1. Dylan M. Djani
Mentor: Nikolay M. Filipov
Department of Physiology and Pharmacology
College of Veterinary Medicine
University of Georgia
2. Presentation Outline
• Obesity and Autism Spectrum Disorder (ASD)
– Prevalence, key features
• ASD Etiology
– Maternal environment
• Early Brain Monoamines
What are
monoamines?
(Bear et. al., 2015)
3. Obesity Trends in the U.S.
National Health and Nutrition Examination Survey (NHANES)
• Obesity Prevalence in U.S. Adults ≥ 25 years [2007 – 2012]
– Men: 35.04% Data published June 22, 2015 (Yang and Colditz, 2015)
– Women: 36.84%
Prevalence consistently high [2003 – 2010]
Data published February 26, 2014 (Ogden et. al., 2014)
Obesity – chronic inflammatory condition:
risk mortality, physical, emotional, and mental conditions
OVER 1 in 3 AMERICANS
Regardless of sex or race
(Monteiro and Azevedo, 2010; Nijhuis et. al., 2009)
4. Maternal Obesity Trends in the U.S.
Schlaff et. al., 2014
*Gestational weight gain; 1Archive for Research on Child Health
Overweight and obese women are more likely to gain excess weight during pregnancy.
>50% women gained excess weight during gestation. (2009 Institute of Medicine)
5. Maternal Obesity
Adversely Impacts
Offspring
Rivera et. al., 2015
BMI:
Body mass index
GWG:
Gestational weight gain
HFD:
High-fat diet
Rivera et. al., 2015
Human research studies
Animal model research studies
6. Autism-Spectrum Disorder: Overview
DSM-V: Neurodevelopmental Disorder
Early onset and lifelong impact (APA, 2013)
Core symptoms: deficits in communication, social interaction, behavior
http://www.autismspeaks.org/
Last updated January 6, 2015
Prevalence Facts:
*1 in 68 children in the U.S.
*Male bias (M/F Sex Ratio 4:1)
*Positive correlation with obesity
123% increase in ASD
prevalence since 2002.
Analysis of 2010 data; published in 2014; studies ongoing (ADDM)
CDC Autism and Developmental Disabilities Monitoring Network
9. Maternal Obesity in the Perinatal Period
Maternal obesity
(Rivera et. al., 2015; Sullivan et. al., 2015; Mehta et. al., 2014; Bolton and Bilbo, 2014)
Immunologic dysregulation
Altered placental function
Maternal circulation
Pro-inflammatory cytokines
Glucose and triglycerides
Hormones (i.e. leptin)
Serotonin (5-HT)
Altered fetal exposure
during perinatal period
Altered brain connectivity and neurotransmitter systems
Epigenetic, metabolic, neurobehavioral programming
10. Monoaminergic Involvement in ASD: Serotonin
Jaiswal et. al., 2015
Neuroimmunologic dysregulation in ASD involves serotonin.
11. Monoaminergic Involvement in ASD
Dopaminergic System Noradrenergic System
ASD Psychiatric comorbidities
Cross-talk between DA and NE
Brainstem (VTA, LC)
Dorsal hippocampus
(Hara et. al., 2015; Kriete and Noelle, 2015) (Jellinger, 2011; Guiard et. al., 2008)
Prefrontal Cortex
Valproic-acid mouse model: PND21
Computerized developmental modeling
Rivera et. al., 2015
12. Experimental Objectives
Evaluate the effects of maternal obesity and sex on
monoamine systems in the early post-natal life of mice.
Hypothesis
Significant neurochemical differences will be observed
in selected brain regions of post-natal day 10 mice due
to maternal high fat diet and sex.
13. Experimental Design and Timeline
Brain Collection: PND10
N = 7; 1m/1f per group
Brain Region Collection:
500 μm coronal sectioning; dry ice
Regional micropunches obtained
Animals:
C57BL/6 female mice, 6-7 weeks
Assigned Diet*:
High fat: 60% kcal from fat HFD
Low fat: 10% kcal from fat LFD
PND = post-natal day; *Diets balanced for simple sugars and micronutrients.
*Assigned diets maintained throughout weaning (PND21).
WEEK 0
Maternal
diets
assigned
WEEK 6
Mating with
control males.
PND 0
Pups born.
PND 10
Pups selected for
brain collection.
Brain region collection
Neurochemical analysis
14. Materials and Methods
Monoamine and Metabolite Analytes:
Dopaminergic System: DA, DOPAC, HVA, 3-MT
Serotonergic System: 5-HT, 5-HIAA
Noradrenergic System: NE, MHPG
Brain regions collected:
Prefrontal cortex (PFC)
Striatum (STR)
Dorsal Hippocampus (dHIPP)
Ventral Hippocampus (vHIPP)
Cerebellum (CER)
Neurochemical Analysis: HPLC-ECD + Bradford Assay
Data normalized per mg protein prior to statistical analysis
Data Processing and Presentation:
Performed with Microsoft Excel, SigmaPlot, GraphPad Prism 5 software
15. Comparative Mammalian Neurodevelopment
“Translating Time” Across Mammals – Why PND10?
Assumptions:
Mouse gestation length: 18.5 days
Human gestation length: 270 days (~38.5 weeks)
(Workman et. al., 2013)
Including synaptogenesis!
Mouse PND10 equivalent to late third trimester in
terms of neurodevelopment.
16. Critical Windows – Why Post-Natal Day 10?
Significantly increased rates of AXONAL GROWTH and SYNAPTOGENESIS
during the late third trimester of human gestation.
Vertes and Bullmore, 2015
Critical window for cortical wiring of neuronal circuitries and neurotransmitter systems.
Added benefit: offspring nutritional source is maternal lactation.
17. RESULTS
Dopaminergic
dysregulation.
Possible behavioral
consequences
(i.e. hyperactivity).
*Statistically significant main effect of diet. aStatistically significant effect of diet within females.
#Statistical trend for main effect of diet. ^Statistical trend for effect of diet within females.
Prefrontal Cortex: Dopamine
LFD
H
FD
LFD
H
FD
0.0
0.1
0.2
0.3
0.4
Males Females
DA(ng/mgprotein)
*
a
Dorsal Hippocampus: Dopamine
LFD
H
FD
LFD
H
FD
0.000
0.125
0.250
0.375
0.500
Males FemalesDA(ng/mgprotein)
*
^ LFD
HFD
Ventral Hippocampus: Dopamine
LFD
H
FD
LFD
H
FD
0.00
0.75
1.50
2.25
3.00
Males Females
DA(ng/mgprotein)
*
^
Ventral Hippocampus: DOPAC
LFD
H
FD
LFD
H
FD
0.0
0.5
1.0
1.5
2.0
Males Females
DOPAC(ng/mgprotein)
#
18. RESULTS
Serotonergic
dysregulation
(female-restricted)
Possible
communication or
social interaction
deficits.
*Statistically significant main effect of sex. aStatistically significant effect of diet within females.
#Statistical trend for main effect of diet. ^Statistical trend for effect of diet within females.
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)
^
*
Prefrontal Cortex: 5-HIAA
LFD
H
FD
LFD
H
FD
0.0
0.5
1.0
1.5
2.0
Males Females
5-HIAA(ng/mgprotein)
^
Ventral Hippocampus: 5-HT
LFD
H
FD
LFD
H
FD
0
2
4
6
8
Males Females
5-HT(ng/mgprotein)
a
Cerebellum: 5-HT
LFD
H
FD
LFD
H
FD0
2
4
6
8
Males Females
5-HT(ng/mgprotein)
*
^ LFD
HFD
19. RESULTS
Sex-specific
noradrenergic
differences.
*Statistically significant main effect of sex.
#Statistical trend for main effect of sex.
Prefrontal Cortex: NE
LFD
H
FD
LFD
H
FD
0.0
0.5
1.0
1.5
2.0
Males Females
NE(ng/mgprotein)
#
Cerebellum: NE
LFD
H
FD
LFD
H
FD
0
2
4
6
8
Males Females
NE(ng/mgprotein)
*
Dorsal Hippocampus: NE
LFD
H
FD
LFD
H
FD
0.0
0.5
1.0
1.5
2.0
2.5
Males Females
NE(ng/mgprotein)
*
Ventral Hippocampus: NE
LFD
H
FD
LFD
H
FD
0
1
2
3
Males Females
NE(ng/mgprotein)
*
Female:
NE PFC, vHIPP, CER
NE dHIPP
Positive correlation between
female-restricted maternal HFD
effects on 5-HT and female-
specific NE differences.
20. Conclusion
Maternal HFD disrupts monoamine systems at PND10 in mice in a
sex-specific manner, consistent with altered brain neurochemistry
and connectivity.
Data suggests maternal HFD may put offspring on a trajectory
towards ASD.
Future Research:
Monoamine turnover rate analysis via NT/metabolite ratios.
Biomarker analysis of dopaminergic dysfunction in PFC, dHIPP, vHIPP
Western Blot + qPCR (qPCR samples obtained for HIPP)
Integration with neurochemical data at other time points.
Integration with behavioral data and immunologic assessment.
21. General and Funding Acknowledgements
Department of Physiology and Pharmacology
Saritha Krishna John J. Wagner Sadie E. Nennig Nikolay M. Filipov
Department of Infectious Diseases
Donald A. Harn
Department of Foods and Nutrition
Claire B. de La Serre
Miscellaneous
Annika Carter
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.
Funding was also provided by through a grant
from the University of Georgia’s Obesity Initiative
(http://obesity.ovpr.uga.edu).
Editor's Notes
Neurochemical deficits in offspring of dams (mothers) fed high-fat diets.
In particular, we will focus on the effects on brain monoamines.
“Biogenic amines” – AA neurotransmitters
Synaptic transmission – form brain circuits.
Diffuse projections throughout forebrain/striatum
Focus on DA, 5-HT, NE
M. F. Bear, B. W. Connors, M. A. Paradiso, Neuroscience : exploring the brain. (Lippincott Williams & Wilkins, ed. 4, 2015).
Chronic inflammatory condition: multifactorial, mechanisms include adipocyte-sourced pro-inflammatory cytokines, involvement of other pro-inflammatory mediators, and cell rupture and ensuing inflammation.
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This epi study assessed GWG via two methods – the ARCH method was more robust.
Obese trend slightly less strong than overweight trend – suggests threshold of sort.
2014 cohort study re: pre-pregnancy BMI, body size, GWG, and physical activity.
Data collected 2008-2012 via women participating in ARCH1 study in Michigan.
ARCH calculations more robust than birth certificate calculations.
Schlaff RA, Holzman C, Maier KS, Pfeiffer KA, Pivarnik JM. Associations among gestational weight gain, physical activity, and pre-pregnancy body size with varying estimates of pre-pregnancy weight. Midwifery. 2014 11//;30(11):1124-31.
ARCH = Archive for Research on Child Health – study to collect and store record information re: pregnancy, perinatal urine/blood/placenta samples, etc.
Birth certificate information depends on weight from records and hospital variation in such procedures makes it less robust (self-reporting vs. actually weighing etc.)
ARCH calculated BMI from information collected systematically (kg weight / m^2 height upon patient reporting) as well as using weight recorded on birth certificates
2015 publication describing neuropsychiatric risk and maternal obesity factors.
Increased risk of ASD, anxiety/depression, and schizophrenia were associated with increased gestational weight gain.
H. M. Rivera, K. J. Christiansen, E. L. Sullivan, The role of maternal obesity in the risk of neuropsychiatric disorders. Frontiers in Neuroscience 9, 194 (2015).
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Prevalence of autism spectrum disorder among children aged 8 years - autism and developmental disabilities monitoring network, 11 sites, United States, 2010. MMWR Surveill Summ. 2014 Mar 28;63(2):1-21.
https://www.autismspeaks.org/science/science-news/can-rise-autism-be-explained-broadened-diagnosis
Accessed June 25, 2015.
Demonstrates neuroimmunologic implications.
Recall maternal obesity increasing risk for ASD and some of the psychiatric comorbidities listed above.
Chen M-H, Wei H-T, Chen L-C, et al. Autistic spectrum disorder, attention deficit hyperactivity disorder, and psychiatric comorbidities: A nationwide study. Research in Autism Spectrum Disorders. 2015 2//;10(0):1-6.
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OUR EXPERIMENTAL DESIGN INTENDS TO FOCUS IN ON PERINATAL MONOAMINERGIC EFFECTS OF MATERNAL OBESITY using mice models in a controlled experiment.
Tying together information from animal models and epidemiological studies, two key characteristics of autism include altered brain connectivity, ultimately from dysregulation at the level of the synapse, as well as alterations in the innate immune system particularly of a pro-inflammatory nature.
Teasing apart etiological factors for ASD involves identifying genetic and environmental contributions, as well as how their interactions contribute to ASD etiology.
Here we see the narrow-sense heritability for ASD is estimated around 52%, thus environmental factors and gene-environment interactions play roughly an equally important role in ASD. Furthermore, the largest contribution to this 52% is from common genetic variation in the population, with only ~2.6% coming from rare mutations in genes involved with synaptic regulation, among other fundamental intracellular processes (cytoskeletal function, cell adhesion/growth)
The maternal environment, in particular the perinatal intrauterine environment, has emerged as a key environmental factor impacting offspring development and behavior. Furthermore, the role of epigenetics in synaptic and immunologic regulation has also emerged as a key factor through mediating gene-environment interactions. What this means is that an individual’s epigenetic make-up is influenced by the maternal environment and affects neurodevelopment. What about maternal obesity in particular?
Genes involved include those that directly and epigenetically regulate several aspects of synaptic homeostasis and immune pathways, as well as other basic intracellular processes. Some studies suggest involvement of genes involved in toxicant excretion from the CNS, though these overlap with immune pathways. (Rusu et. al., 2015; Medzhitov, 2008).
C. Rusu, C. Preda, A. Sireteanu, C. Vulpoi, RISK FACTORS IN AUTISM SPECTRUM DISORDERS: THE ROLE OF GENETIC, EPIGENETIC, IMMUNE AND ENVIRONMENTAL INTERACTIONS. Environmental Engineering & Management Journal (EEMJ) 14, 901-917 (2015).
R. Medzhitov, Origin and physiological roles of inflammation. Nature 454, 428-435 (2008).
Environmental toxicants
Disruption of perinatal uterine environment leads to programming of offspring metabolically and neurobehaviorally.
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Serotonin and the enteric nervous system may also help explain gastrointestinal alterations in ASD.
Jaiswal P, Mohanakumar KP, Rajamma U. Serotonin mediated immunoregulation and neural functions: Complicity in the aetiology of autism spectrum disorders. Neuroscience & Biobehavioral Reviews. 2015 8//;55(0):413-31.
Dopaminergic dysregulation
Meth-induced DA release
Meth-induced hyperactivity
D1 and D2 mRNA levels
PND21 mice showed no changes in monoamine levels after maternal valproic acid administration on post-conception day 12.5
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.
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Average American diet is roughly 33% kcal from fat (CDC; http://www.cdc.gov/nchs/fastats/diet.htm)
Upon extraction, brains were rinsed with ice-cold Hank’s buffer, sliced midsagitally, and half was later frozen over dry ice and coronally sectioned into 500 micron segments in order to obtain brain regions via micropunches.
DOPAC: 3,4-dihydroxyphenylacetic acid
HVA: homovanillic acid
3-MT: 3-methoxytyramine
5-HT: 5-hydroxytryptamine
5-HIAA: 5-hydroxyindoleacetic acid
MHPG: 3-methoxy-4-hydroxyphenylglycol
Critical window since altered connectivity is seen in autism. Furthermore, post-natal day 10 is prior to weaning (PND21); thus, the offspring’s sole nutritional source is via maternal lactation, allowing us to better factor out environmental impacts on our study.
So the question of why we chose post-natal day 10 in the mouse naturally arises. Looking at critical windows in neurodevelopment, significant increases in rates of axonal growth and synaptogenesis occur in the late third trimester of human gestation. This window is integral for physiologic “wiring” of the brain, including structural formation of cortical regions, as well as functional development, as related to levels of NTs, receptor density in post-synaptic targets, and receptor sensitivity.
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Sullivan EL, Riper KM, Lockard R, Valleau JC. Maternal high-fat diet programming of the neuroendocrine system and behavior. Horm Behav. 2015 Apr 24.
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Monoamine system most affected by maternal HFD: dopaminergic system.
Brain region most affected: ventral hippocampus; least affected: striatum.
Sex-independent increases in DA levels in PFC, dHIPP, vHIPP. Stat. sig in PFC, dHIPP females, trend in vHIPP fema les.
Sex-independent increase in DOPAC in vHIPP – suggests increased DA vHIPP tone at PND10 due to maternal HFD.
MATERNAL HFD EFFECTS:
Sex-independent increased DA levels in the PFC, dHIPP, and vHIPP.
Statistically significant in female PFC and vHIPP.
EFFECTS OF SEX (not shown)
*HVA levels lower in female PFC, higher in female dHIPP.
*Striatal 3-MT levels higher in females.
Both appear to be driven by maternal diet in females (interaction approaching significance).
Maternal HFD leads to disrupted dopaminergic homeostasis in the PND10 mouse, which may relate to ASD-like behaviors involving behavioral dysregulation or hyperactivity.
Female-restricted increases in 5-HT noted in PFC, vHIPP, CER, but specifically not the dHIPP.
Statistical trend for increased 5-HIAA in female PFC, suggests increased serotonergic tone in PFC in female PND10 mice.
MATERNAL HFD EFFECTS:
(i) sex-dependent female-specific increased 5-HT levels.
Statistically significant in female vHIPP, but not the dHIPP*
Statistical trend in female PFC, CER.
(ii) Increased 5-HIAA levels in female PFC (statistically significant).
Suggests increased serotonergic tone in female PND10 PFC.
EFFECTS OF SEX (not shown) – appear to be driven by maternal diet in females.
*5-HT levels higher in female PFC and CER – appear to be driven by diet.
*5-HT levels lower in female dHIPP – statistically and biologically significant.
Maternal HFD leads to disrupted serotonergic homeostasis in the PND10 female mouse, which may relate to ASD-like behaviors involving communication or social deficits.
There were no statistically significant effects of maternal HFD or sex on MHPG levels in PND10 mouse.
Consistent with other studies.
L.-J. Kepser, J. R. Homberg, The neurodevelopmental effects of serotonin: A behavioural perspective. Behavioural Brain Research 277, 3-13 (2015).
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.
Loke YJ, Hannan AJ, Craig JM. The Role of Epigenetic Change in Autism Spectrum Disorders. Frontiers in Neurology. 2015 05/26. 02/28/received. 04/28/accepted;6:107.
Recall the valproic-acid mouse study previously mentioned, where no differences were found in monoamine levels at PND21.
