimulation induced normal endocannabinoid-mediated long-term depression responses in the pyramidal neurons in the prelimbic area of the prefrontal cortex in animals consuming the n-3 PUFA adequate diet. The long-term depression is a sustained reduction in the response of neurons which can last several hours or longer. In the n-3 PUFA deficient animals, long-term depression was completely abolished (Figure 2). A similar response was observed for the nucleus accumbens area (Figure 1), which has also been linked to reward and pleasure as well as mood disorders. The prelimbic area of the prefrontal cortex communicates with the nucleus accumbens via excitatory projections. These observations suggest that n-3 PUFA deficiency abolished endocannabinoid-mediated synaptic plasticity in different regions of the brain.

The researchers also investigated whether the effects of n-3 PUFA deficiency were observed in other forms of synaptic plasticity, such as those mediated via AMPA and NMDA glutamate receptors, which are also involved in long-term synaptic plasticity. Results from these experiments revealed no differences between the n-3 PUFA sufficient and deficient groups. These findings plus the demonstration that n-3 PUFA deficiency had no effect on downstream transduction pathways suggested that the observations on endocannabinoid-mediated synaptic plasticity were specific to this type of synaptic process and were not a generalized effect.

To determine whether n-3 PUFA deficiency affected the ability of the cannabinoid receptor CB1 to couple with its effector G-proteins, the investigators used a CB1 agonist to assess presynaptic function. They observed that the agonist inhibited synaptic transmission in the n-3 PUFA sufficient animals in a manner similar to that seen in chow-fed mice, but that inhibition of synaptic transmission was greatly reduced in the n-3 PUFA deficient animals. When the researchers stimulated CB1 receptor binding to its isotope-labeled G-protein binding protein, they found that binding was significantly reduced in specific layers of the prelimbic prefrontal cortex in the n-3 PUFA deficient animals compared with the n-3 PUFA sufficient animals. Receptor binding was not different in the motor cortex andCB1 expression was not affected by the dietary deficiency.

Circulating levels of the two major endocannabinoids were similar in both n-3 PUFA groups. As this observation contrasts with previous results, the authors suggested that homeostatic adaptations may have occurred in the n-3 PUFA deficient animals or high concentrations of endocannabinoids near synapses may not be accurately measured with bulk measurements.

Finally, the research team investigated whether there were behavioral changes in the two groups of animals that were related to their n-3 PUFA status. Using a forced swimming test, a model of depressive behavior, they observed that the n-3-deficient mice spent more time immobile and less time swimming compared with the n-3 PUFA sufficient animals. They also observed other behaviors in the deficient animals that were indicative of impaired exploratory behavior and greater anxiety, including fewer social explorations, more litter-scratching, less time spent in the center of the exploratory arena and a greater tendency to remain close to the arena wall.

In summary, these experiments demonstrated that n-3 PUFA deficient diets:

- led to decreased DHA, EPA and docosapentaenoic acid levels in the whole brain and prefrontal cortex;
- abolished endocannabinoid-dependent synaptic plasticity, but not other types of synaptic plasticity;
- desensitized and uncoupled CB1 binding without affecting the expression of CB1 receptors;
- may have altered endocannabinoid-CB1 binding at synapses, which diminishes endocannabinoid synaptic plasticity;
- were associated with increased anxiety and impaired social exploration behaviors.

In discussing these findings, the authors suggested that Western diets high in n-6 PUFAs and low in n-3 PUFAs, especially the latter, might create conditions where CB1 function in the brain is sufficiently impaired so that mood disorders develop more readily. Ligands for these receptors differ by type and the class of PUFA from which they are derived. For example, animals fed diets containing ARA and DHA had multi-fold increases in the endocannabinoids in the brain, while supplementation with DHA enhanced the production of n-3-PUFA-derived endocannabinoids at the expense of those derived from ARA. Over-activation of the endocannabinoid system, especially the CB1 receptors, is associated with obesity and reduced insulin sensitivity. On the other hand, antagonists of the CB1 receptor are associated with reduced food intake and lower abdominal adiposity. Further, a clinical trial of a drug that blocks CB1 receptors designed to control food intake in obese individuals was halted because of the development of psychiatric problems. These results support the evidence that dysregulation of the endocannabinoid system is associated with mood disorders and other brain functions. Further elucidation of the different effects of n-3 and n-6 LC-PUFAs in the regulation of the endocannabinoid system is likely to be increasingly important.

Source, Graphs and Figures: Articles on Health Nutrition Research : Benefits of Fish, Omega-3s, Fish Oil and Polyunsaturated Fats
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