1. 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 studies1-3. 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 behavior6-8, 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 RESULTSINTRODUCTION
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. Djani1, Saritha Krishna1, Donald A. Harn2, John J. Wagner1, Claire B. de La Serre3, and Nikolay M. Filipov1
1Department of Physiology and Pharmacology, 2Department of Infectious Diseases, College of Veterinary Medicine, 3Department 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).
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Female
mice/dams
maintained
on
HFD
or
LFD.
Brain
slicing
Sample
collec0on
Neurochemical
analysis
Prefrontal Cortex: 5-HT
LFD
H
FD
LFD
H
FD
0.000
0.375
0.750
1.125
1.500
Males Females
5-HT(ng/mgprotein)
^
*
*Statistically significant main effect of sex
^Statistical trend for effect of diet within females
Ventral Hippocampus: 5-HT
LFD
H
FD
LFD
H
FD
0
2
4
6
8
Males Females
5-HT(ng/mgprotein)
a
aStatistically significant effect of diet within females
Cerebellum: 5-HT
LFD
H
FD
LFD
H
FD
0
2
4
6
8
Males Females
5-HT(ng/mgprotein)
*
*Statistically significant main effect of sex
^Statistical trend for effect of diet within females
^ LFD
HFD
Prefrontal Cortex: Dopamine
LFD
H
FD
LFD
H
FD
0.0
0.1
0.2
0.3
0.4
Males Females
DA(ng/mgprotein)*Statistically significant main effect of diet
aStatistically significant effect of diet within females
*
a
Dorsal Hippocampus: Dopamine
LFD
H
FD
LFD
H
FD
0.000
0.125
0.250
0.375
0.500
Males Females
DA(ng/mgprotein)
*
*Statistically significant main effect of diet
^Statistical trend for effect of diet within females
^
Ventral Hippocampus: Dopamine
LFD
H
FD
LFD
H
FD
0.00
0.75
1.50
2.25
3.00
Males Females
DA(ng/mgprotein)
*
*Statistically significant main effect of diet
^Statistical trend for effect of diet within females
^
HFD
LFD