Hippocampal neurogenesis and Arc expression are enhanced in high-fat fed prepubertal female pigs by a diet including omega-3 fatty acids and Bifidobacterium breve CECT8242

Neural plasticity is essential to accommodate new experiences and learn throughout our lives [1]. Once embryonic development of the brain is completed, neurogenesis and synaptic plasticity help to maintain a high degree of environmentally induced neural plasticity. The dentate gyrus of the hippocampus represents one of the adult neurogenic areas which shows synaptic plasticity related to memory and learning processes. Prepubertal age and adolescence mark a crucial period for the maturation of the hippocampus, denoting this stage as a sensitive period during which interventions, such as diets, could have a significant impact on neural plasticity and cognitive function [2].

Different dietary components have been reported to modulate neural plasticity. Excessive intake of certain macronutrients, such as simple carbohydrates and saturated fatty acids, can lead to obesity and attendant metabolic dysfunction. Moreover, excess body weight in midlife has been associated with lower cognitive ability and steeper cognitive decline in late life [3, 4]. Specifically, high-fat (HF) diet has been shown to impair cognitive function both in rodent models and humans [5‐8]. Research in rodents has revealed that HF-induced reduction of neurogenesis may be obesity independent, suggesting that dietary fat per se impairs hippocampal neurogenesis [9]. In addition, HF diets reduce the levels of brain-derived neurotrophic factor (BDNF) in both rodents and humans [7, 10]. HF diets also reduce other plasticity-related proteins, such as activity-regulated cytoskeleton-associated protein (Arc) [11], cAMP response element-binding protein (CREB) and synapsin I [8], as well as impair N-methyl-d aspartate receptor (NMDAr)-related glutamate transmission [12].

In contrast, other dietary components have been reported to positively modulate neural plasticity. Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid that has become increasingly interesting as a dietary component that may potentiate cognitive performance. A greater intake of DHA and eicosapentaenoic acid (EPA) by preschool-aged children has been associated with better cognitive test outcomes [13]. DHA supplementation has also demonstrated to induce slight improvements in learning capacities of children with attention-deficit/hyperactivity disorder [14]. Kawakita et al. [15] proved that dietary DHA supplementation stimulates hippocampal neurogenesis in rats and is an essential molecule for neuronal differentiation. However, other interventional studies in clinical trials with patients with mild cognitive impairment or Alzheimer disease find minor or nonexistent cognitive improvements following the treatment with omega-3 [5, 16], suggesting that its combined use with other dietary factors could enhance DHA effects on cognitive function. For instance, DHA has previously shown synergistic effects with probiotics in regard to metabolic and inflammatory regulation [17, 18]. Interestingly, probiotics are novel nutritional candidates for potentiating and protecting cognition. Different studies have found protective effects against memory deficit in mice, as well as improved learning and memory ability in both aging rats and Alzheimer disease rodent models [19‐22]. Recent studies demonstrate that Bifidobacterium breve suppresses cognitive deficits and synaptic dysfunction while restoring the levels of BDNF and postsynaptic protein PSD95 in an Alzheimer disease rodent model [23]. Moreover, a study examining the effects of B. breve supplementation demonstrated a reduction in cognitive decline in older adults with mild cognitive impairment [24].

Despite the encouraging findings regarding diet effects on hippocampal plasticity shown in rodent models, there is great evolutionary distance between rodents and humans and their translational value is far from perfect. Instead, the pig serves as a good model because of its similarity to human nutrition requirements, comparable gastrointestinal development [25] and similar gyrencephalic brain development [1]. Moreover, similar to humans, the major brain growth spurt in pigs extends from the late prenatal to the postnatal period [1, 26] and their neurodevelopment is also susceptible to dietary factors [27, 28].

The aim of this work was to analyze the effects of two dietary treatments including omega-3 fatty acids and/or probiotic B. breve CECT8242 on hippocampal synaptic plasticity and neurogenesis in prepubertal female pigs on a HF diet, and to compare them with the effects of both normal and HF diets. For this purpose, we performed immunolabeling of two widely accepted neural plasticity markers, doublecortin (DCX), a microtubule-associated protein which is expressed in both lineage-restricted neuronal progenitors and immature granule cells of the DG [29], and Arc, a synaptic plasticity-related protein that interacts with specific effector proteins in neuronal compartments and is critical to memory consolidation [30].

Results of the present study show that a diet with high fat content but including the probiotic B. breve CECT8242 and omega-3 increased neural plasticity in female prepubertal piglets compared to animals following a conventional HF diet. This diet increased the total number of DCX-positive cells and Arc protein levels in the DG region of the dorsal hippocampus, suggesting a potentiation of neural plasticity in the prepubertal brain. This outcome is especially remarkable since prepubertal age and adolescence mark a crucial period for the development of higher-order cognition through enhanced plasticity of brain circuits [34], and it points to this stage as a sensitive period during which interventions, such as diet, could impact on neural plasticity and cognitive function [2]. Moreover, it has been established that the group at the highest risk of obesity during adulthood are children and young people of prepubertal age with overweight [35], and that attendant metabolic dysfunction has important implications for pubertal development in girls [36]. In light of this, strategies that could influence neural plasticity aimed at normalizing body weight and improving dietary habits before the onset of puberty, gain relevance.

Considering that high-fat diets have been reported to induce alteration of neurogenesis [9, 35] and neural plasticity [7, 8, 10], our initial hypothesis was that administration of the HF would cause a decrease in DCX-positive cells and Arc levels in the prepubertal pigs. Our work is the first study analyzing hippocampal protein levels of Arc in pigs, and to our knowledge, there is only one other study examining the effect of HF diets on DCX cells in pigs. However, in the aforementioned study, the HF diet was administered to sows during gestation and lactation, causing reduction of hippocampal DCX cells in their piglets [27]. Regarding the molecular relationships between DCX and lipid demand, it has recently been show that lipid droplets are highly abundant in adult mouse neural stem/progenitor cells (NSCP), which is significant given that lipid droplet availability could affect NSCP proliferation [38]. On the other hand, it has been demonstrated that the 27-hydroxycholesterol, a circulating cholesterol derivative that can cross the brain–blood barrier, down-regulates hippocampal Arc expression in mice with dietary cholesterol [11]. Additionally, changes in the lipid composition of the diet could have an impact on Arc function since membrane budding events, such as endocytic internalization of AMPA receptors interacting with “lipid rafts”, could be sensitive to differences in the lipidic membrane constituents [39]. Nevertheless, the HF diet used in this study did not lead to quantitative changes in both the analyzed neural plasticity markers in the DG of female piglets. Our results lend support to the assertion that there is an absence of an established metabolic syndrome in these animals at 19 weeks of age, as reported in recently published reports of Jove et al. [32], Ballester et al. [31] and Valent et al. [33]. Their findings integrate with the present results into a multidisciplinary study analyzing the effect of the four dietary treatments and show that the administration of this HF diet caused some anomalies that could trigger the emergence of the metabolic syndrome. The results of these studies showed changes in the circulating and tissular lipidome, transcriptomic changes in adipose tissue related with proinflammatory pathways, upregulation of obesity-related pathways as well as the alteration of appetite-regulatory neuropeptides and neurotransmitters due to the HF diet, which were partially reversed by supplementation with the proposed nutrients [31, 33].

We cannot rule out the possibility that the absence of negative effects of the HF diet on neurogenesis and synaptic plasticity could also be due to a higher availability of nutrients, required in this high-plasticity stage. In this regard, hippocampal neurogenesis had been analyzed in adult macaques showing positive correlations of number of DCX-positive cells in DG with TC/HDL ratio in serum, suggesting that increased neurogenesis is associated with elevated metabolic activity [40]. In support of this hypothesis, we also observed a positive correlation between neurogenesis and lipid ratio TC/HDL, both in T1 and T2 groups. Moreover, we observed a negative correlation between HDL serum levels and the number of hippocampal young neurons for all groups, except for T4 group. Since HDL participates in reverse cholesterol transport from peripheral cells to the liver for further catabolism and excretion [41], these results reinforce the hypothesis that neurogenesis was related to higher lipidic demands. Importantly, inadequate or dysfunctional brain-derived HDL are also implicated in cerebrovascular dysfunctions, neurodegeneration, or neurovascular instability, as shown in a recent review [42]. Moreover, although both HF and cholesterol enriched diets have been shown to reduce Arc expression [11] in rodents, it has also been reported that HF diets increase Arc expression in mice in the early stages of obesity [43], suggesting the involvement of compensatory mechanisms in the response of neural plasticity to HF diet.

Interestingly, T4 exerted a positive effect on neurogenesis. The increase in DCX-positive hippocampal cells was independent of the TC/HDL lipid ratio or of the HDL levels, suggesting that the bioactive ingredients triggered alternative mechanisms to induce this neurogenic effect. Thus, despite the growing evidence in support of nutritional lipids playing an important role of in the regulation of neural stem cell niche, the mechanism of action nor the critical exposure time are clear [44] and further studies are required to establish the exact relationships between dietary lipids and adult neurogenesis.

On the other hand, the DG protein Arc levels were also found increased in T4 experimental group, despite no correlations being observed between Arc levels and the physiological parameters. This neural plasticity marker has been associated with the improvement of HF diet-induced cognitive impairment in mice. Deficiency of Cyp1b1 has been found to protect the animals from HF diet-induced learning and memory deficits, and has also been shown to increase Arc expression [45]. Considering this relation to cognitive improvement, it is particulary interesting that the T4 dietary treatment increased DG protein levels of Arc in pigs, including in a HF dietary context.

Therefore, this study shows that dietary supplementation with bioactive T4 compounds not only reverses HF-induced lipidomic and appetite-related neurological abnormalities observed in the previous studies [31, 33], but can also enhance neuronal plasticity.

These analyses were performed in the dorsal hippocampus, but a previous analysis of the ventral hippocampus did not detect any changes in neurogenesis (data not shown). This suggest that neurogenic potentiation by this dietary supplementation was specific for the dorsal hippocampus (denominated anterior in rodents), an area that plays a preferential role in cognition and memory, whereas the posterior (ventral) hippocampus regulates emotion [29].

It has been reported that supplementation of preterm pigs with B. breve, plus fructo-oligosaccharide and glutamine, improved cognitive performance [28]. Our results show that T3 diet, including the probiotic, did not increase the number of DCX-positive cells nor Arc protein levels in DG. However, it did alter the correlation of lipid TC/HDL ratio and the neurogenesis marker. Interestingly, Bernier et al. recently demonstrated that niacin production by B. breve could protect against lipid droplet alterations and neuroinflammation [46]. As mentioned above, lipid droplet availability could affect NPSC proliferation [38]. Considering that T4 diet only differs from T3 in the addition of omega-3 fatty acids, the neurogenic and plasticity effects of T4 diet could be attributed to the DHA. Despite DHA having been previously shown to enhance neurogenesis [15, 37, 47, 48] and regulate the expression of neuronal plasticity markers including Arc [49, 50], synergistic effects with the other components of the diet such as B. breve CECT8242 could not be discarded. In this regard, it has been reported that either omega-3 fatty acid or a mixture of probiotics is able to regulate lipidic profile, insulin sensitivity and inflammatory markers, but it was the addition of the two components together which had more pronounced effects [17, 18]. One limitation of our study is that it was not designed to analyze the effect of the individual components of the formulated diets, and so their synergics could not be explored. As far as we know, the synergistic effects of omega-3 fatty acids and probiotics on neural plasticity or on cognition have not been studied yet, and our results particularly encourage further exploration of whether DHA and the probiotic B. breve CECT8242 act synergistically to potentiate these processes.