Abstract
Background
Recent studies have shown a relationship between the composition of the maternal diet and acquiring a risk of mental illnesses through changes in the offspring’s brain. This study assessed the role of a modified maternal diet on the levels of serotonin (5-HT)2C and 5-HT2A receptors in the offspring brain.
Methods
Wistar rat dams during gestation and lactation were maintained either on a standard (SD) or special diets: high-fat (HFD), high-carbohydrate (rich in sucrose, HCD) or mixed (MD). Offspring were weaned to SD after lactation, and at postnatal days (PNDs) 28 and 63 changes in the 5-HT2C and 5-HT2A receptor levels were evaluated in their prefrontal cortex (PFCx), nucleus accumbens (NAc), dorsal striatum (DSTR) and hippocampus (HIP).
Results
Maternal HFD reduced the expression of 5-HT2C receptors in male rats at PND 28 in the PFCx, NAc, and DSTR but increased it at PND 63 in male animals in the NAc and DSTR. HCD induced a decrease in the expression of 5-HT2C receptors in male offspring at PND 28 but increased it in female rats at PND 63 in the PFCx. MD reduced 5-HT2C receptor expression in males at PND 28 in the PFCx and increased it in male and female offspring at PND 28 in the HIP. Moreover, maternal HFD reduced 5-HT2A receptor levels within the PFCx in adolescent male offspring.
Conclusion
Our findings indicate that a modified maternal diet induces age- and sex-specific adaptive changes mainly in 5-HT2C receptors, which may contribute to disturbances in the offspring brain.
Graphic abstract
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Introduction
The global epidemic of obesity increasingly occurring among women of reproductive age and around conception is due to excessive food intake and an unhealthy lifestyle. In many countries, more than one-third of pregnant women are overweight or obese, which has numerous negative effects on the health of the offspring [1]. Currently, much evidence indicates that not only obesity but also maternal feeding patterns during pregnancy and lactation may be responsible for fetal developmental adaptations that permanently affect the offspring’s metabolism, neurodevelopment, and behavior throughout their life. This phenomenon is called “fetal programming” and could be associated with epigenetic changes during early life (see review [2]). Epidemiological and experimental studies suggest a link between early exposure to an unbalanced maternal diet (e.g., high-fat or high-carbohydrate) and susceptibility to morphological, molecular and functional changes in the offspring’s brain as well as neurodevelopmental or mental disorders, such as attention-deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), anxiety, depression, schizophrenia and eating disorders [2,3,4].
Serotonin (5-hydroxytryptamine, 5-HT) is an important neurotransmitter in the central nervous system and is an endogenous ligand for seven types of 5-HT receptors (5-HT1–5-HT7). One of the interesting links to mental illness from the nutritional and pathological points of view are the 5-HT2A and 5-HT2C receptors, seven-transmembrane, G-protein linked receptors positively coupled to phospholipase C. 5-HT2A receptors are involved in the regulation of learning and memory [5], and 5-HT2C receptor signaling is related to physiological brain functions, such as regulation of appetite, food intake and energy balance, stress response or sleep regulation [6]. The 5-HT2C receptor has structural similarity to the 5-HT2A receptor, as well as similar pharmacological profiles and cellular signaling pathways [7]. Given the important role of 5-HT neurotransmission in brain development, function, and mental disorders, disturbances in 5-HT2A and 5-HT2C receptor levels resulting from abnormal conditions of intrauterine development may have critical implications for the behavior and mental health of offspring during their entire lifetime [8].
In light of the above information and recent results from our group showing that maternal modified diets limited to periods of pregnancy and lactation predispose offspring to the development of depressive-like behavior [9, 10] or changes in the strength of cocaine-seeking behavior [11, 12], in this study, we investigated the role of early exposure to maternal high-fat (HFD), high-carbohydrate (rich in sucrose, HCD) and a mixed diet (rich in fat and carbohydrate, MD) on 5-HT2A and 5-HT2C receptors profiles within the brain structures (prefrontal cortex, dorsal striatum, nucleus accumbens, and hippocampus) associated with mental disorders during offspring development (adolescence and adulthood).
Materials and methods
Animals and diets
This study was performed in accordance with the guidelines of the EU Directive 2010/63/EU with the approval of the Ethical Committee at the Maj Institute of Pharmacology Polish Academy of Sciences (approval number 1270/2015; December 17, 2015). Every effort was made to minimize suffering and the number of animals used.
Wistar rats from Charles River (Sulzfeld, Germany) were housed in standard cages in an animal room maintained at 22 ± 2 °C and 55 ± 10% humidity under a 12 h light–dark cycle (lights on at 6.00 a.m.). The animals had free access to water and food.
Female virgin rats (200–240 g), after the acclimatization period and during the proestrus phase (smears from females were assessed to determine the estrous cycle phase) were mated with males. The pregnancy was confirmed by examining the vaginal smears for the presence of sperm. Then, the pregnant females were individually housed and randomly assigned to four groups: standard diet (SD, 65% carbohydrate, 13% fat, 22% protein, 3.4 kcal/g; VRF1; Special Diets Services, Witham, UK) or modified diets purchased from Altromin (Lage, Germany): HFD (24% carbohydrate, 60% fat, 16% protein, 5.31 kcal/g; C1057 mod.), HCD (70% carbohydrate: rich in sucrose, 12% fat, 18% protein, 3.77 kcal/g; C1010) or MD (56% carbohydrate, 28% fat, 16% protein, 3.90 kcal/g; C1011). Dams were fed these diets ad libitum during pregnancy (21 days) and lactation (21 days). The special diets used in this study did not affect the litter size or birth weight of the offspring [11]. Litter sizes were normalized to 9–12 pups with a sex ratio as close to 1:1 as possible. To reduce “litter effects” [13], animals for each group were selected from 3–4 different dams. After weaning, offspring at postnatal (PND) 22 were separated according to sex and kept on a SD. Male and female offspring were used in this study. Biochemical tests were performed at PNDs 28 and 63.
Brain tissue isolation
Groups of male and female offspring (n = 8 in each group) were sacrificed through decapitation at PNDs 28 and 63. The prefrontal cortex (including the infralimbic, prelimbic and cingulate cortices; bregma: 5.2–2.7 mm), nucleus accumbens, dorsal striatum, and hippocampus, were dissected according to The Rat Brain Atlas [14] and were immediately frozen on dry ice and stored at − 80 °C until analysis. All tissues were collected between 9.00 and 12.00 a.m.
Enzyme-linked immunosorbent assay (ELISA)
Frozen brain structures were homogenized using a sonicator (EpiShear™ Probe Sonicator; Active Motif, Carlsbad, CA, USA) in 10% (w/v) of 0.32 M sucrose HEPES buffer (containing 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose and 5 mM HEPES) with a protease inhibitor cocktail (Complete, Roche Diagnostics, Mannheim, Germany). Isolation of the synaptosomal fraction was performed to observe molecular changes in the protein composition at the synapses and to determine the 5-HT2C and 5-HT2A receptor concentrations as described previously [12]. For protein determination, a bicinchoninic acid (BCA) protein assay kit (Pierce™ BCA Protein Assay Kit, Thermo Scientific, Rockford, IL, USA) was used. The concentrations of 5-HT2C and 5-HT2A receptors in the selected brain structures were measured using ELISA (Cat. No E1793Ra, Cat. No E1799Ra; BT LAB, Shanghai, China) according to the manufacturer’s instructions. Forty microliters of each sample (n = 8 for the prefrontal cortex and nucleus accumbens; n = 7 for the dorsal striatum and hippocampus) were transferred to 96-well ELISA plates in duplicate with standards. The absorbance was measured at a wavelength (λ) of 450 nm using a Multiskan Spectrum spectrophotometer (Thermo Labsystems, Philadelphia, PA, USA). The concentrations of the 5-HT2C and 5-HT2A receptors were calculated from a standard curve and are expressed as ng/mg of protein and pg/mg of protein, respectively. The precision was intra-assay: CV < 8% and interassay: CV < 10%.
Statistical analyses
Statistical analyses were performed using GraphPad Prism 9.1.0 software (GraphPad Software, San Diego, CA, USA). All data are expressed as the mean (± SEM). Statistical analysis was performed using a two-way analysis of variance (ANOVA, diet × sex). Post hoc Tukey’s tests were used to examine differences between group means. p < 0.05 was considered statistically significant.
Results
Adolescence
The effects of modified maternal diets on 5-HT2C receptor levels in the brains of male and female offspring at PND 28 are shown in Fig. 1. In adolescent offspring, two-way ANOVA indicated a significant effect of diet on 5-HT2C receptor levels within the prefrontal cortex (F3,56 = 11.50, p < 0.001), dorsal striatum (F3,48 = 6.20, p < 0.01) and hippocampus (F3,48 = 24.39, p < 0.001) and a diet × sex interaction in the prefrontal cortex (F3,56 = 6.03, p < 0.01), dorsal striatum (F3,48 = 4.27, p < 0.01) and nucleus accumbens (F3,56 = 5.50, p < 0.01). Male offspring from the HFD group showed decreased 5-HT2C receptor concentrations in the prefrontal cortex (p < 0.001), nucleus accumbens (p < 0.05) and dorsal striatum (p < 0.01), while maternal HCD and MD during pregnancy and lactation reduced 5-HT2C receptor levels within the prefrontal cortex (p < 0.001 and p < 0.01, respectively). Moreover, upregulation of the hippocampal 5-HT2C receptor was observed in both male and female offspring from the MD group (p < 0.001).
We determined the level of 5-HT2A receptors in the HFD group, in which the most numerous changes in the levels of the 5-HT2C receptors were determined. At PND 28, two-way ANOVA indicated a significant effect of diet × sex interaction on 5-HT2A receptor levels only within the prefrontal cortex (F1,28 = 5.66, p < 0.05). In fact, a maternal HFD decreased the cortical 5-HT2A receptor concentration in male offspring (p < 0.05) (Fig. 2).
Adulthood
In adult animals, statistical analysis also showed the importance of the dietary effect on the expression of the 5-HT2C receptor in the prefrontal cortex (F3,56 = 3.78, p < 0.05), nucleus accumbens (F3,56 = 24.15, p < 0.001), and dorsal striatum (F3,48 = 11.47, p < 0.001) but not within the hippocampus (F3,48 = 1.05, p = 0.38), as well as a diet × sex interaction in the prefrontal cortex (F3,56 = 7.87, p < 0.001), nucleus accumbens (F3,56 = 22.13, p < 0.001), and dorsal striatum (F3,48 = 12.75, p < 0.001) (Fig. 3). A maternal HFD led to increased levels of 5-HT2C receptors in the nucleus accumbens (p < 0.001) and dorsal striatum (p < 0.001) only in male offspring. On the other hand, exposure to maternal HCD during pregnancy and lactation at PND 63 increased the cortical 5-HT2C receptors concentration in female offspring (p < 0.01). Moreover, we noted that maternal HFD did not affect the levels of 5-HT2A receptors in either male or female adult offspring (Fig. 4).
Discussion
Brain plasticity during the fetal and early postnatal period is essential for proper brain development and adaptation to a changing environment, but this also means that external factors, such as maternal diet, stress or infections, can lead to permanent changes in the central nervous system in offspring [15].
In the current study, we investigated the effect of modified maternal diets (HFD, HCD, and MD) during pregnancy and lactation on the protein level of 5-HT2C receptors in the brain structures of male and female offspring. Here, we report that maternal HFD reduced the level of 5-HT2C receptors in the synaptosomal fraction of the prefrontal cortex, dorsal striatum, and nucleus accumbens in adolescent male rats. On the other hand, in adult males exposed to maternal HFD, we observed significantly increased levels of 5-HT2C receptors within the nucleus accumbens and dorsal striatum but not in the prefrontal cortex. Moreover, in contrast to the males exposed to maternal HFD, we did not observe any changes in the protein level of 5-HT2C receptors in adolescent and adult females. It is worth emphasizing that maternal HFD reduced the 5-HT2A receptor level in the prefrontal cortex in adolescent males (the same direction of changes as for 5-HT2C receptors) but did not induce significant changes in other structures at any of the time points examined. Therefore, it appears that the disturbed signaling mediated by 5-HT2C receptors is not cognized by 5-HT2A receptors.
Although the current literature does not provide data on the impact of modified maternal nutrition during pregnancy and lactation on 5-HT2C and 5-HT2A receptors in the offspring brain, there are some reports indicating the effect of prolonged (15–20 weeks) feeding of adult male rodents a HFD (inducing obesity) on these receptors. Thus, Lopez-Esparza et al. [16] found a reduction in the expression of 5-HT2C receptors in the hippocampus in rats, while Huang and colleagues [17] reported an increased mRNA expression of these receptors in the mouse hypothalamus and increased expression of 5-HT2A receptors within the olfactory nucleus.
In the present study, for the opposite effects of maternal HFD on 5-HT2C receptors in limbic structures at PNDs 28 and 63 in male rats, one should take into account that such a maternal diet also affects other elements of the 5-HT system, which could lead to the differences in protein levels of 5-HT2C receptors in adolescent and adult offspring. It has been shown that maternal HFD evoked an increase in tryptophan hydroxylase 2 (TPH2, an enzyme involved in 5-HT synthesis) expression at PND 28 in mice [18] and increased mRNA of 5-HT1A inhibitory autoreceptors at PND 95 in rats [19] in some brain structures, effects that could correspondingly lead to an increase or reduction of 5-HT tone, and consequently to a reduction or increase of the 5-HT2C receptor expression, respectively.
At the same time, the lack of an effect of maternal HFD on 5-HT2C receptors in the female offspring may be related to the importance of sex hormones, as has been suggested by Gugusheff et al. [20], who examined the effect of modified maternal diets on mu-opioid receptors in rat offspring.
In some behavioral experiments, we have also demonstrated that maternal HFD (but not HCD and MD) evokes depression-like behavior in offspring as it prolongs immobility time in the forced swimming test (FST) and induces anhedonia manifesting as a decrease in sucrose preference [9, 10]. The above observations and our present results, taken together with some preclinical findings and the results of postmortem studies of the human brain in suicide victims, could suggest the involvement of 5-HT2C and 5-HT2A receptors in the pathogenesis of depression [6, 21]. However, such a suggestion seems doubtful since, in contrast to changes in the expression of 5-HT2C and 5-HT2A receptors, maternal HFD-induced depressive-like behaviors were dependent on neither the age nor the sex of the offspring [9, 10]. Moreover, the brain regions tested did not come directly from the animals used in the behavioral experiments; therefore, we cannot directly correlate the results for individual animals.
There are also a number of preclinical and clinical reports demonstrating that maternal HFD can lead to several neurodevelopmental or other mental disorders, such as ADHD, ASD, anxiety, schizophrenia, eating disorders, and cocaine-seeking behavior [3, 4, 11]. However, again, there are no data, which may indicate that they could be related to HFD-induced changes in the expression of brain 5-HT2C or 5-HT2A receptors.
In contrast to HFD, other diets used in the present study during pregnancy and lactation did not evoke coherent changes in the expression of 5-HT2C receptors. In fact, maternal HCD reduced their expression in the adolescent male offspring but increased it at PND 63 in female animals in the prefrontal cortex only. At the same time, maternal MD decreased the expression of the 5-HT2C receptors in the prefrontal cortex of males at PND 28 but increased it in the hippocampus of males and females at PND 28. Whether these changes in 5-HT2C receptors have any functional meaning remains to be elucidated.
Taken together, the present study provides evidence that modified prenatal and early postnatal diets (mainly HFD) modulate the expression of 5-HT2C and 5-HT2A receptors in offspring brain structures. Further studies are necessary to elucidate their functional and/or pathological importance, as well as their potential impact on the efficacy of drugs (e.g., antidepressants) for which 5-HT2C and 5-HT2A receptors are one of their molecular targets.
Abbreviations
- ADHD:
-
Attention-deficit hyperactivity disorder
- ASD:
-
Autism spectrum disorder
- FST:
-
Forced swimming test
- HCD:
-
High-carbohydrate diet
- HFD:
-
High-fat diet
- MD:
-
Mixed diet
- PND:
-
Postnatal day
- SD:
-
Standard diet
- 5-HT:
-
Serotonin (5-hydroxytryptamine)
References
Chen C, Xu X, Yan Y. Estimated global overweight and obesity burden in pregnant women based on panel data model. PLOS ONE. 2018;13(8):e0202183. https://doi.org/10.1371/journal.pone.0202183.
Gawlińska K, Gawliński D, Filip M, Przegaliński E. Relationship of maternal high-fat diet during pregnancy and lactation to offspring health. Nutr Rev. 2021;79(6):709–25. https://doi.org/10.1093/nutrit/nuaa020.
Edlow AG. Maternal obesity and neurodevelopmental and psychiatric disorders in offspring. Prenat Diagn. 2017;37(1):95–110. https://doi.org/10.1002/pd.4932.
Rivera HM, Christiansen KJ, Sullivan EL. The role of maternal obesity in the risk of neuropsychiatric disorders. Front Neurosci. 2015;9:194. https://doi.org/10.3389/fnins.2015.00194.
Zhang G, Stackman RW. The role of serotonin 5-HT2A receptors in memory and cognition. Front Pharmacol. 2015;6:225. https://doi.org/10.3389/fphar.2015.00225.
Chagraoui A, Thibaut F, Skiba M, Thuillez C, Bourin M. 5-HT2C receptors in psychiatric disorders: a review. Prog Neuro-Psychopharmacol Biol Psychiatry. 2016;66:120–35. https://doi.org/10.1016/j.pnpbp.2015.12.006.
Bécamel C, Gavarini S, Chanrion B, Alonso G, Galéotti N, Dumuis A, Bockaert J, Marin P. The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins. J Biol Chem. 2004;279(19):20257–66. https://doi.org/10.1074/jbc.M312106200.
Brummelte S, Mc Glanaghy E, Bonnin A, Oberlander TF. Developmental changes in serotonin signaling: implications for early brain function, behavior and adaptation. Neuroscience. 2017;342:212–31. https://doi.org/10.1016/j.neuroscience.2016.02.037.
Gawlińska K, Gawliński D, Przegaliński E, Filip M. Maternal high-fat diet during pregnancy and lactation provokes depressive-like behavior and influences the irisin/brain-derived neurotrophic factor axis and inflammatory factors in male and female offspring in rats. J Physiol Pharmacol. 2019;70(3):407–11. https://doi.org/10.26402/jpp.2019.3.07.
Gawlińska K, Gawliński D, Korostyński M, Borczyk M, Frankowska M, Piechota M, et al. Maternal dietary patterns are associated with susceptibility to a depressive-like phenotype in rat offspring. Dev Cogn Neurosci. 2021;47:100879. https://doi.org/10.1016/j.dcn.2020.100879.
Gawliński D, Gawlińska K, Frankowska M, Filip M. Maternal diet influences the reinstatement of cocaine-seeking behavior and the expression of melanocortin-4 receptors in female offspring of rats. Nutrients. 2020;12(5):1462. https://doi.org/10.3390/nu12051462.
Gawliński D, Gawlińska K, Frankowska M, Filip M. Maternal high-sugar diet changes offspring vulnerability to reinstatement of cocaine-seeking behavior: role of melanocortin-4 receptors. FASEB J. 2020;34(7):9192–206. https://doi.org/10.1096/fj.202000163R.
Festing MF. Design and statistical methods in studies using animal models of development. ILAR J. 2006;47(1):5–14. https://doi.org/10.1093/ilar.47.1.5.
Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 4th ed. San Diego: Academic Press; 1998.
Shah R, Courtiol E, Castellanos FX, Teixeira CM. Abnormal serotonin levels during perinatal development lead to behavioral deficits in adulthood. Front Behav Neurosci. 2018;12:114. https://doi.org/10.3389/fnbeh.2018.00114.
Lopez-Esparza S, Berumen LC, Padilla K, Miledi R, García-Alcocer G. Expression of hippocampal serotonin receptors 5-HT2C and 5-HT5A in a rat model of diet-induced obesity supplemented with tryptophan. Int J Dev Neurosci. 2015;42:80–5. https://doi.org/10.1016/j.ijdevneu.2015.02.010.
Huang XF, Han M, Storlien LH. Differential expression of 5-HT2A and 5-HT2C receptor mRNAs in mice prone, or resistant, to chronic high-fat diet-induced obesity. Mol Brain Res. 2004;127(1–2):39–47. https://doi.org/10.1016/j.molbrainres.2004.05.008.
Dias CT, Curi HT, Payolla TB, Lemes SF, Betim Pavan IC, Torsoni MA, et al. Maternal high-fat diet stimulates proinflammatory pathway and increases the expression of tryptophan hydroxylase 2 (TPH2) and brain-derived neurotrophic factor (BDNF) in adolescent mice hippocampus. Neurochem Int. 2020;139:104781. https://doi.org/10.1016/j.neuint.2020.104781.
Paradis J, Boureau P, Moyon T, Nicklaus S, Parnet P, Paillé V. Perinatal western diet consumption leads to profound plasticity and GABAergic phenotype changes within hypothalamus and reward pathway from birth to sexual maturity in rat. Front Endocrinol (Lausanne). 2017;8:216. https://doi.org/10.3389/fendo.2017.00216.
Gugusheff JR, Bae SE, Rao A, Clarke IJ, Poston L, Taylor PD, et al. Sex and age-dependent effects of a maternal junk food diet on the mu-opioid receptor in rat offspring. Behav Brain Res. 2016;301:124–31. https://doi.org/10.1016/j.bbr.2015.12.027.
Underwood MD, Kassir SA, Bakalian MJ, Galfalvy H, Dwork AJ, Mann JJ, Arango V. Serotonin receptors and suicide, major depression, alcohol use disorder and reported early life adversity. Transl Psychiatry. 2018;8(1):279. https://doi.org/10.1038/s41398-018-0309-1.
Funding
This study was supported by the statutory funds of the Maj Institute of Pharmacology Polish Academy of Sciences and research grant UMO-2016/21/B/NZ4/00203 (to M.F.) from the National Science Centre, Kraków, Poland.
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KG, MF, and EP conceptualized and designed the study; KG and DG performed the experiments and analyzed the data; KG and EP wrote the manuscript. All authors approved the final manuscript.
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Gawlińska, K., Gawliński, D., Filip, M. et al. Maternal feeding patterns affect the offspring’s brain: focus on serotonin 5-HT2C and 5-HT2A receptors. Pharmacol. Rep 73, 1170–1178 (2021). https://doi.org/10.1007/s43440-021-00298-0
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DOI: https://doi.org/10.1007/s43440-021-00298-0