Effect of hypercaloric and isocaloric diets different in fatty acid content on the endo cannabinoid system in pregnant dams and their offspring.

Lipid molecules are the building blocks of all cell membranes and provide essential secondary metabolites. The central nervous system is enriched in polyunsaturated fatty acids (PUFAs): arachidonic acid (AA) and docosahexaenoic acid (DHA). During brain development, PUFAs play a critical role in determining neuronal structure, particularly axonal outgrowth. In mammals, AA and DHA cannot be synthesized de novo and must be obtained largely from dietary sources. Moreover, during pregnancy and lactation, fetuses and infants rely on PUFAs from their mothers through the placenta and breast milk, respectively. Thus, the maternal diet needs to contain an appropriate omega-3/omega-6 PUFAs ratio to help child development. Based on these premises, we investigated the consequences of maternal malnutrition, defined as a shifted dietary ratio of omega-3 or omega-6 PUFAs on the brain of the offspring. In particular, we modeled human relevance by manipulating both the content and time of daily diets, with a keen focus on endocannabinoid (eCB) system given the critical roles of this AA-derived neuromodulator system during pre- and postnatal brain development. In the first approach of this project we fed female mice with hypercaloric diets, rich in omega-3 or in omega-6 PUFA, for two (short-term diet) or nine weeks (long-term diet) before mating and during gestation. We found that, levels of endocannabinoids (AEA, 2-AG) and AEA-like mediators (PEA, OEA) did not change with short-term diet in pregnant dams. Moreover, only omega-3 diet induced a significant increase of 2-AG levels, after long-term protocol. In embryos, we found that the short-term exposure of two weeks of omega-3 and omega-6 diet feeding is already sufficient to allow alteration of endocannabinoid system, especially FAAH and CB1R. In fact, in female embryos, we found down-regulation of CB1R and increase of FAAH after treatment with both diets. Interestingly, both enzyme and receptor levels are normalized after high-fat diet administration for a long period, where AEA levels were found decreased. By contrast, in males the alterations found in CB1R after the shorter protocol persist also after the prolongation of the treatment. In line with this, these data suggest a relationship among AEA, CB1R and FAAH, and an important link between PUFAs and endocannabinoid system. The second approach of this project was based on the administration of isocaloric diets, different in omega-3 levels but not in omega-6 PUFAs throughout gestation and until adulthood. We discovered that during gestation, not only the increase but also the decrease of omega-3 levels markedly affected the eCB system in the hippocampus of embryos. Furthermore, our in vivo results strongly suggest that omega-3 diets enriched and deficient affect the principal presynaptic marker in embryonic hippocampus but not in adult, where maternal malnutrition leads to long-term behavioral alterations in adult rats characterized by the presence of recognition memory deficits. As a whole, the second approach of this study supports our hypothesis about a relationship between PUFAs and the endocannabinoid system and provides further evidence on the importance of omega-3 PUFA on hippocampal development and functioning. All together, our results suggest that changes in dietary omega-3/omega-6 PUFAs ratio during gestation affect the endocannabinoid system in the brain of the offspring and the major effects of diets are present at the beginning of the treatment (i.e during gestation). Moreover, these data suggest that not only lipids but also fat can have a role in these changes