INDUCED COGNITIVE DECLINE/REDUCTION IN NEUROGENESIS

FIELD OF THE INVENTION

The present invention is in the field of nutritional compositions that have a beneficial effect on reducing early-life stress induced disorders, for example nutritional compositions that have a beneficial effect on reducing early-life stress induced cognitive decline, in particular early-life stress experienced by a human subject.

BACKGROUND OF THE INVENTION

Early life stress is known to negatively affect cognition throughout the entire lifespan of the subject. Cognitive decline induced by early life stress usually cannot or only to a limited extent be remedied. During early life, the developing human brain is highly sensitive to modulation by environmental influences and any adaptation of structure and function therefore is programmed by these early environmental influences for the remaining lifespan. A direct association between early life stress and cognitive impairment has been suggested by clinical data. This association has been reviewed by Lucassen et al. in Trends in Neurosciences, 2013, 36, 621-631.

The hippocampus is one of the very few brain regions that maintain the ability to generate new neurons throughout adult life. This process is named adult neurogenesis. The newly formed neurons are crucial to hippocampal functioning and are involved in specific aspects of hippocampus-dependent learning and memory. Naninck et al. in an article entitled "Chronic early life stress alters developmental and adult neurogenesis and impairs cognitive function in mice" in Hippocampus (2015) vol 25(3): 309-328, describe that early-life stress (ES) induces alterations in neurogenesis and also a reduction in survival of neurons formed in adulthood and that altered levels of neurogenesis are functionally relevant for cognitive impairments in adulthood. In short, early-life stress impairs cognitive functions and hippocampal neurogenesis in adulthood, also named 'adult neurogenesis. The authors express the hope that reaching understanding of the basis of this early-life stress induced alterations is profoundly important to mental health and disease and should provide the foundation of future therapeutic interventions. SUMMARY OF THE INVENTION

During early-life, the developing human brain is highly sensitive to modulation by environmental influences and any adaptation of structure and function therefore is programmed by these early environmental influences for the remaining lifespan. The present invention seeks to provide a way of influencing brain structure and functioning by means of nutritional intervention to relieve impairments due to early-life stress. The inventors surprisingly found that administration of a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a low LA/ ALA weight ratio is effective in preventing early life stress induced decline in cognitive functioning. Mice that were subjected to early life stress and which were fed the nutritional composition according to the invention outperformed mice that were subjected to the same stress but which were fed the nutritional composition with a higher LA/ ALA ratio in object recognition test, object location tests and Morris Water Maze test executed in their adulthood, which indicated a significant preventive effect on the decline in cognitive functioning. Hence, surprisingly, by dietary intervention, negative consequences of stress early in life can be prevented.

Also the inventors surprisingly found that administration of a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a low LA/ ALA weight ratio is effective in preventing early-life stress (ES) induced reduction in neurogenesis. It was found that the dietary intervention with a nutritional composition comprising a low LA/ ALA weight ratio restored neurogenesis in adulthood after being exposed to early-life stress. Mice that were subjected to early-life stress and which were fed the nutritional composition according to the invention showed an unexpected hippocampal cell survival in their adulthood compared to mice that were subjected to the same stress but which were fed the nutritional composition with a higher LA/ ALA, which indicated a significant preventive effect on the reduction in neurogenesis. Hence, surprisingly, by dietary intervention, negative consequences of stress early in life can be prevented. Applying a low dietary LA/ALA weight ratio early in life protects against early-life stress induced impairments, in particular protects against reduction in neurogenesis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention thus concerns a method for preventing early life stress induced decline in cognitive functioning in a human subject, comprising administering to the subject a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a LA/ ALA weight ratio in the range of 0.1 - 12. In one embodiment, the method for preventing early life stress induced decline in cognitive functioning is a non-medical method.

In other words the invention concerns a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a LA/ ALA weight ratio in the range of 0.1 - 12 for use in preventing early life stress induced decline in cognitive functioning in a human subject.

The invention can also be worded as the use of linoleic acid (LA) and alpha-linolenic acid (ALA) in the manufacture of a nutritional composition comprising LA and ALA in a LA/ ALA weight ratio in the range of 0.1 - 12 for preventing early life stress induced decline in cognitive functioning in a human subject.

The invention can also be worded as the use of a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a LA/ ALA weight ratio in the range of 0.1 - 12 for preventing early life stress induced decline in cognitive functioning in a human subject.

The present invention also concerns a method for preventing early-life stress (ES) induced reduction in neurogenesis in a human subject, comprising administering to the human subject a nutritional composition comprising linoleic acid (LA) and alpha- linolenic acid (ALA) in a LA/ ALA weight ratio in the range of 0.1 - 12. In one embodiment, the method for preventing early-life stress (ES) induced reduction in neurogenesis is a non-medical method.

In an alternative wording, the present invention concerns a method for restoring neurogenesis in a human subject in adulthood after early-life stress, comprising administering to the human subject a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a LA/ ALA weight ratio in the range of 0.1 - 12. In one embodiment, the method for restoring neurogenesis in adulthood is a non-medical method. In other words the invention concerns a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a LA/ ALA weight ratio in the range of 0.1 - 12 for use in preventing early-life stress (ES) induced reduction in neurogenesis in a human subj ect.

In yet other words, the invention concerns a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a LA/ ALA weight ratio in the range of 0.1 - 12 for use in restoring neurogenesis in a human subject in adulthood after early-life stress.

The invention can also be worded as the use of linoleic acid (LA) and alpha-linolenic acid (ALA) in the manufacture of a nutritional composition comprising LA and ALA in a LA/ ALA weight ratio in the range of 0.1 - 12 for preventing early-life stress (ES) induced reduction in neurogenesis in a human subject.

The invention can also be worded as the use of linoleic acid (LA) and alpha-linolenic acid (ALA) in the manufacture of a nutritional composition comprising LA and ALA in a LA/ALA weight ratio in the range of 0.1 - 12 for restoring neurogenesis in a human subject in adulthood after early-life stress

The invention can also be worded as the use of a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a LA ALA weight ratio in the range of 0.1 - 12 for preventing early-life stress (ES) induced reduction in neurogenesis in a human subject.

The invention can also be worded as the use of a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a LA/ ALA weight ratio in the range of 0.1 - 12 for restoring neurogenesis in a human subject in adulthood after early-life stress.

In a preferred embodiment, the prevention of early-life stress (ES) induced reduction in neurogenesis occurs via preventing a decline in hippocampal cell survival in a human subject. In one aspect the present invention concerns a method for preventing early-life stress (ES) induced decline in hippocampal cell survival in a human subject, comprising administering to the human subject a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a LA/ALA weight ratio in the range of 0.1 - 12. In one embodiment, the method for preventing early-life stress (ES) induced decline in hippocampal cell survival is a non-medical method.

In other words the invention concerns a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a LA/ ALA weight ratio in the range of 0.1 - 12 for use in preventing early-life stress (ES) induced decline in hippocampal cell survival in a human subject.

The invention can also be worded as the use of linoleic acid (LA) and alpha-linolenic acid (ALA) in the manufacture of a nutritional composition comprising LA and ALA in a LA/ ALA weight ratio in the range of 0.1 - 12 for preventing early-life stress (ES) induced decline in hippocampal cell survival in a human subj ect.

The invention can also be worded as the use of a nutritional composition comprising linoleic acid (LA) and alpha-linolenic acid (ALA) in a LA/ ALA weight ratio in the range of 0.1 - 12 for preventing early-life stress (ES) induced decline in hippocampal cell survival in a human subject.

All said below for the method according to the invention equally applies to the use according to the invention and to the composition for use according to the invention, and vice versa.

Nutritional composition

The nutritional composition that is to be administered according to the method of the invention comprises linoleic acid (LA) and alpha-linolenic acid (ALA) in a LA ALA weight ratio in the range of 0.1 - 12. This composition is referred to as "the nutritional composition according to the invention" or "the nutritional composition".

The nutritional composition according to the invention may take any form of nutrition suitable to be administered to human subjects, preferably suitable to be administered to human subjects during early-life. The nutritional composition according to the invention may be a nutritional supplement, e.g. as an additive to a normal diet, a fortifier, to add to a normal diet, a complete nutrition or an infant nutrition suitable for feeding infants (e.g. infant formula or follow-on formula). Preferably, the nutritional composition is suitable for feeding infants and/or toddlers. In a preferred embodiment, the nutritional composition is selected from an infant formula, a follow-on formula, a growing up milk and a nutritional supplement for pregnant women, most preferably the nutritional composition is an infant formula. Depending on the form of the nutritional composition, it may constitute a complete nutrition or a supplement. Preferably, the nutritional composition is a complete nutrition. It is preferred that the composition contains a lipid component, a digestible carbohydrate component and a protein component. It may further contain ingredients such as dietary fibres, minerals, vitamins, organic acids, nucleotides and flavouring agents. The lipid component preferably provides 2.9 to 6 g lipid per 100 kcal, the protein component preferably provides 1.8 to 5.5 g per 100 kcal, suitably 1.8 to 2.5 g per 100 kcal and the digestible carbohydrate component preferably provides 9 to 14 g per 100 kcal, of the composition. The amount of total calories is determined by the sum of calories derived from protein, lipids, digestible carbohydrates and non-digestible oligosaccharides

The nutritional composition of the invention is typically an enteral composition, i.e. intended for oral administration. It is suitably administered in liquid form. For instance, the composition may comprise water in which the further components are dissolved or suspended. The composition is thus suitably a liquid, or a solid, typically a powder or tablet, which is reconstitutable with a liquid, suitably with water or other food grade aqueous liquids, to obtain a liquid composition, or in a liquid concentrate form that is to be diluted with water. Preferably, the liquid composition has a viscosity below 100 mPa.s, more suitably below 60 mPa.s, more suitably below 35 mPa.s, even more suitably below 6 mPa.s as measured in a Brookfield viscometer at 20°C at a shear rate of 100 s ^"1 . In order to meet the caloric requirements of the infant, the composition preferably comprises 45 to 200 kcal/100 ml liquid, more preferably 60 to 90 kcal/100 ml liquid, even more preferably 60 to 75 kcal/100 ml liquid. This caloric density ensures an optimal ratio between water and calorie consumption. The osmolarity of the present composition is preferably between 150 and 420 mOsmol/1, more preferably 260 to 360 mOsmol/1. Lipid

The nutritional composition comprises a lipid component, suitably a lipid component suitable for infant nutrition as known in the art. The lipid component of the present composition suitably provides 2.9 to 6.0 g, preferably 4 to 6 g per 100 kcal of the composition. When in liquid form, the nutritional composition preferably comprises 2.1 to 6.5 g lipid per 100 ml, more preferably 3.0 to 4.0 g per 100 ml. The lipid provides preferably 30 to 60 % of the total calories of the composition. More preferably the present composition comprises lipid providing 35 to 55 % of the total calories, even more preferably the present composition comprises lipid providing 40 to 50 % of the total calories. When in liquid form, e.g. as a ready-to-feed liquid, the composition preferably comprises 2.1 to 6.5 g lipid per 100 ml, more preferably 3.0 to 4.0 g per 100 ml . In one embodiment, the nutritional composition comprises at least 15 wt.% lipid based on dry weight of the composition. Based on dry weight the nutritional composition preferably comprises 10 to 50 wt.%, more preferably 12.5 to 40 wt.% lipid, even more preferably 15 to 35 wt.% lipid, even more preferably 19 to 30 wt.% lipid.

The lipid component comprised in the nutritional composition comprises linoleic acid (LA) and alpha-linolenic acid (ALA) Herein, "linoleic acid" or "LA" refers to linoleic acid and/or acyl chain (18:2 n6). Herein, "alpha-linolenic acid" or "ALA" refers to oc- linolenic acid and/or acyl chain (18:3 n3). LA preferably is present in a sufficient amount in order to promote a healthy growth and development. The composition therefore preferably comprises less than 20 wt.% LA based on total fatty acids, preferably from 5 to 15 wt.%. Preferably the composition comprises over 5 wt.% LA based on fatty acids, preferably at least 10 wt.% based on total fatty acids. Preferably ALA is present in a sufficient amount to promote a healthy growth and development. The nutritional composition therefore preferably comprises at least 0.5 wt.% ALA based on total fatty acids preferably comprises at least 1.0 wt.% ALA based on total fatty acids. Preferably the composition comprises at least 1.4 wt.% ALA based on total fatty acids, more preferably at least 1.5 wt.%. Preferably the nutritional composition comprises less than 10 wt.% ALA, more preferably less than 5.0 wt.% based on total fatty acids. In one embodiment, the nutritional composition comprises at least 10 wt.% LA and at least 1 wt.% ALA, preferably from 10 to 20 wt.% LA and from 1 to 5 wt.% ALA. Surprisingly it has been found that a reduced LA/ ALA weight ratio, when compared to conventional nutritional compositions, in particular conventional infant formulae, beneficially affects the cognition of the subject upon exposure to early life stress and prevents early life stress induced cognitive decline. The LA/ALA weight ratio of the nutritional composition according to the present invention is the range of 0.1 - 12, preferably in the range of 0.5 - 11, more preferably in the range of 0.8 - 10, more preferably in the range of 1 - 9, more preferbly in the range of 1 - 8.

The lipid components such as LA and ALA, as well as the optionally present further components such as (LC-)PUFAs, may be provided as free fatty acids, in triglyceride form, in diglyceride form, in monoglyceride form, in phospholipid form, or as a mixture of one of more of the above. Preferably, the nutritional composition comprises triglycerides Preferably the composition comprises at least 70 wt.%, more preferably at least 80 wt.%, more preferably at least 85 wt.% triglycerides, even more preferably at least 90 wt.% triglycerides based on total lipids. The lipids may further comprise one or more of free fatty acids, monoglycerides and diglycerides.

Also, surprisingly it has been found that a reduced LA/ ALA weight ratio, when compared to conventional nutritional compositions, in particular conventional infant formulae, beneficially affects the hippocampal cell survival of the subject upon exposure to early- life stress and prevents early-life stress induced reduction in neurogenesis. The LA/ ALA weight ratio of the nutritional composition according to the present invention is the range of 0.1 - 12, preferably in the range of 0.2 - 1 1, preferably in the range of 0.4 - 10, preferably in the range of 0.5 - 10, more preferably in the range of 0.6 - 10, more preferably in the range of 0.8 - 10, more preferably in the range of 1 - 8.

In a preferred embodiment, the nutritional composition comprises triglycerides derived from vegetable fat. In a further preferred embodiment, the nutritional composition, preferably additionally, comprises phospholipids. Preferably the nutritional composition comprises phospholipids derived from non-human mammalian milk. The presence of vegetable lipids advantageously enables an optimal fatty acid profile, high in polyunsaturated fatty acids and more reminiscent to human milk fat. Using lipids from ruminant milk in particular cow's milk alone, or other domestic ruminant mammals, does not provide an optimal fatty acid profile. This less optimal fatty acid profile, such as a large amount of saturated fatty acids, is known to be not beneficial. Preferably the present composition comprises at least one, preferably at least two lipid sources selected from the group consisting of linseed oil (flaxseed oil), rape seed oil (such as colza oil, low erucic acid rape seed oil and canola oil), salvia oil, perilla oil, purslane oil, lingonberry oil, sea buckthorn oil, hemp oil, sunflower oil, high oleic sunflower oil, safflower oil, high oleic safflower oil, olive oil, black currant seed oil, echium oil, coconut oil, palm oil and palm kernel oil. Preferably the present composition comprises at least one, preferably at least two lipid sources selected from the group consisting of linseed oil, canola oil, coconut oil, sunflower oil and high oleic sunflower oil. Preferably the composition comprises 30 to 99.5 wt.% vegetable lipids based on total lipids, more preferably 35 to 99 wt.%, even more preferably of 40 to 95 wt.%, based on total lipids.

Preferably, the nutritional composition comprises fat or lipids from mammalian milk, more preferably from ruminants milk, even more preferably cow's milk, goat milk, sheep milk, buffalo milk, yak milk, reindeer milk, and camel milk, most preferably cow's milk. Preferably, the mammalian milk is not human milk. Hence in one embodiment, the nutritional composition comprises non-human mammalian milk fat. Preferably, the mammalian milk fat component comprises at least 70 wt.% triglycerides, more preferably at least 90 wt.%, more preferably at least 97 wt.%.

Preferably the mammalian milk fat is derived from the group consisting of butter, butter fat, butter oil, and anhydrous milk fat, more preferably anhydrous milk fat and butter oil. Such milk fat lipid sources are high in triglyceride levels. Furthermore these lipid sources are in the form of a continuous fat phase or in a water-in-oil emulsion form. Using these sources of milk fat during the manufacture of the nutritional composition according to the invention enables the formation of lipid globules, wherein each globule comprises a mixture of vegetable fat and milk fat.

Preferably the nutritional composition comprises 5 to 70 wt.% non-human mammalian milk lipids based on total lipids. In one embodiment the composition comprises 10 to 65 wt.%), even more preferably 15 to 60 wt.%, even more preferably 25 to 55 wt.% non- human mammalian milk lipids based on total lipids. Preferably these milk lipids are selected from the group consisting of butter, butter fat, butter oil, and anhydrous milk fat. Preferably the ratio of vegetable fat to milk fat ranges from 3/7 to 20/1. The composition also may comprise non-vegetable lipids and non-milk fat, such as animal fat other than milk fat, such as fish oil, and egg lipid, and microbial, algal, fungal or single cell oils. Preferably the non-vegetable, non-milk fat is present in an amount of at most 10 wt.% based on total lipid, more preferably at most 5 wt.%. Preferably the lipid in the nutritional composition according to the invention comprises a fat source comprising long chain poly-unsaturated fatty acids (LC-PUFA), selected from the group consisting of fish oil, marine oil, algal oil, microbial oil, single cell oil and egg lipid in an amount of 0.25 to 10 wt.% based on total lipid, preferably in an amount of 0.5 to 10 wt.%.

In a particular preferred embodiment, the lipid is the form of lipid globules. When in liquid form, the lipid globules are emulsified in the aqueous phase. Alternatively, the lipid globules are present in a powder and the powder is suitable for reconstitution with water or another food grade aqueous phase. Typically and preferably, the lipid globules comprise a core and a surface. The core preferably comprises vegetable fat and milk fat and preferably comprises at least 80 wt.%, more preferably at least 90 wt.% triglycerides and more preferably essentially consists of triglycerides. Not all triglyceride lipids that are present in the composition need necessarily be comprised in the core of lipid globules, but preferably a major part is, preferably more than 50% wt.%, more preferably more than 70 wt.%, even more preferably more than 85 wt.%, even more preferably more than 95 wt.%), most preferably more than 98 wt.% of the triglyceride lipids that are present in the composition are comprised in the core of lipid globules. According to a preferred embodiment, the lipid globules have a mode diameter based on volume of above 1.0 μπι, preferably above 2.0 μιη, preferably above 2.5 μπι, more preferably above 3.0 μιη. The lipid globules preferably have a mode diameter based on volume of below 6.0 μιη, preferably below 5.5 μιη, more preferably of below 5.0 μιη. Preferably, the lipid globules have a mode diameter based on volume from 2 to 6 μιη, preferably form 2.0 to 6.0 μιη, more preferably from 2.5 to 6.0 μιτι, more preferably from 3.0 to 6.0 μηι, even more preferably from 3.0 to 5.5 μιη, even more preferably from 3.0 to 5.0 μηι. The mode diameter relates to the diameter which is the most present based on volume of total lipid, or the peak value in a graphic representation, having on the X-as the diameter and on the Y-as the volume (%). A suitable method to determine the volume of lipid globules and their size distribution is by using a Mastersizer particle size analyzer (Malvern Instruments, Malvern, UK), for example by the method described in Michalski et al, 2001, Lait 81 : 787-796. The specific surface area of lipid globules is the surface area per weight of lipid and decreases as the size of globules increases. The specific surface area of the lipid globules can thus be calculated from the particle size distribution of the lipid globules and the concentration and density of the lipid. The lipid globules comprised in the nutritional composition preferably have a specific surface area of 0.5 to 15 m ² /g lipid, preferably from 1.0 to 10.0 m ² /g, more preferably from 1.5 to 8.0 m ² /g, even more preferably from 2.0 to 7.0 m ² /g lipid.

The nutritional composition preferably comprises phospholipids derived from (or "originating from") non-human mammalian milk. Phospholipids derived from non- human mammalian milk include glycerophospholipids and sphingomyelin. The phospholipids are preferably comprised in a coating on the surface of the lipid globule. By 'coating' is meant that the outer surface layer of the lipid globule comprises phospholipids, whereas these phospholipids are virtually absent in the core of the lipid globule. Not all phospholipids that are present in the nutritional composition need necessarily be comprised in the coating, but preferably a major part is. Preferably more than 30 wt.%, preferably more than 50 wt.%, more preferably more than 70 wt,%, even more preferably more than 85 wt.%, most preferably more than 95 wt.% of the phospholipids that are present in the composition are comprised in the coating of lipid globules.

In one embodiment, the nutritional composition comprises at least 0.5 wt.% phospholipids based on total lipid. Preferably 0.5 to 20 wt.% phospholipids based on total lipid, more preferably 0.5 to 10 wt.%, more preferably 1 to 10 wt.%, even more preferably 1.0 to 5 wt.% even more preferably 1.0 to 2.0 wt.% phospholipids based on total lipid. Preferably at least 80 wt.% of the phospholipids is derived from non-human mammalian milk, more preferably at least 90 wt.%, even more preferably at least 95 wt.%) or 99% wt. or preferably all of the phospholipids is derived from non human mammalian milk.

The phospholipids preferably comprise sphingomyelin. Sphingomyelins have a phosphorylcholine or phosphorylethanolamine molecule esterified to the 1 -hydroxy group of a ceramide. Preferably the nutritional composition comprises 0.05 to 10 wt.% sphingomyelin based on total lipid, more preferably 0.1 to 5 wt.%, even more preferably 0.2 to 2 wt.%. Preferably, the nutritional composition comprises at least 15 wt.%, more preferably at least 20 wt.% sphingomyelin based on total phospholipids. Preferably the amount of sphingomyelin is below 50 wt.% based on total phospholipids.

The phospholipids preferably comprise glycerophospholipids. Glycerophospholipids are a class of lipids formed from fatty acids esterified at the hydroxyl groups on carbon- 1 and carbon-2 of the backbone glycerol moiety and a negatively-charged phosphate group attached to carbon-3 of the glycerol via an ester bond, and optionally a choline group (in case of phosphatidylcholine, PC), a serine group (in case of phosphatidyl serine, PS), an ethanolamine group (in case of phosphatidylethanolamine, PE), an inositol group (in case of phosphatidylinositol, PI) or a glycerol group (in case of phosphatidylglycerol, PG) attached to the phosphate group. Preferably the nutritional composition contains PC, PS, PI and/or PE, more preferably at least PS. Preferably the nutritional composition comprises at least 1 wt.%, preferably at least 2 wt.% phosphatidyl serine based on total phospholipids. Preferably the amount of phosphatidylserine is below 10 wt.% based on total phospholipids. Phospholipids derived from non-human mammalian milk include phospholipids isolated from milk lipid, cream lipid, cream serum lipid, butter serum lipid beta serum lipid, whey lipid, cheese lipid and/or buttermilk lipid. Preferably the phospholipids are obtained from milk cream. The phospholipids are preferably derived from milk of cows, mares, sheep, goats, buffalos, horses and camels, most preferably from cow's milk. It is most preferred to use a lipid extract isolated from cow's milk. A suitable source of phospholipids derived from non human mammalian milk is the fraction that can be isolated from milk called milk fat globule membrane (MFGM). Hence in one embodiment, the phospholipids that are comprised in the nutritional composition according to the invention are provided as MFGM.

The phospholipids are conveniently located on the surface of lipid globules, i.e. comprised in a coating or outer layer. In one embodiment, the lipid globules comprise a monolayer comprising phospholipids derived from milk fat. A suitable way to determine whether the polar lipids are located on the surface of the lipid globules is laser scanning microscopy or trans-electron microscopy. The concomitant use of polar lipids in particular phospholipids, derived from domestic animals milk and triglycerides derived from vegetable lipids therefore enables to manufacture coated lipid globules with a coating more similar to human milk, while at the same time providing an optimal fatty acid profile. Methods for obtaining lipid globules with such diameters and/or phospholipid-coatings are disclosed in WO 2010/ 0027258 and WO 2010/0027259.

In one embodiment, the nutritional composition comprises at least 0.3 wt.% butyric acid and/or acyl chain (BA), based on weight of total fatty acids. In one embodiment the nutritional composition comprises from 0.3 to 4.0 wt.%, preferably 0.3 to 3 wt.%, BA based on weight of total fatty acids. The presence of relatively high amounts of BA is characteristic for triglycerides derived from ruminant milk, such as cow's milk. It is not present in vegetable fat or MCT rich fat such as coconut oil and it is also not found in polar lipids derived from milk fat. So one alternative way of describing the presence of milk fat triglycerides in a composition is to define the fatty acid profile in having a BA content from 0.3 to 4.0 wt.% based total fatty acids. Based on total weight of fatty acids, the composition preferably comprises at least 0.3 wt.% BA, preferably at least 0.5 wt.%, more preferably at least 0.6 wt.%, more preferably at least 0.8 wt.%. Preferably the composition has a wt.% of BA below 4 wt.% based on weight of total fatty acids more preferable below 3 wt.%, more preferable below 2.5 wt.%.

Preferably, the nutritional composition comprises at least 5 wt.% medium chain fatty acids and/or acyl chains (MCFAs) based on total fatty acids, more preferably at least 7 wt.%). In the context of the present invention, MCFA refer to fatty acids and/or acyl chains with a chain length of 8 to 12 carbon atoms. The composition advantageously comprises less than 15 wt.% MCFA based on total fatty acids, more preferably less than 10 wt.%.

Preferably the nutritional composition comprises 10 to 25 wt.% polyunsaturated fatty acids and/or acyl chains (PUFAs) based on total fatty acids. Amounts above 25 wt.% are higher than present in human milk, and cause technological problems such as stability in the nutritional composition. Preferably, the nutritional composition comprises long chain polyunsaturated fatty acids and/or acyl chains (LC-PUFAs), more preferably n-3 LC-PUFA. In the context of the present invention, PUFAs comprise at least 20 carbon atoms in the fatty acyl chain and have 2 or more unsaturated bonds. More preferably, the present composition comprises eicosapentaenoic acid and/or acyl chain (20:5 n3, EPA), docosapentaenoic acid and/or acyl chain (22:5 n3, DPA) and/or docosahexaenoic acid and/or acyl chain (22:6, n3, DHA), even more preferably DHA. Since a low concentration of DHA, DPA and/or EPA is already effective and normal growth and development are important, the content of n- 3 LC-PUFA in the nutritional composition, more preferably DHA, preferably does not exceed 5 wt.% of the total fatty acid content. Preferably the nutritional composition comprises at least 0.15 wt.%, preferably at least 0.35 wt.%, more preferably at least 0.75 wt.%), n-3 LC-PUFA, more preferably DHA, of the total fatty acid content. The nutritional composition preferably comprises at least 0.25 wt% LC-PUFA based on total fatty acids. Preferably the lipid in the nutritional composition comprises a fat source comprising 0.25 wt.% to 5 wt.% LC-PUFA based on total fatty acids of which at least 0.15 wt% n-3 LC-PUFA based on total fatty acids selected from the group consisting of DHA, EPA, and DPA, more preferably DHA.

As the group of n-6 fatty acids, especially arachidonic acid and/or acyl chain (20:4 n6, ARA) and LA as its precursor, counteracts the group of n-3 fatty acids, especially DHA and EPA and ALA as their precursor, the nutritional composition preferably comprises relatively low amounts of ARA. The n-6 LC-PUFA content, more preferably ARA content, preferably does not exceed 5 wt.%, more preferably does not exceed 2.0 wt.%, more preferably does not exceed 0.75 wt.%, even more preferably does not exceed 0.5 wt.%), based on total fatty acids. Since ARA is important in infants for optimal functional membranes, especially membranes of neurological tissues, the amount of n-6 LC-PUFA, preferably of ARA, is preferably at least 0.02 wt.% more preferably at least 0.05 wt.%, more preferably at least 0.1 wt.% based on total fatty acids, more preferably at least 0.2 wt.%). The presence of preferably low amounts of ARA is especially beneficial in nutrition to be administered to infants below the age of 6 months, since for these infants the infant formulae is generally the only source of nutrition. Preferably the n-6 LC- PUFA/n-3 LC-PUFA weight ratio, more preferably ARA DHA weigh ratio is below 3, more preferably 2 or below, even more preferably 1 or below. Protein

The nutritional composition preferably comprises a protein component. The protein component preferably provides 5 to 15% of the total calories. Preferably the composition comprises protein that provides 6 to 12% of the total calories. More preferably protein is present in the composition below 9% based on calories. Human milk comprises a lower amount of protein based on total calories than cow's milk. The protein concentration in a nutritional composition is determined by the sum of protein, peptides and free amino acids. Based on dry weight the composition preferably comprises less than 12 wt.% protein, more preferably from 9.6 to 12 wt.%, even more preferably from 10 to 11 wt.%. Based on a ready-to-drink liquid product, the composition preferably comprises less than 1.5 g protein per 100 ml, more preferably from 1.2 to 1.5 g, even more preferably from 1.25 to 1.35 g.

The source of the protein component should be selected in such a way that the minimum requirements for essential amino acid content are met and satisfactory growth is ensured. Hence protein sources based on cows' milk proteins such as whey, casein and mixtures thereof and proteins based on soy, potato or pea are preferred. Preferably, the nutritional composition according to the invention comprises casein and whey protein. In case whey proteins are used, the protein source is preferably based on acid whey or sweet whey, whey protein isolate or mixtures thereof and may include a-lactalbumin and β- lactoglobulin. More preferably, the protein source is based on acid whey or sweet whey from which caseino-glyco-macropeptide (CGMP) has been removed. Preferably the composition comprises casein, preferably it comprises at least 3 wt.% casein based on dry weight. Preferably the casein is intact and/or non-hydrolyzed. For the present invention protein includes peptides and free amino acids.

Digestible carbohydrate

Preferably, the nutritional composition comprises a digestible carbohydrate component. The digestible carbohydrate component preferably provides 30 to 80% of the total calories of the composition. Preferably the digestible carbohydrate component provides 40 to 60% of the total calories. When in liquid form, e.g. as a ready-to-feed liquid, the composition preferably comprises 3.0 to 30 g digestible carbohydrate per 100 ml, more preferably 6.0 to 20, even more preferably 7.0 to 10.0 g per 100 ml. Based on dry weight the present composition preferably comprises 20 to 80 wt.%, more preferably 40 to 65 wt.% digestible carbohydrate.

Preferred digestible carbohydrate sources are lactose, glucose, sucrose, fructose, galactose, maltose, starch and maltodextrin. Lactose is the main digestible carbohydrate present in human milk. Lactose advantageously has a low glycemic index. The present composition preferably comprises lactose. The present composition preferably comprises a digestible carbohydrate component, wherein at least 35 wt.%, more preferably at least 50 wt.%, more preferably at least 75 wt.%, even more preferably at least 90 wt.%, most preferably at least 95 wt.% of the digestible carbohydrate is lactose. Based on dry weight the present composition preferably comprises at least 25 wt.% lactose, preferably at least 40 wt.%.

Further components

In one embodiment, the nutritional composition preferably comprises non-digestible oligosaccharides. Preferably the present composition comprises non-digestible oligosaccharides with a degree of polymerization (DP) from 2 to 250, more preferably from 3 to 60. Preferred non-digestible oligosaccharides are selected from fructo- oligosaccharides, galacto-oligosaccharides and/or galacturonic acid oligosaccharides, more preferably galacto-oligosaccharides, most preferably transgalacto- oligosaccharides. In a preferred embodiment the composition comprises a mixture of transgalacto-oligosaccharides and fructo-oligosaccharides. Suitable non-digestible oligosaccharides are for example Vivinal GOS (FrieslandCampina DOMO), Raftilin HP or Raftilose (Orafti).

Preferably, the composition comprises of 80 mg to 2 g non-digestible oligosaccharides per 100 ml, more preferably 1 0 mg to 1.50 g, even more preferably 300 mg to 1 g per 100 ml. Based on dry weight, the composition preferably comprises 0.25 wt.% to 20 wt.%, more preferably 0.5 wt.% to 10 wt.%, even more preferably 1.5 wt.% to 7.5 wt.%.

The composition preferably comprises other ingredients, such as vitamins, minerals according to international directives for infant nutrition, in particular infant formulae. Application

The inventors surprisingly found that the nutritional composition according to the invention is effective in preventing early-life stress induced decline in cognitive functioning in a human subject. The method according to the invention thus concerns administration of the nutritional composition according to the invention during the early life of the human subject. When the composition is in a liquid form, the preferred volume administered on a daily basis is in the range of about 80 to 2500 ml, more preferably about 450 to 1000 ml per day. The inventors surprisingly found that the nutritional composition according to the invention is effective in preventing early-life stress induced reduction in neurogenesis in a human subject. The method according to the invention thus concerns administration of the nutritional composition according to the invention during the early-life of the human subject. When the composition is in a liquid form, the preferred volume administered on a daily basis is in the range of about 80 to 2500 ml, more preferably about 450 to 1000 ml per day.

In the context of the present invention, "preventing" may also be referred to as "reducing", "reducing the risk or occurrence of, "prophylaxis of or "(prophylactic) treatment of. The present use of "preventing early life stress induced decline in cognitive functioning" may also be worded as "stimulating cognitive functioning after exposure to early life stress" or "improving cognitive functioning after exposure to early life stress". The present use of "preventing early-life stress induced reduction in neurogenesis" may also be worded as "protecting against early-life stress induced reduction in neurogenesis" or "restoring neurogenesis after exposure to early-life stress" or "stimulating neurogenesis after exposure to early-life stress" or "improving neurogenesis after exposure to early-life stress". Further, the present use of "neurogenesis in a human subject in adulthood" may also be worded as "adult neurogenesis". In the context of the present invention, "early life" typically lasts up to and including adolescence, e.g. up to the age of 18 years, preferably up to the age of 12 years, more preferably up to the age of 5 years, even more preferably up to the age of 36 months, most preferably up to the age of 12 months. Early life also includes the prenatal stage of the human subject, preferably the foetal stage, more preferably the foetal stage in the third trimester of pregnancy. In one embodiment, early life starts at birth.

The human subject is preferably selected from the group of unborn foetuses, infants, toddlers, children, preadolescents and adolescents. In a more preferred embodiment, the human subject is selected form unborn foetuses, infants and toddlers, more preferably from infants and toddlers, i.e. human subjects having an age of 0 - 36 months, most preferably the human subject is an infant, i.e. having an age of 0 - 12 months. The unborn foetus is preferably in the third trimester of pregnancy. Administration to the unborn foetus occurs via the pregnant mother, and the nutritional composition preferably takes the form of a nutritional supplement for pregnant women. Since the lipid composition of a mother's diet is reflected in her breastmilk, the nutritional composition can also be in the form of a nutritional supplement for lactating women. Human subjects up to the age of 36 months, or even up to the age of 12 months, are especially preferred, since generally speaking brain plasticity is highest in this early stage of existence, and administration of the nutritional composition thus has the highest impact at this age.

In a preferred embodiment, the human subject is at risk or even at high risk of experiencing early life stress or is experiencing early life stress Examples of such at (high) risk subjects are subjects, in particular children, from a lower socioeconomic status (e.g. children from families with an income level below average or a low level of education, e.g. not exceeding high school), children whose parents are separated or going through a separation, children separated from one of both parents, children whose parent are both working, neglected children, abused children, children whose parent(s) are suffering from stress, depression, depressed children, ill children, malnurtured children and children exposed to violence. Herein, "children" includes infants and toddlers, preferably the child is an infant.

Particular subjects that experience physical stress and/or mental stress are preterm infants and infants small for gestational age (SGA) infants. A preterm infant relates to an infant born before the standard period of pregnancy is completed, thus before 37 weeks pregnancy of the mother, i.e. before 37 weeks from the beginning of the last menstrual period of the mother. Preterm infants are also referred to as premature infants. SGA infants are those whose birth weight lies below the 10th percentile for that gestational age. Reasons for SGA can be several; for example, term or preterm infants can be born SGA because they have been the subject of intrauterine growth restriction (IUGR). Many preterm infants are also small for gestational age. Premature and/or SGA infants include low birth weight infants (LBW infants), very low birth weight infants (VLBW infants), and extremely low birth weight infants (ELBW infants). LBW infants are infants with a birth weight below 2500 g; this group includes term infants born SGA. VLBW and ELBW infants are almost always born preterm and are defined as infants with a birth weight below 1500 g or 1000 g, respectively. Hence in one embodiment in the method or use according to the present invention, the human subject is a preterm infant, an infant born small for gestational age or both.

There appears to be an indication that male subjects suffer to a greater extent of early life stress induced decline in cognitive functioning and consequently male human subjects may experience more benefit from the present dietary intervention. Hence in one embodiment the human subj ect is a male.

Thus, administration of the nutritional composition according to the invention typically occurs when the human subject is 18 years old or younger, preferably the human subject has an age below 12 years, more preferably up to 5 years, even more preferably up to 36 months, most preferably up to 12 months. Although early life typically halts after adolescence, the beneficial effects on cognition and/or neurogenesis may last beyond early life, such as up to or even beyond adulthood. The effects may even last the entire lifespan of the subject. According to a preferred embodiment, the prevention of early life stress induced cognitive decline and/or reduction in neurogenesis prolongs into adulthood. Preferably, the prevention of declined cognitive functioning and/or reduced neurogenesis is manifest when the subject has reached adulthood or when the subject has reached an age of 18 years or higher, or even an age of 25 years or higher. Hence, it is preferred that the nutritional composition according to the invention exhibits a later-in- life effect on cognition and/or neurogenesis. Herein, the prevention of early life stress induced decline in cognitive functioning and/or reduction in neurogenesis is manifest later-in-life, preferably when the subject has reached an age of 5 years. According to a preferred embodiment, the beneficial effect on cognition and/or neurogenesis prolongs at least 3 months, preferably at least 1 year, more preferably at least 5 years after administration of the nutritional composition according to the invention has halted. Exemplary of such a later-in-life effect, which is an especially preferred embodiment of the present invention, is administration of the composition according to the invention to a foetus, infant or toddler, most preferably to an infant, while the preventive effect on early life stress induced declined cognition and/or reduction in neurogenesis is observed at an age of 5 years or older, more preferably at an age of 12 years or older, most preferably at an age of 18 years or older.

Early life stress may take any known form. Typical stressors include psychological stressors, physiological stressors and physical stressors. The early life stress may the form of mental stress, physical stress, metabolic stress or any combination thereof. Exemplary stressors include disrupted families (e.g. divorce, separation, blending of families), separation from one or both parents (e.g. incarceration of a parent), maternal depression, both parents working, lack of attention by parent(s), exposure to violence, abuse (e.g. physical, mental, sexual), neglect (e.g. emotional, physical), death of a loved one (in particular death of a parent), illness (e.g. mental, physical), malnutrition. Herein, "parent(s)" may also refer to "care-taker(s)" or "guardian(s)". Most suitably, the early life stress is selected from malnutrition, maternal stress, depression and abuse.

Early life stress induced cognitive decline is a specific type of cognitive decline, which is associated with many complex structures of the brain, such as the cerebral cortex, frontal cortex, hypothalamus, hippocampus and the perirhinal cortex. These structures in the brain play important roles in both cognition and regulating stress responses. The hippocampus for instance contains many stress hormone receptors and is highly plastic. Since these structures develop rapidly in the last trimester of pregnancy and postnatally until the age of about 16 years or beyond, they are particularly sensitive towards early life stress.

In a preferred embodiment, the declined cognitive functioning preferably includes declined hippocampal-dependent cognitive functioning, or the declined cognitive functioning preferably is declined hippocampal-dependent cognitive functioning. Particularly preferred embodiments of cognitive functioning in the context of the present invention are (a) novel object recognition; (b) the preference for novelty; (c) attention for (change in) surroundings; (d) non-spatial (object) memory; (e) spatial memory and (f) spatial reference memory, but also include novel object recognition and/or novel object location, or hippocampal-dependent memory (especially long term spatial memory).

In a preferred embodiment, the prevention of the reduction in neurogenesis is via preventing a decline in hippocampal cell survival. Alternatively, the invention also concerns as such preventing early-life stress induced decline in hippocampal cell survival by administration of the nutritional composition according to the invention during the early-life of the human subject. The present use of "preventing early-life stress induced decline in hippocampal cell survival" may also be worded as "stimulating hippocampal cell survival after exposure to early-life stress" or "improving hippocampal cell survival after exposure to early-life stress".

EXAMPLES Example 1:

Female C57/BL6 mice with litters were exposed to early life stress paradigm consisting of limited nest/ bedding material as described by Naninck et al., Hippocampus 2015, 25:309-328. The stress paradigm was initiated at 2 days after birth of litter (PN2), and lasted until PN9. Litters were weaned at PN21

From PN2 onwards the offspring lactating dam were exposed to experimental diets until PN42 (male offspring kept on respective diet after weaning). From PN42 until end of experiment all male offspring were fed the same AIN-93M diet.

The experimental diets were semisynthetic diets with a macronutrient and micronutrient composition according to American Institute of Nutrition formulation of ATN93-G purified diets for laboratory rodent (Reeves 1993), and differed only in fatty acid composition with respect to linoleic (LA) and alpha linolenic (ALA) content, see table 1. In adulthood, male offspring were tested for cognitive performance using the novel object recognition test, object location test and Morris water maze test. These test are widely used in the art of behavioural neuroscience. Table 1 : Fatty acid composition of the experimental diets.

fatty acids (% in the diet)

Diet A Diet B

C6:0 0.01 0.01

C8:0 0.13 0.13

C10:0 0.13 0.13

C12:0 1.07 1.08

C14:0 0.48 0.48

C16:0 0.67 0.64

C18:0 0.42 0.44

C20:0 0.07 0.06

C18: l 1.51 1.55

C20: l 0.04 0.03

C18:2n6 (LA) 2.14 1.21

C18:3n3 (ALA) 0.14 1.07

LA ALA ratio 15.29 1.13

Novel object recognition and Novel object location test

The mice were subjected to a novel object recognition test (ORT) at day 120. This test is a behavioural procedure to study preference for non-spatial object memory based on the innate preference of mice for novelty. In the test mice had to discriminate between a novel object and an object that they had explored previously (familiar). During three days, mice were habituated for five minutes/day to the testing arena that consisted of a rectangular plastic box. On the training day, mice had five minutes to explore two identical objects (9,5 cm high glass bottles) that were placed in the box 12 cm from each other and 11 cm from the wall. On the testing day 24hpost-training, one object was exchanged by a novel object (constructed of yellow Lego Duplo bricks) placed in exactly the same position, and mice were reintroduced into the arena for five min to explore the novel and the familiar object. The time spent exploring each object was measured, and the ratio of novel/familiar object exploration time calculated. The results are shown in table 2. Object location tests (OLT): This test is a behavioural procedure to study spatial object memory based on the innate curiosity of mice for novel locations. The test was conducted at PN 130 day For the OLT, the arena and habituation protocol were identical to those used in the ORT; two completely novel identical objects (white coffee cups) were placed 12 cm from each other and 11 cm from the wall. On the training day, mice had five minutes to explore the objects and their location. On the testing day, 24h post-training one object was moved to a novel position in the arena and mice were reintroduced into the arena for five min to explore. Time spent exploring was recorded as before and the ratio of novel/familiar object exploration time was calculated. The results are shown in table 2.

Table 2: Ratio novel/old object (location) exploration time

As can be seen in table 2, the ratio novel/old exploration time during ORT is reduced by previous exposure to early life stress in the animals that were fed Diet A indicating that the animals were not able to distinguish between the object they have already seen and the new one that they are seeing for the first time. This index is not reduced by the stress paradigm in animals that were fed Diet B. The difference between the two stressed diet group is statistically significant and indicate that Diet B can prevent stress induced impairments in cognitive performance, in particular novelty preference and non spatial memory performance.

The ratio novel/old exploration time during OLT is also reduced by previous exposure to early life stress in the animals that were fed Diet A, indicating that the animals were unable to distinguish between the object that is in the location compared to the object that has been moved to a new location. In contrast, this difference is not present in animals that were fed Diet B. This indicates that Diet B can prevent stress induced impairments in cognitive performance, in particular novelty preference and spatial memory performance. Morris Water Maze

Mice were also subjected to a Morris Water Maze test at PN 140 also known as Morris Water Maze navigation task, is a behavioral procedure to study cognitive function, in particular spatial learning and memory. In this experiment animals were trained to locate a hidden platform based on environmental cues. In short a platform was submerged just below water surface at a fixed position in a round water tank. The tank was in a room with fixed cues on the walls that were visible from the water surface in the tank. During 7 consecutive days, mice were trained twice daily (inter trial interval 10 minutes) to locate the hidden platform. During each trial, animals were placed in the water of the tank and had to locate the platform within 60 seconds. Between trials the starting position of the animal was varied between one of the three quadrants without the platform. On day eight, the platform was removed from the pool for a single probe trial, in which the time spent in the target quadrant was recorded. This trial is referred to as probe trial. The results are shown in table 3.

Table 3 : time (%) spent in the target quadrant in the probe trial of the Morris Water Maze.

As can be deduced from table 3, the percentage of time spent in the target quadrant is reduced by previous exposure to early life stress in the animals that were fed Diet A indicating that the animals did not remember as well where the platform was located. Exposure to Diet B, however, prevented the stress- induced impairment in time spent in the target quadrant. This is indicative for an improved cognitive performance, in particular long term (spatial) memory performance.

Example 2

Cell survival of adult-born neurons was assessed using 5-bromo-2'-deoxyuridine (BrdU; Sigma- Aldrich Chemie BV, Zwijndrecht, The Netherlands). At 7 months after birth, mice were injected intraperitoneally with 100 mg/kg BrdU (10 mg/mL dissolved in sterile saline + 0.007M NaOH) for 2 times a day on 4 consecutive days. Four weeks after the last injections, mice were transcardially perfused at P230.

Tissue collection was performed as follows: mice were anesthetized by intraperitoneal inj ection of pentobarbital (Euthasol®, 120 mg/kg) and transcardial perfused with initially 0.9% saline, which was followed by 4% paraformaldehyde (PFA) in phosphate buffer (PB 0.1M, pH 7.4). Whole perfused brains were carefully removed, post-fixated with 4% PFA in O. l .M PB at 4°C for 24 h and stored in PB with 0.01% sodium azide at 4°C. Prior to brain slicing, perfused brains were cryoprotected in 30% sucrose in 0.1M PB. Subsequently, frozen brains were sliced in 40 μΜ thick coronal sections and divided over 6 parallel series by a microtome and stored in antifreeze (30% Ethylene glycol, 20% Glycerol, 50% 0.05M PBS; company) at -20°C.

BrdU-labeled cells were detected using fluorescent immunohistochemistry. Brain slices (from one of the 6 parallel series) were mounted on glass (Superfrost Plus slides, Menzel, Braunschweig, Germany), followed by heat-induced antigen retrieval in 0.1M citrate buffer (pH 6.0) using a microwave (Samsung M6235) at ~95°C for 15 min (5 min at 800 Watt, 5 min at 400 Watt, 5 min at 200 Watt). Subsequently, premounted sections were treated with a blocking buffer for 30 min (1% bovine serum albumin in TBS), followed by the incubation with the primary antibody monoclonal rat anti-BrdU (1 :200; Accurate Chemical and Scientific Corporation, OBT 0030) in incubation mix (1% bovine serum albumin, 0.3% Triton X-100 in 0.05M TBS) for 24 h. Afterwards, sections were incubated with the secondary antibody donkey anti-rat, alexa fluor 488 conjugate (1 : 1000, Invitrogen) for 2 h and covered with DAPI-containing Vectashield.

To determine the volume of the granular zone of the denate gyrus (DG) boundary contour tracings were set using a 20x magnification on a Zeiss Axiophot light microscope using the Stereoinvestigator software (MicroBrightField, Germany) for eight bregma' s, multiplied to 6 (to correct for the parallel series) and by 40 μπι (for the thickness of the sections). Forthe BrdU (40x objective, Leica CTR5500) quantification, cell-counts were made in the subgranular and granular zone of the DG in eight matched bregma' s and multiplied by 6 for a total number of immunoreactive cells per DG. The results are shown in the figure. The figure represents the hippocampal cell survival measured as labeled BrdU cells in the denate gyrus (DG) for diet A and diet B of the control group not having been subjected to early-life stress (open bars on the left side) and the group that has been subjected to early-life stress (black filled bars on the right). The hippocampal cell survival after having been subjected to early-life stress is significantly lower for diet A compared to diet B. The hippocampal cell survival for diet B after having been subjected to early-life stress is the same as that for both diets after not after having been subjected to early-life stress. From these results it is concluded that nutritional intervention with a diet having a low LA/ ALA ratio has a preventive effect on reducing neurogenesis after early-life stress or in other words protects against early-life stress induced reduction in neurogenesis. In addition it can be concluded that nutritional intervention with a diet having a low LA/ALA ratio restores neurogenesis in adulthood after early-life stress.