Adverse effects of gestational ω‐3 and ω‐6 polyunsaturated fatty acid imbalance on the programming of fetal brain development

Abstract Obesity is a key medical challenge of our time. The increasing number of children born to overweight or obese women is alarming. During pregnancy, the circulation of the mother and her fetus interact to maintain the uninterrupted availability of essential nutrients for fetal organ development. In doing so, the mother's dietary preference determines the amount and composition of nutrients reaching the fetus. In particular, the availability of polyunsaturated fatty acids (PUFAs), chiefly their ω‐3 and ω‐6 subclasses, can change when pregnant women choose a specific diet. Here, we provide a succinct overview of PUFA biochemistry, including exchange routes between ω‐3 and ω‐6 PUFAs, the phenotypes, and probable neurodevelopmental disease associations of offspring born to mothers consuming specific PUFAs, and their mechanistic study in experimental models to typify signaling pathways, transcriptional, and epigenetic mechanisms by which PUFAs can imprint long‐lasting modifications to brain structure and function. We emphasize that the ratio, rather than the amount of individual ω‐3 or ω‐6 PUFAs, might underpin physiologically correct cellular differentiation programs, be these for neurons or glia, during pregnancy. Thereupon, the PUFA‐driven programming of the brain is contextualized for childhood obesity, metabolic, and endocrine illnesses.


| INTRODUCTION
Since David Barker first postulated a direct link between inadequate nutrition in early life and increased susceptibility to the onset of cardiometabolic pathologies in adulthood, the "fetal origins of disease" concept has evolved, and become broadly applied to a variety of conditions, including central nervous system (CNS) development. 1This is because the developing CNS is particularly vulnerable during intrauterine development to metabolic compromise given the exceptional energy demands of its many cell types that are being generated (including neurons, astroglia, microglia, oligodendroglia, vasculature), their protracted movements (migration), morphogenesis, and assembly into functional circuits.Thus, disruption of these processes by environmental factors will likely provoke long-lived modifications to brain structure and, ultimately, function. 2,3In principle, two models of developmental "programming" (that is, a lasting change to cell type identity and function) are distinguished: (i) direct "fetal programming", when an intrauterine insult directly alters cellular identity to an extent that it manifests in somatic and/or mental illnesses later in life.Such diseases include schizophrenia, depression, and even the emergence of neurodegenerative diseases. 4(ii) An intrauterine insult evokes subthreshold changes only.Nevertheless, exposure to the same or a similar disruptor postnatally will provoke a disease phenotype.This scenario, often termed "dual-hit hypothesis" can underpin the pathobiology of schizophrenia, psychosis, and panic disorder with maternal exposure to psychostimulants and/or illicit drugs recognized as lead candidates to evoke the "first hit" when taken during pregnancy.A second exposure to the same class of drugs (e.g., cannabis or synthetic cannabinoids) during adolescence can then be liable for neuropsychiatric diseases to manifest. 5ven the magnitude and pace of nutrient supply to and turnover within the developing CNS, the efficient exchange of nutrients with the maternal circulation is required to maintain the expansion and maturation of the neuronal connectome.Multiple gestational factors could affect CNS development including maternal psychosocial stress and infection, 6 alcohol, 7 illicit and/or prescription drugs, 8 and maternal diet. 9Restrictive societal views, legislation, and even medications prevent fundamental changes to many of these maternal conditions during pregnancy.The foremost exception to these concerns is maternal food preference, that is, both the type of nutrition and its amount.
Given the global obesity epidemic, which has by now spilled from the industrialized world to even developing countries, the study of how maternal nutrition affects fetal organ development has become a focus for public health regulators worldwide. 10e number of overweight and obese adults exceeded 1.9 billion by 2018, including $42.5% of the US population alone. 11The percentage of women with pre-pregnancy obesity also continues to rise.It is estimated that, globally, there are close to 39 million pregnancies per year complicated by maternal obesity and, in some countries, the estimated prevalence of overweight and obesity in pregnancy is over 60%. 12Notably, a simple mismatch between maternal supply versus fetal nutritional demand (that is, the gross level of energy and macronutrients) does not account for the harmful effects of maternal obesity on child development.Instead, obesity is closely linked to compositional changes in the diet, which are particularly pronounced when considering the amount and molecular identity of polyunsaturated fatty acids (PUFAs) in Western diets. 13Anecdotally, the oriental/Japanese diet is considered as the opposite of the Western diet and taken as healthy.It is not surprising, therefore, that children born to obese mothers have a high risk of being exposed to the undesired bioactive properties of fatty acids during prolonged periods of their intrauterine life.
Here, we provide a snapshot of contemporary concepts on the function of PUFAs during physiological brain development, and present examples on how they affect either neurons or (astro-)glia, or both.We emphasize that the disbalance, rather than an absolute change, of any of the ω-6 (n-6)/ω-3 (n-3) PUFAs can adversely impact the developing CNS.Thus, we highlight a significant risk through imbalanced maternal nutrition given existing data to causally link the mother's nutritional status during pregnancy with the health status and long-term postnatal prospect of their offspring (Table 1).The notion that disrupted fetal development might transcend to second and even third generations highlights the medical and socioeconomic importance of controlling obesity in pregnant women.Further mechanistic insights into the modulation of fetal brain development could advance the early diagnosis of negative outcomes, and the design of preventive and treatment measures.

| PUFAS: BIOCHEMISTRY IN A NUTSHELL
The human diet is the source of fatty acids, many of which are "essential fatty acids" that the human body is unable to produce.Once ingested, free fatty acids are absorbed from the gastrointestinal tract and transported by the bloodstream.At the cellular level, fatty acids do not only serve as energy sources but also as integral constituents of the cell membranes, thus affecting cell shape, size, protein localization, signal transduction, and survival.[16] Fatty acids are composed of a hydrocarbon chain with a methyl group and a terminal carboxyl group.A fatty acid is considered saturated if each carbon is joined to its neighbor by a single bond.If one or more double bonds exist, the fatty acid is considered unsaturated.Amongst unsaturated fatty acids, monounsaturated (one double bond) or polyunsaturated (more than one double bond; PUFA) entities are distinguished. 17 # expressive language skills Saros et al. 36 # memory performance DeBoer et al. 37 " ADHD, ASD, schizophrenia symptomatology/ risk, as well as of cognition impairment Rivera et al. 39 ω-6/ω-3 PUFA imbalanced diet " risk of cognitive impairments Bernard et al. 40 Lopez-Vicente et al. 41 " ADHD symptomatology/ risk Lopez-Vicente et al. 41 Galera et al. 44 position of their unsaturated carbon bonds at either their n-3 or n-6 position.The series of ω-3/n-3 and ω-6/n-6 PUFAs share much of their biosynthetic enzymes (Figure 1), hence conversion can occur.Linoleic acid (LA; 18:2 n-6) and α-linolenic acid (ALA; 18:3 n-3) are the principal essential unsaturated fatty acids.LA and ALA differ in their physiological roles and can act in competition.LA can be converted into arachidonic acid (AA; 20:4 n-6), which then serves as a precursor to n-6 endocannabinoids/endovanilloids, prostaglandins, thromboxane, and leukotrienes, as well as their derivatives. 18ALA can be converted to two somewhat longer n-3 PUFAs, eicosapentaenoic acid (EPA; 20:5 n-3) and docosahexaenoic acid (DHA; 22:6 n-3), which are both physiologically significant, 18 and whose ethanolamine derivatives (DHEA, EPEA) are recognized as ω-3 endocannabinoids. 19e mammalian brain contains large amounts of fatty acids, of which 50% are PUFAs. 20AA and DHA are particularly efficacious to alter the biophysical properties of cell membranes, transporters, and receptors, thus modulating neurotransmitter action ubiquitously in neural networks. 21During antenatal life, maternal nutrient intake, including that of dietary fatty acids, is the single-most determinant of the formation of the brain's cytoarchitecture. 22Notably, the rate of PUFA conversion in the placenta and the fetus is limited. 23Therefore, mechanisms have evolved for preformed DHA and AA that F I G U R E 1 A summary of synthesis and metabolic pathways for both ω-3 and ω-6 PUFAs.Intermediates for both ω-3 and ω-6 PUFAs are produced through elongation and desaturation steps of their respective essential precursors, α-linolenic acid (α-LA) and linoleic acid (LA), respectively.Rate-limiting enzymes were color-coded in light blue.circulate in the mother's blood to be transported across the placenta into fetal venous blood. 23,24It is therefore not at all surprising that any change in alimentary PUFA intake correlates with neurochemical alterations (e.g., neurotransmitter, neuromodulator availability, surface abundance of receptors) in both the maternal and fetal brains, 25 and exert lasting impact on cognitive performance in adult offspring. 26In the following sections, these observations are considered within the context of brain development, including physiological and disease states.

| MATERNAL DIET AS A RISK FACTOR FOR MENTAL DISORDERS IN THEIR OFFSPRING
Both maternal obesity and undernourishment carry the risk of gestational complications, [27][28][29] and affect fetal development as early as determining the number and differentiation stage of the cell mass in the blastocyst well before implantation. 30Here, we thematically review data from both human studies (Table 1) and rodent models (Table 2-4) on brain structure and behavioral outcomes in the offspring.We note that human obesity is modeled by feeding rodents with a high-fat diet (HFD), generally resulting in 40%-60% of its energy equivalent from fat.When mimicking Western diets, fat content is enriched in ω-6 PUFAs at the expense of some ω-3 PUFAs.
Conversely, oriental diets are mimicked by a disproportionate increase in ω-3 PUFAs. 31

| Human data on maternal diet affecting the offspring's intellectual ability
Weight gain is an essential part of a healthy pregnancy to support fetal growth, placental development, and the production of amniotic fluid.Therefore, it is only chronic overweight and obesity, defined by the World Health Organization as a body mass index (BMI) of 25-29.9kg/m 2 and≥30 kg/ m 2 , respectively, that are seen as health risks.High BMI in pregnant women is associated with gestational diabetes (hyperglycemia, insulin resistance), and pre-eclampsia, pathologies that increase both maternal and infant mortality and morbidity. 32,33During pregnancy, the fetus(-es) of obese mothers are particularly prone to overgrow, have altered body composition, and display neural tubes defects. 27,34Long-term consequences for children born to obese mothers include cardiovascular disease, metabolic syndrome, diabetes, cancer, chronic inflammatory disorders, and psychiatric diseases. 35Longitudinal follow-up of 1-and 2-year-old children born to overweight or obese mothers substantiated a link between gestational diabetes versus reduced memory performance and delayed language skills, maternal obesity versus cognitive, language, and motor skills 36,37 (Table 1).Developmental deficits of the CNS persisted in school age children and were classified as mild cognitive impairment. 38However, food addiction, anxiety, depression, attention deficit hyperactivity disorder (ADHD), autism spectrum disorders (ASD), and schizophrenia are amongst the mental health disorders that children of obese (and/or diabetic) mothers most frequently display. 39ilst maternal BMI is a predictor of the outcome of pregnancy and even lactation; recent studies highlighted that a shifted maternal dietary ω-6/ω-3 PUFA ratio is closely associated with reduced cognitive performance in the offspring. 40,41Based on evolutionary principles, an "ideal ratio" of ω-6/ω-3 PUFAs ranges between 1:1 and 2:1 in the brain.This is also suggested to be the correct target ratio for daily intake. 31,42When this ratio deviates in either direction, adverse effects might ensue.For example, the Norwegian Mother and Child Cohort Study demonstrated that the intake of a diet classified as "unhealthy" during pregnancy because of its high content of processed meat, refined cereals, salty snacks, and sweet drinks increased the prevalence of externalizing problems among infants at 12-18 months of age. 43Likewise, results from a French mother-child cohort revealed that maternal preference for a Western diet positively correlated to the offsprings' ADHD symptoms at ages ranging from 2 to 8 years. 44When the prenatal ratio of ω-6:ω-3 PUFAs was found shifted towards ω-6 PUFAs in cord plasma, subclinical symptoms of ADHD appeared at 7 years of age. 41Strikingly, symptom scores for ADHD increase by $13% per unit change in the ω-6: ω-3 PUFA ratio; an association that is likely driven by low DHA levels. 41Conversely, maternal intake of a diet enriched in fruit, whole-grains, vegetable, and fish was associated with improved cognitive development (e.g., visual spatial skills), intelligence, and executive function at both early and mid-childhood. 45t maternal effects of the diet are not restricted to intrauterine life.Alike the cellular reprogramming effects of psychoactive drugs, for example, Δ 9 -tetrahydrocannabinol (THC), 46 delivered by breast milk to infants, a shifted (1:8.4)ratio of ω-6 and ω-3 PUFAs negatively influenced the outcome of the parent-reported Communicative Development Inventory and Ages and Stages Questionnaire for 2-year-old infants against never-breastfed children. 47,48ese data suggest that maternal dietary preferences significantly affect both pre-and postnatal offspring development.Here, we first discuss maternal determinants of PUFA-driven fetal outcomes, such as obesity-induced comorbidities, changes in placental transfer, and the altered composition of breast milk before providing mechanistic insights in cellular programming events for neurons and astroglia.

| Obesity-related obstetric complications
In obese individuals, increased food intake alters the circulating levels of nutrients, with the resultant increase in the amount of adipose tissue affecting the plasma levels of hormones controlling energy homeostasis, growth factors, and inflammatory mediators.
In overweight and obese pregnant women, plasma levels of adiponectin are lower than in normal weight women (reported as 8.4 ± 5.3 vs. 12.6 ± 6.0 ng/mL), thus increasing fat mass, insulin resistance, glucose production, and birth weight. 49,50In contrast, the plasma concentration of leptin, a peptide hormone encoded by the obese (ob) gene, is significantly increased in both overweight and obese mothers, as compared to normal weight women (33.4 ± 14.8 vs. 23.0 ± 10.8 ng/mL).This increase is suggested to predict the development of pre-eclampsia. 49ring pregnancy, obese women are prone to develop hyperinsulinemia, glucose tolerance, and an altered lipid profile. 51Even though the levels of circulating lipids are per se increased in healthy pregnancies in normal weight women, obesity lowers the levels of high-density lipoprotein (HDL), particularly in the first trimester (13.0 ± 0.9 mg/mL; rage: 3.8-30.8).Conversely, obese pregnant women display elevated triglyceride (TG) levels in both the second and third trimesters, which is a known risk factor for post-partum hypertriglyceridemia. 52 TGs are hydrolyzed to non-esterified (free) fatty acids, whose levels remain elevated in the plasma of obese pregnant women. 51ternal obesity also exacerbates the mild pro-inflammatory state known to be associated with any normal pregnancy. 535][56] Cumulatively, these observations suggest that obesity adversely affects pregnancy outcomes.

| Placental function in obese mothers
The placenta acts as an essential interface for the transport of nutrients and hormones necessary for fetal growth.Given the poor ability of the fetus to synthesize non-essential PUFAs, the levels of AA and DHA in the fetus strictly depend on active transport across the placenta. 57To do so, the functional unit of the placenta is the trophoblast villous tree, which incorporates fetal blood vessels, and covered by syncytiotrophoblasts, which provide the primary plasmalemmal interface of the placenta with transporters used for the maternal-fetal active exchange of glucose, amino acids, and lipids.
During gestation, TGs and fatty acids are transported through specific placental transport systems, and binding proteins.In normal weight pregnancies, fetal fatty acid levels are significantly lower than in maternal plasma (fetal/maternal ratio: $1:3). 58In particular, the PUFA profile is different between the maternal and fetal circulation, with AA and DHA increased versus LA and ALA decreased in fetal plasma as compared to the maternal counterpart. 58ternal obesity alters the supply of fatty acids through the placenta by affecting its transport mechanisms. 59Notably, Segura et al. 60 reported elevated PUFA content in the placenta in obese pregnant women versus normal weight women.In particular, EPA was found T A B L E 2 Outcomes of dietary manipulations with ω-6 PUFA enrichment and/or ω-3 PUFA deficiency; in vivo experiments.
• • " anxiety-and depression-like symptoms Larrieu et al. 92 increased in the phospholipid fraction of placental tissues of obese women.This increase is likely a result of the higher expression of both FATP6, a fatty acid transport protein, and FAT/CD36, a fatty acid translocase.ALA, LA, AA, and DHA remained unchanged, suggesting the selective mobilization and utilization of specific PUFAs.
It is generally acknowledged that higher fetal adipogenesis shall be the result of an increased cross-placental lipid transfer in obesity. 61wever, when maternal obesity reaches extreme severity, the ability of the placenta to transport any nutrient might become severely compromised or fail, causing the cessation of delivering PUFAs to the fetus. 62Thus, one might suggest a correlation between maternal obesity and placental function, which can though progress from increased transfer to an acute impairment of nutrient supply.Identification of the molecular trigger(s) of this phenomenon will benefit from further research.

| Maternal diet and breastfeeding
The composition and availability of breast milk are the single-most determinants of neonatal development. 63Breastfeeding is viewed as healthy for both the baby and the mother, with its health benefits likely enduring into childhood and manifesting as improved cognitive development. 64As such, medical guidelines recommend breastmilk to be the only source of nutrients for the first 6 months of life; and continued up to 2 years when possible. 63This is because human breastmilk covers most of the energy (ketogenic source) and fatty acid requirements of the developing infant(s).The fat content of human breastmilk is $3.8-3.9g/100 mL, 65 yet with a composition changing over time due to the type of milk produced (that is colostrum [produced for $5 days after giving birth], transitional milk [TM], mature milk [MM]), the feeding stages (foremilk, hindmilk), and the dietary habits of the mothers during pregnancy and lactation. 66e overall PUFA content of breastmilk changes throughout lactation: it is highest in the colostrum (21.47 g/100 mL) and gradually decreases as the milk matures (TM: 18.82 g/100 mL, MM: 18.53 g/100 mL).Likewise, the mean ω-6 PUFA content becomes gradually reduced (colostrum: 19.88 g/100 mL, TM: 17.56 g/100 mL, MM: 17.28 g/100 mL). 66Among the ω-6 PUFAs, LA is the most prevalent, amassing $10% of the total fatty acid content of breast milk.
Approximately 30% of LA present in the milk is directly transferred from the diet, while the rest (70%) is generated by adipose tissue.
9][70] Both AA and DHA have a propensity to accumulate in the tissues of both fetuses and infants during the third trimester of pregnancy and in the first months of life, respectively. 23,24The latter observation is despite that • " aggressiveness, • " exploratory activity.
their concentration is gradually reduced in human milk as lactation progresses. 66One way to increase the conversion of ALA to DHA is to supplement the mothers' diet.2][73] This phenomenon is likely due to the low efficiency of the human body to convert ALA into bioactive derivatives, such as DHA and EPA.Accordingly, clinical studies in Greece, China, and US reported a positive correlation between dietary DHA quantities during lactation and the amount of DHA excreted into the breast milk, [74][75][76][77] and led to a daily recommended dose of 250-500 mg/day for both DHA and EPA during pregnancy. 78,79These guidelines are on the backdrop of high geographical variations in DHA intake, ranging from 30 to 180 mg/day in women living in inland and coastal areas, respectively. 80In sum, the above data show that dietary preferences can influence the composition of breast milk.
The impact of maternal weight on the fat content of breastmilk is an important aspect of early postnatal tissue programming.Maternal obesity skews the ω-6: ω-3 PUFA ratio as early as in the colostrum, with a discrepancy that persists until the MM. 81In addition, a close correlation exists between delayed lactogenesis and the maternal BMI categories: from 31% among women in the normal BMI range, to 43% among women in the overweight BMI range and 52% among women in the obese BMI range. 82This acutely increases the risk of excess neonatal weight loss.Obese women are also more likely to have shortened periods of breastfeeding. 83Thus, we conclude that maternal obesity poses a significant risk for the success of adequate nutrition for newborns when considering breastfeeding as the primary route of nutrition.

| Modified experimental diets during pregnancy and their developmental outcomes
Animal studies are critical in interrogating the mechanisms by which maternal diets program the brain, and consequently trigger behavioral dysfunction in affected offspring.Animal models using imbalanced ω-6: ω-3 PUFA diets are well documented (Table 2À4) to produce long-lasting changes in the brain functions of affected rodent offspring. 84As early as 1998, the effect of a HFD enriched in ω-6 PUFAs (43% energy derived from fat, high in ω-6 vs. 16% in control diet) on offspring behavior was documented, including altered escape behaviour, aggressiveness, and locomotor activity. 85Ever since, a series of studies showed (Table 2À4) that the consumption of experimental diets high in ω-6 but low in ω-3 PUFAs by pregnant mice (i) increases the consumption of palatable foods by their offspring by impinging on mesolimbic dopamine neurotransmission, 86 (ii) provokes anxiety and depression-like behavior traits 9 and (iii) autistic-like sociability deficits 87 in adult offspring.9][90][91] Indeed, maternal diets particularly high in LA and poor in ALA were detrimental for long-term synaptic plasticity, particularly synaptic depression mediated by AAderived endocannabinoids, in, among other areas, the prefrontal cortex, nucleus accumbens, 90,92 and hypothalamus 92 of adult offspring.
These changes behaviorally manifested as impaired emotional behavior, cognition, and memory. 93perimental models also reinforced the health benefits of ω-3 PUFA supplements (e.g., diet of 6% fat enriched in ALA) during gestation: ω-3 PUFAs rescued social and cognitive deficits of ASD-like traits in rats. 94More specifically, EPA (and also DHA though more moderately) was efficacious in attenuating chronic unpredictable mild stress-induced weight loss and depression-like behaviors, and increased exploratory drive in novel environments. 95Thus, the physiologically adequate brain and cognitive development of the offspring are programmed in utero by maternal nutrients.
Alike for humans, both the quantity and quality of maternal care during early life stages determines emotional behavior in adult animals. 96,97Pups look for and respond to their parents by ultrasonic vocalizations (USV). 98Experimental diets enriched in either LA (ω-3-deficient diet) or ALA (ω-3-enriched diet) decreased maternal care behaviors (e.g., nest building, arched, blanket, and passive nursing, licking, pup retrieval, breast grooming) whilst promoting non-maternal behaviors (feeding, exploring, self-grooming), and changed the pattern and subtypes of USVs of pups deprived of their mothers.Thus, longlasting behavioral disturbances ensued in the experimental offspring. 99

| Imbalanced ω-6: ω-3 PUFA diets impact brain lipid composition
Experimental models allow unprecedented analytical accuracy in determining PUFA composition of the developing brain at both prenatal and postnatal time-points.To manipulate the ω-3 PUFA content of the brain, an experimental diet deficient in ω-3 PUFAs (LA: 74.4% of total lipids; ALA: 0.3% of total lipids; ω-6/ω-3 ratio 121.4 ± 1.9) decreased its DHA content, 90,92 and allowed associations between low DHA levels and anxiety-and depressive-like symptoms, 90,92 as well as impaired learning. 91,100It is noteworthy that a lowered maternal intake of DHA directly led to a reduction in DHEA ("synaptamide") in the fetal brain, 101 which could exceed an 80% loss when ω-3 PUFAs were removed from the maternal diet. 69,102In contrast, enrichment of the maternal diet in ω-3 PUFAs increased DHA, DHEA, and EPEA levels in the cerebrum of neonatal offspring, 102 and improved their memory performance later in life. 103These data corroborate observations in humans on a direct link between maternal and fetal circulation and placental fatty acid transfer, and the ability of PUFAs to modify adult behaviors with ω-6 PUFAs and ω-3 PUFAs being antagonistic (risk vs. resilience) factors, respectively.

| Sex-specific sensitivity and outcome severity in fetal brains
Environmental factors (be these illicit drugs, 104,105 stress, 106,107 maternal immune activation, 108,109 or prenatal malnutrition 110 ) can have sex-specific outcomes, particularly when priming for neurodevelopmental vulnerabilities.Do dietary PUFAs also act in a sexspecific manner?
As discussed above, HFD during pregnancy can increase tissue levels of pro-inflammatory cytokines in utero.This is a fundamental feature of developmental pathobiology, mediated by the pattern recognition receptor toll-like receptor (Tlr)-4 in the placenta and fetal brain, equally affecting male and female offspring.Changes in USVs emitted by infant offspring were also changed in both sexes, and compromised maternal pup retrieval and care behaviors in HFD dams. 111Yet, the specific behavioral phenotypes that adult offspring exhibit are sex-specific: females have significantly decreased sociability scores, whereas males had diminished non-social reward behaviors, and increased escape responses. 112Equally sexdetermined is the increased vulnerability of male offspring because of the altered epigenetic control of gene transcription in the developing brain, 113 which could limit spatial learning and memory. 114In turn, female offspring show more pronounced stress pathobiology in adulthood, with altered gene expression seen in both the hypothalamus and amygdala. 115,116n added level of differential responses are seen when using specified diets: an ω-6 PUFA-enriched diet (>6% of energy from LA vs. $1.4% in control) offered during pregnancy, lactation, and/or the post-weaning period increased depression-like behavior in male but not female offspring, 117 as measured in the forced swim and open field tests.These data also support the notion that the life-long intake of LA can pose a significant risk for depression onset, with a greater susceptibility in males.Although mechanistic understanding of these phenomena is as yet fragmented, sex-specific differences in the expression of genes for lipid metabolism might underpin the adverse effects of LA administered in utero and neonatally. 118However, in a mouse model of fragile X syndrome and ASD, the loss of the Fmr1 gene recapitulated behavioral alterations in humans, including hyperactivity, anxiety, cognitive deficits, and and changes in sensorimotor gating. 119,120When exposing Fmr1 knock-out mouse dams to an ω-3 PUFA-rich diet, the rescue of ASD-like deficits in female but not male offspring was recorded, including improved emotionality, social interaction, and non-spatial memory. 121,122Cumulatively, these data suggest that ω-6 and ω-3 PUFAs can differentially impact the behaviors of female and male offspring born to dams on experimental diets.

| Epigenetic mechanisms underpin PUFA action
Epigenetic regulation is a versatile and powerful mechanism to control cellular differentiation programs, and to maintain altered cell-states at the long term.A robust line of research highlights epigenetic mechanisms, e.g., DNA methylation, histone modifications, and non-coding RNAs, as central to brain development. 123,124Indeed, the maternal diet can re-model epigenetic patterns during prenatal development, with the incorporation of repressive marks being likely negative for longterm outcomes in the offspring. 125For ω-3 PUFAs, the activation of transcription factor cascades is likely 126,127 as a result of the modulatory action of epigenetic regulators, such as DNA (de-)methylation globally or through gene-specific promoter modifications. 128,129As such, reduced DNA methyltransferase (DNMT) 3A and 3B activity was proposed as a potential molecular underpinning of variations in placental DNA methylation upon insufficient ω-3 PUFA availability.Particularly, ω-3 PUFAs could sustain or even increase Bdnf transcription (encoding brain-derived neurotrophic factor), a hypothesis derived from loss-of-function studies with ω-3 PUFA-deficient diets increasing the methylation of the Bdnf promoter. 129Since Bdnf signaling is central to neurogenesis and neuronal differentiation (including neuronal survival and synaptogenesis), diet-induced modifications to the availability of this neurotrophin can indeed alter brain architecture.Alternatively, ALA and DHA affect neurogenesis by modulating miRNA expression. 130nally, ω-6 PUFA-derived endocannabinoids can disrupt neuronal differentiation by desensitizing CB 1 cannabinoid receptors (CB 1 Rs), with a subsequent hypermethylation and reduced chromatin accessibility of target genes. 9Overall, epigenetic mechanisms seem fundamental for the sex-specific and long-lasting ability of dietary PUFAs to reprogram cellular identity and differentiation trajectories.(including both neuritogenesis and synaptogenesis), axonal myelination, and synapse pruning by microglia 84,131 (Figure 2 and Table 5).Moreover, a rapidly evolving field focuses on the liberation of PUFAs by exosomes as a means of itnercellular communication, for which we refer to a recent review. 132Notwithstanding, ω-3 and ω-6 PUFAs were contrasted as "good" versus "bad" for decades, a description we view as misleading because it does not accurately reflect their dietary ratios but emphasizes their effect sizes instead.

| ω-3 PUFAs
By lay terms, ω-3 PUFAs are seen as fundamental for a healthy lifestyle, promoting leanness, reducing cardiovascular risk, and increasing longevity. 133,134In developmental neurobiology, ω-3 PUFAs are known to mediate, and even directly control, the balance between self-renewal ("pro-survival" effects) and differentiation in embryonic neural stem (NSCs) and progenitor cells. 126Particularly, DHA has been reported to affect NSC proliferation, cell-fate decision, and the survival of newly-born progeny by being a major structural constituent of plasma membranes. 126,127,135,136During neuronal differentiation, DHA is particularly enriched in growth cones and synaptosomal membranes, whereby the dynamics of DHA availability and exchange with other PUFAs can affect directional growth processes, as well as the excitability of synapses 127,137 through tuning membrane fluidity and receptor accessibility. 137DHA also affects the transcriptional control of the cell-cycle through Hes1, p27kip1, NeuroD, and Map2, which positively affect the expression of terminal targets (e.g., Camk2, Bdnf, Syt1, AMPA/NMDA receptor subunits). 126,136,137Consensus view implies that DHA interrupts the cell-cycle, thus driving NSC-to-neuroblast transition. 126At the same time, DHA possesses anti-apoptotic and antioxidant properties, thus increasing the survival of differentiating progeny (alike neuroprotection in adult brain). 129,131,138A acts on a pool of transcription factors overlapping with those activated by DHA, yet with its effects being markedly different: for example, while DHA significantly decreases Hes1 expression to promote neuronal differentiation, EPA upregulates Hes1 instead.Moreover, Hes6, which promotes neuronal differentiation by cooperating with Hes1 in a positive-feedback loop, 139 is sensitive to EPA but not DHA.Even though these observations are, to a large extent, limited to in vitro cellular models (Table 5), we note that the levels of EPA and DHA present in embryonic tissues at any time during the expansion of the neuronal pool (that is, from gestational week 5 in humans, and embryonic day 8/9 in mouse), can contribute to the control of complex and fate-restricting transcription factor matrices to regulate NSCs.126,136 It is worth noting that ω-3-to-ω-6 PUFA conversion, or recruitment, is a prevailing phenomenon affecting NSC turnover and fate choices.We illustrate this by the finding that the effect of EPA on NSC proliferation can be abolished by prior exposure of NSCs to antagonists of either the CB 1 R or CB 2 cannabinoid receptor (CB 2 R).90,127 This is because EPA administration increases tissue levels of AA, which are then partly converted into 2-arachidonoylglycerol (2-AG), an efficacious endocannabinoid, whose action at CB 1 R or CB 2 R triggers MAPK signaling.This mechanism is attenuated by both AM251 and AM630, respective CB 1 R and CB 2 R antagonists (see below).Likewise, DHA exposure increases endocannabinoid levels and downstream MAPK activity.Yet, this coupling is ineffective to drive NSC proliferation, and insensitive to either CB 1 R or CB 2 R antagonists.127 Here, a crosstalk between DHA and endocannabinoid-mediated signaling path- molecules (designated as "n-3 endocannabinoid" 19 ), including a shared biosynthetic mechanism with arachidonoylethanolamine.69,140 DHEA induces neuronal differentiation (neuritogenesis, synaptogenesis) 138 by activating protein kinase A (PKA)/cAMP response element binding (CREB) signaling through GPR110.141 Thus, the observations available to date suggest that the availability of ω-3 PUFAs is beneficial for neuronal maturation.

| ω-6 PUFAs
"Western" diets are particularly enriched in ω-6 PUFAs, with their absolute amounts directly linked to obesity. 142AA is one of the most abundant ω-6 PUFAs in the nervous system along the entire lifespan.
As the brain evolves, AA concentrations rapidly rise.This is not surprising if one considers the many modes of AA action during cell division, 18 and intercellular signaling (e.g., synaptic neurotransmission) by itself or through its metabolites that affect protein kinases, ion channels, and SNARE proteins (e.g., syntaxin-3) that modulate neurite outgrowth, repair, and neurotransmitter exocytosis. 68,143 is a precursor to many bioactive lipids, including endocannabinoids. 68,143Endocannabinoids control each of the seven consecutive steps of neuronal development (neurogenesis, survival of progeny, neuroblast migration, neurite outgrowth, axon fasciculation, synaptogenesis, astroglial recruitment and (metabolic) interplay) [144][145][146][147][148][149] through CB 1 R and/or CB 2 R, and likely GPR55. 143en though multiple endocannabinoids exist (e.g., 2-AG, anandamide), their redundancy is essential to maintaining an "endocannabinoid tone" to allow neurons (but also glia) to progress through their developmental programs.The fundamental nature of endocannabinoid availability and signaling during brain development is best exemplified by their ligand and/or receptor switches in proliferative vs. differentiating niches in the brain, 144,146,147,150 the ubiquitous upregulation of Cnr1/CB 1 R expression upon neurogenic fate decisions in all prospective neurons, 147,151 and the partitioning of endocannabinoids to advancing growth cones in a metabolically-controlled fashion. 144,152Besides neurons, AA (as the precursor of adrenic acid, one of the most abundant fatty acids found early in brain development, 153 and 2-AG) is also involved in axonal myelination 154,155 , a process whose successful completion determines the speed of action potential propagation along each axon.

Rat
• AA and DHA promote the maintenance of neurogenic potential, • DHA promotes the maintenance of gliogenic differentiation.
Sakayori et al. 135 DHA, AEA, and DHEA treatment of primary cortical neurons and neural stem cells
Lee et al. 141 Pyramidal cell cultures and in vivo (transgenic mice) Rat and mouse • AA-derived endocannabinoids regulate neural progenitor proliferation and lineage commitment.
Galve-Roperh et al. 148 Molina-Holgado et al. 150 Mulder et al. 147 Keimpema et al. 70 Cultured interneurons and pyramidal cells and in vivo (transgenic mice) Rat and mouse • AA-derived endocannabinoids regulate the migration and differentiation of immature neurons.
Berghuis et al. 146 Berghuis et al. 136 DHA treatment of primary hippocampal neurons
is a target for acetaminophen (paracetamol), 156 as well as non-steroidal anti-inflammatory drugs 157 (Figure 3).The above pathways are therefore likely to be sensitive to the maternal use of "over-the-counter" medication to fight, for example, infections, during pregnancy.Thus, care should be exercised when setting dosage regimens for pregnant women because of potential unwanted effects on fetal development, with drug sensitivity (tissue enrichment/accumulation in brain, metabolism, intermediates) likely different from that in the mother.Overall, we suggest that ω-6 PUFAs are as important as ω-3 PUFAs to physiological developmental processes, with their actions being complementary to those of ω-3 PUFAs.

| ω-6:ω-3 PUFA disbalance in neuronal differentiation
The above mechanisms outline why both ω-3 and ω-6 PUFAs are critical for cellular differentiation, and suggest that their gross imbalance in maternal diets during pregnancy could be detrimental for fetal brain development. 88We have recently shown that excess ω-6 PUFA intake during gestation is harmful because of the ensuing deregulation of endocannabinoid signaling, which yields CB 1 R loss-of-function by desensitization. 9Notably, this mechanism is phenotypically similar to Cnr1 À/À mice and to gestational THC exposure, 158 with axonal wiring defects, and anxiety and depression-like traits in adult offspring. 9e importance of keeping a balanced diet is best explained by the existence of compensatory and convergent metabolic pathways to safeguard PUFA availability (Figure 1).Accordingly, when nutritional uptake of ω-3 PUFAs is particularly poor, surplus ω-6 PUFA derivatives are produced by the endoplasmic reticulum and peroxisomes to replace ω-3 PUFAs in, for example, biological membranes. 18nsequently, neurons with reduced somatic sizes appear, 159 with their polarized dendritic structures also significantly shrunken. 160At the molecular level, insufficient ω-3 PUFAs reduce the expression of vesicular neurotransmitter transporters, thus depleting the content of presynaptic vesicles and limiting synaptic neurotransmission. 25,160reover, changing the dietary EPA:DHA ratio (noting that DHA is likely unable to cross the placenta and is in situ regenerated 24 ) directly impacts EPHA:DHEA levels, 69,138 whose misalignment (given a significant excess of EPA) could lead to neuronal misplacement and the arrest of neuritogenesis.Overall, we propose that balanced ω-3: ω-6 PUFA intake is necessary for correct neuronal development instead of disproportionately increasing nutritional ω-3 PUFA availability for pregnant women.

| BEYOND NEURONS: PUFAS IN ASTROGLIA SAFEGUARD METABOLIC HOMEOSTASIS
Neurons develop in the proximity of glial cells.Astrogliogenesis is largely subsequent to neurogenesis, peaking at the early neonatal period in mice. 161Astrocytes are considered to provide circuit-specific metabolic support to neurons and are integral to "tripartite synapses" with their leaflets enriched in neurotransmitter transporters and enzymes to maintain the temporal and spatial fidelity of synaptic neurotransmission.
Microglia are immune lineage-derived cells, considered fundamental for immune surveillance, pruning of synapses, and modulating inflammatory responses upon disease. 161Both astrocyte metabolism and microglial immunosurveillance are affected by dietary PUFAs.Here, we focus on astrocytes because of the prevalence of mechanistic and developmental biology data (Table 6).For microglia, most studies on their ω-3/ω-6 PUFA-selective transformation, ligands/mediators (e.g., resolvins, protectins, and maresins; Figure 3), receptor-mediated signaling events, and cytokine cascades are available from adult models (including adult F I G U R E 3 Metabolic pathways converting PUFAs into pro-, as well as anti-inflammatory mediators.Cyclooxygenase (COX) and lipoxygenase (LOX)-dependent enzymatic processes are in grey.
[164][165][166][167] Astrocytes are the only cell type in the brain with a capacity to synthesize DHA 168 which, once produced, can be used to supply neurons. 168The role of DHA in astrocytes during brain development is incompletely understood, with recent reports claiming that DHA facilitates astrocyte coupling by gap junctions, 169 glutamate 170 and glucose uptake, 171,172 the regulation of Ca 2+ signaling, 173 and the inhibition of endoplasmic reticulum stress. 174Moreover, cell biology studies point out the ability of DHA to help astrocytes to resist stress, 175 and to promote their differentiation. 176Astrocyte differentiation is also facilitated by EPA. 177Our data suggest that slowed protein turnover through the unfolded protein response (UPR) pathway is central for EPA (and/or EPEA)-mediated effects, and guides astroglia-to-neuron signaling.
Cannabinoid receptors in astroglia 178,179 provide partial, yet important, signaling routes for AA metabolites.Most notably, endocannabinoids define cell fate decisions, that is, if NSCs generate neurons or astrocytes.
Herein, the activation of CB 1 Rs promotes NSC fate choice towards astrocytes. 149,180Moreover, astrocytic CB 1 Rs maintain lactate flow from astroglia to neurons, thus directly impinging on synaptic signaling. 178,179In NSCs in vitro, LA and practically all downstream ω-6 PUFAs upregulate genes relevant for endocannabinoid signaling, which results in a sustained enhancement of 2-AG bioavailability.Considering that 2-AG stimulates astrogliogenesis in a CB 1 R-dependent manner, 180 it is plausible that ω-6 PUFA-enriched maternal diets during gestation and lactation can exert notable impact on the number of astrocytes and their recruitment to functionally-devolved neuronal micro-, meso-, and macrocircuits.

| THE EFFECT OF PUFAS ON CELLULAR BIOENERGETICS
Proliferation and differentiation are energy-consuming processes for NSCs during both embryonic and adult neurogenesis.Likewise, the Champeil-Potokar et al. 169 AA and derivatives in vitro and in vivo
Aguado et al. 149 LA treatment of neural progenitors
migration, differentiation, synapse maintenance, and signaling of postmitotic neurons are amongst the peak energy-demanding cellular features of the body.
The main cellular organelles providing bioenergy are the mitochondria, which use the mitochondrial oxidative phosphorylation (Oxphos) cascade to produce ATP. 181There is a substantial body of clinical epidemiological data demonstrating that neurodevelopmental disorders are accompanied by mitochondrial dysfunction. 181,182Maintaining mitochondrial function is also important for NSC commitment and fate decision. 183A reduction of NSC self-renewal and proliferation, defects in cell cycle exit, and ensuing neuronal differentiation have been found following mitochondrial dysfunction. 184variety of cellular stressors can compromise mitochondrial function and integrity, with PUFA-imbalanced diet during pregnancy considered a significant risk factor.185 ω-3 PUFA intake could improve mitochondrial functions, including their fusion, and reduces the production of reactive oxygen species (ROS).In particular, DHA and EPA promote mitochondrial biogenesis and improve the expression of genes associated with ATP production and energy metabolism.185,186 Moreover, DHA from dietary sources is rapidly incorporated into mitochondrial membranes, where it participates in the Oxphos cascade to produce ATP.187 Thus, ω-3 PUFA deficiency could alter the course of brain development not only through the perturbation of the biophysical integrity of cellular membranes but also by suppressing mitochondrial energy yield.
In neurons, CB 1 Rs partitioned to mitochondria reduce mitochondrial respiration by inhibiting PKA signaling, and Oxphos activity (at the level of complex I), resulting in lower cellular ATP reserves. 189Most recently, CB 1 Rs were also localized to astrocytes, 190 in which endocannabinoid mobilization promoted mitochondrial Ca 2+ signaling, and facilitated astrocyte-to-neuron lactate transport 191 to maintain synaptic activity and plasticity.Thus, ω-3/ω-6 PUFAs could affect mitochondrial integrity and functions in both neurons and astrocytes, and shape intercellular communication by metabolic coupling.

| FUTURE PERSPECTIVES
Considering the ever-increasing prevalence of being overweight or obese during pregnancy, and the mechanistic data that had accumulated during the past decades, we await a series of molecular breakthroughs to dissect how maternal nutrition shapes the many cellular contingents of the fetal brain.From a clinical perspective, we propose a focus on the ω-6: ω-3 PUFA balance of maternal diets, rather than the dominance of either ω-3 or ω-6 PUFAs.Novel insights will not only justify the rationale of weight control during pregnancy but also aid the identification of when, where, additional pathways of behavioral transfer of traits of social, emotional, and cognitive functions. 194Therefore, novel insights in PUFA action on neurodevelopment could have a beneficial impact on the quality of life of affected children.
Two main classes of PUFAs exist based on the T A B L E 1 The impact of maternal diets imbalanced in ω-6 PUFAs:ω-3 PUFAs on child health.

4 |
MECHANISTIC INSIGHTS IN PUFA ACTION ON NEURONAL DEVELOPMENT While the exact mechanisms by which ω-3 and/or ω-6 PUFAs affect specific stages of brain development are often incompletely understood, consensus exists on the many neurodevelopmental processes regulated by PUFAs, including, but not limited to, neural progenitor proliferation, neuroblast migration, differentiation ways could be suggested by DHA being converted, at least in part, into N-docosahexaenoylethanolamide (DHEA).DHEA belongs to the N-acetylated amino acid or neurotransmitter class of lipid signaling F I G U R E 2 Neural progenitor subtypes and neurogenic stages during development of the cerebral cortex in mice.Cortical neurogenesis commences with the intense proliferation of progenitor cells in the ventricular zone (VZ) by embryonic day 10.5.During an initial phase, stem cells expand symmetrically, thus allowing the clonal expansion of radial glia.These progenitors then divide asymmetrically to generate neurons and glia, either directly or indirectly through intermediate progenitor cells or basal radial glia (alike tanycytes in the hypothalamus).Post-mitotic neurons then migrate towards the pial surface and complete their differentiation in the cortical plate (CP).Neurogenesis is followed by a gliogenic phase with the generation of, for example, astrocytes.Arrows indicate lineage relationships as demonstrated by time-lapse imaging and/or retroviral lineage tracing.CP, cortical plate; IZ, intermediate zone; MZ, marginal zone; SP, subplate; SVZ, subventricular zone; VZ, ventricular zone.

T A B L E 5
Cellular processes affected by ω-6 and ω-3 PUFAs during neurogenesis and neuronal differentiation; in vitro experiments.Experimental procedures Species Cellular outcomes/phenotypes References DHA, EPA and AA treatment of neural stem cells Mouse • EPA and DHA increase endocannabinoid levels, • EPA increases stem cell proliferation via endocannabinoid signalling, • " cell fate decision, • " cell survival.Dyall et al. 127 DHA, EPA and AA treatment of neural stem cells Rat • DHA and EPA (but not AA) increase neuronal differentiation, • DHA and EPA regulate the expression of basic helix-loop-helix transcription factors, • DHA controls cell fate.
and how medical interventions can mitigate the adverse effects of gestational dietary PUFA imbalance on the offspring's brain.Among the emerging fields of intergenerational metabolomics, we see great potential in integrating diet-induced alterations in the composition of the maternal gut microbiome with the known effects of microbiome-derived bioactive mediators of neuronal and glial differentiation.Given that the microbiome dynamically drives gene-environment interactions, and exerts a substantial influence on brain function and behavior during all stages of life, 192 understanding how specific PUFAs (and/or their bioactive derivatives) impact the acquisition of cellular identity in the fetal brain appears to be a fascinating endeavor.Regardless of the molecular mediators, persistent epigenetic alterations seem appealing to code life-long and generationspanning consequences of maternal dietary choices.Any such mechanism might involve the early reprogramming of primordial germ cells in the developing fetus, not only somatic lineages, thus influencing subsequent generations.Pharmacological developments and trials with, for example, histone deacetylase inhibitors, might be of medical benefit.Alternatively, non-epigenetic routes possibly affecting offspring neurodevelopment may include differences in the macro-and micronutritional value and composition of the mother's milk, 193 alterations in maternal care, or other/ Outcomes of dietary manipulations with high fat diets and specific PUFA supplements; in vivo experiments.