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The role of the maternal environment in abnormal neurodevelopment represents a relatively recent research focus. Positive association between maternal obesity, partly driven by consumption of high-fat diet (HFD), and increased incidence of autism spectrum disorder (ASD) has been reposted in several epidemiological studies 1-3 . Underlying pathological mechanisms leading to altered brain connectivity in ASD occur during a critical neurodevelopmental window characterized by active axonal growth and synapse formation, as well as the programming of neurotransmitter systems (humans: late third trimester; mice: approx. post-natal day 10 [PND10]) 4-5 . Brain monoamines play important roles in neurodevelopment and behavior 6-8 , but the effects of maternal HFD consumption on offspring brain monoamines during this critical window have not yet been studied up to this date. NEUROCHEMICAL RESULTS INTRODUCTION Serotonergic System: Prefrontal Cortex, Ventral Hippocampus, and Cerebellum Animals : Young adult C57BL/6 female mice (6-7 weeks old, Harlan, Indianapolis, IN, USA) were housed in an environmentally controlled room (4-5/cage, 22-24° C) on a 12-h light/dark cycle in an AAALAC-accredited facility with food and water available ad libitum. After one-week acclimation mice were randomly assigned to a high-fat diet (HFD) or low-fat diet (LFD) and fed their respective diets for 6 weeks prior to mating with controlled males; HFD/LFD feeding continued post-conception until weaning (post-natal day [PND] 21). On PND10 we selected male and female offspring (n=7, 1m/1f each from a litter) from the HFD and LFD dams for whole brain collection and subsequent neurochemical analysis. Sample Collection : Following extraction, brains were rinsed with ice-cold Hank’s buffer, sliced midsagitally, and the half intended for neurochemistry was quickly frozen (-80 ºC). Later on, brains were sectioned coronally (500 μm- thick sections) and tissue samples of the prefrontal cortex (PFC), striatum, dorsal hippocampus (dHIPP), ventral hippocampus (vHIPP), and cerebellum (CER) were obtained via micropunches. Neurochemical Analysis: We used high-performance liquid chromatography with electrochemical detection (HPLC-ED) to quantify levels of dopamine (DA), serotonin (5-HT), norepinephrine (NE), and their major metabolites in each brain region. Measured metabolites of DA included 3,4- dihyroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3- methoxytyramine (3-MT), while the measured 5-HT and NE metabolites were 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG), respectively. We then performed Bradford assays on each sample and used quantified protein levels to normalize the neurochemical data on a per milligram protein basis. Statistical analysis included two-way ANOVA and t- tests to assess the main effects of maternal HFD and sex, and the effects of maternal HFD within males and females, on brain monoamines at PND10. Early neurochemical deficits in offspring of dams fed high fat diets: focus on brain monoamines Dylan M. Djani 1 , Saritha Krishna 1 , Donald A. Harn 2 , John J. Wagner 1 , Claire B. de La Serre 3 , and Nikolay M. Filipov 1 1 Department of Physiology and Pharmacology, 2 Department of Infectious Diseases, College of Veterinary Medicine, 3 Department of Foods and Nutrition, College of Family and Consumer Sciences, University of Georgia, Athens, GA METHODS Dopaminergic System: Prefrontal Cortex and Hippocampus Figure 2. Effects of high-fat diet (HFD) and sex on dopamine (DA) levels in the prefrontal cortex (PFC), dorsal hippocampus (dHIPP), and ventral hippocampus (vHIPP). * Indicates statistically significant main effect of diet (P ≤ 0.05); a Indicates statistically significant effect of diet within sex (P ≤ 0.05); ^ Indicates statistical trend for a diet effect within sex (P ≤ 0.1). CONCLUSION REFERENCES Maternal HFD leads to regionally-specific dopaminergic disruption in the brain regardless of sex, demonstrating that maternal HFD adversely affects male and female offspring; however, female-restricted serotonergic disruption along with cerebellar neurochemical changes suggest that maternal HFD may impact females more broadly. Disruptions of monoamine systems in offspring may relate to ASD-like behaviors, including hyperactivity/behavioral dysregulation (DA) or deficits in communication and social interaction (5-HT). Our data indicate that maternal HFD alters early brain monoamine neurochemistry, consistent with altered brain connectivity, suggesting that maternal HFD may put offspring on a trajectory towards ASD. ACKNOWLEDGEMENTS Week 0: Maternal diets assigned Week 6: Dams bred to control males PND 0: Pups born – maternal nutrition PND 10 • Brains collected from selected offspring PND 21: Weaning, maternal diet discontinued Figure 1. Experimental Timeline Figure 3. Effects of high-fat diet (HFD) and sex on serotonin (5-HT) levels in the prefrontal cortex (PFC), ventral hippocampus (vHIPP), and cerebellum (CER). * Indicates statistically significant main effect of sex (P ≤ 0.05); a Indicates statistically significant effect of diet within sex (P ≤ 0.05); ^ Indicates statistical trend for a diet effect within sex (P ≤ 0.1). DISCUSSION The data demonstrate two major effects of maternal HFD: (i) sex-independent DA increases in PFC, dHIPP, and vHIPP; (ii) female-specific 5-HT increases in PFC, vHIPP, and CER. Maternal HFD consumption also led to sex- independent increases in vHIPP levels of the dopamine metabolite DOPAC. Moreover, the effect of maternal HFD showed a trend for increased 5-HIAA levels in the female PFC, suggesting that maternal HFD leads to increased prefrontal cortical serotonergic tone in PND10 female mice. Sex-dependent regional monoamine and metabolite differences were noted: HVA levels of females were lower in the PFC and higher in the dHIPP. Furthermore, levels of both 5-HT and NE of females were higher in PFC and CER, but lower in dHIPP. Lastly, females showed lower dHIPP NE and higher striatal 3-MT levels. Overall, maternal HFD most strongly affected the dopaminergic system, particularly vHIPP. The striatum, as a brain region, and the noradrenergic system, as a monoaminergic circuitry, were least affected by maternal HFD across both sexes. Compared to the increased 5-HT levels in female HFD offspring in other brain regions, the lack of such effect on 5-HT levels in dHIPP, along with sex-dependent female- specific dHIPP differences in NE and HVA levels, suggest that maternal HFD affects dHIPP and vHIPP monoamine homeostasis differentially. Finally, increased cerebellar 5-HT due to maternal HFD was observed in females alongside sex-dependent increases in cerebellar levels of 5-HIAA and NE in female offspring. Thus, cerebellar monoaminergic projections may also be involved in the sex-specific effects of maternal HFD. The project described was supported by Grant Number 05 T35 OD010433-09 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH) and its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. The project was also funded via a grant from The University of Georgia’s Obesity Initiative (http://obesity.ovpr.uga.edu). 1. E. L. Sullivan, K. M. Riper, R. Lockard, J. C. Valleau, Maternal high-fat diet programming of the neuroendocrine system and behavior. Horm Behav, (2015). 2. S. Y. Kim, A. J. Sharma, W. Sappenfield, H. G. Wilson, H. M. Salihu, Association of maternal body mass index, excessive weight gain, and gestational diabetes mellitus with large-for-gestational- age births. Obstet Gynecol 123, 737-744 (2014). 3. H. Jo et al., Maternal prepregnancy body mass index and child psychosocial development at 6 years of age. Pediatrics 135, e1198-1209 (2015). 4. Workman AD, Charvet CJ, Clancy B, Darlington RB, Finlay BL. Modeling transformations of neurodevelopmental sequences across mammalian species. J Neurosci. 2013 Apr 24;33(17): 7368-83. 5. Herlenius E, Lagercrantz H. Development of neurotransmitter systems during critical periods. Experimental Neurology. 2004;190, Supplement 1:8-21. 6. P. Jaiswal, K. P. Mohanakumar, U. Rajamma, Serotonin mediated immunoregulation and neural functions: Complicity in the aetiology of autism spectrum disorders. Neuroscience & Biobehavioral Reviews 55, 413-431 (2015). 7. Hara Y, Takuma K, Takano E, et al. Reduced prefrontal dopaminergic activity in valproic acid-treated mouse autism model. Behavioural Brain Research. 2015 8/1/;289(0):39-47. 8. McGinnis WR, Audhya T, Edelson SM. Proposed toxic and hypoxic impairment of a brainstem locus in autism. International Journal Of Environmental Research And Public Health. 2013;10(12):6955-7000. Female mice/dams maintained on HFD or LFD. Brain slicing Sample collec0on Neurochemical analysis Prefrontal Cortex: 5-HT LFD HFD LFD HFD 0.000 0.375 0.750 1.125 1.500 Males Females 5-HT (ng/mg protein) ^ * *Statistically significant main effect of sex ^ Statistical trend for effect of diet within females Ventral Hippocampus: 5-HT LFD HFD LFD HFD 0 2 4 6 8 Males Females 5-HT (ng/mg protein) a a Statistically significant effect of diet within females Cerebellum: 5-HT LFD HFD LFD HFD 0 2 4 6 8 Males Females 5-HT (ng/mg protein) * *Statistically significant main effect of sex ^ Statistical trend for effect of diet within females ^ LFD HFD Prefrontal Cortex: Dopamine LFD HFD LFD HFD 0.0 0.1 0.2 0.3 0.4 Males Females DA (ng/mg protein) *Statistically significant main effect of diet a Statistically significant effect of diet within females * a Dorsal Hippocampus: Dopamine LFD HFD LFD HFD 0.000 0.125 0.250 0.375 0.500 Males Females DA (ng/mg protein) * *Statistically significant main effect of diet ^ Statistical trend for effect of diet within females ^ Ventral Hippocampus: Dopamine LFD HFD LFD HFD 0.00 0.75 1.50 2.25 3.00 Males Females DA (ng/mg protein) * *Statistically significant main effect of diet ^ Statistical trend for effect of diet within females ^ HFD LFD
