darryl b. hood, ph.d. department of neurobiology and neurotoxicology center for molecular and...
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Darryl B. Hood, Ph.D.Darryl B. Hood, Ph.D.Department of Neurobiology and NeurotoxicologyDepartment of Neurobiology and Neurotoxicology
Center for Molecular and Behavioral Neuroscience Center for Molecular and Behavioral Neuroscience
““Molecular Dysfunction Following Environmental Molecular Dysfunction Following Environmental Intoxication During Gestation”Intoxication During Gestation”
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Outline of Presentation
• Background
• Rationale
• Hypothesis
• Approach
• Behavioral Dysfunction
• Neuronal Dysfunction
• Dysfunction in Developmental
“MET” Expression
• Model & Conclusions
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Benzo(a)Pyrene
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Benzo(a)pyrene
Polycyclic Aromatic Hydrocarbon (prototype)
•Released as a result of combustion processes •AhR Agonist•1º exposure routes are oral and inhalation•Peripheral neuropathies •Neurobehavioral deficits •Decline in MDI- Perera et al., 2006•Dysregulates MET temporal developmental expression
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For the first time, results presented demonstrate that in utero exposure to PAH affects early
cognitive development
Perera, et al., Environ Health Perspect 114:1287-1292 (2006)
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HYPOTHESIS
Prenatal exposure to B(a)P produces behavioral learning and memory deficits mediated through downregulation of developmental glutamatergic receptor subunit expression at a time when synapses are being formed for the first time.
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Approach:Susceptibility-Exposure Paradigm
Insemination Birth-Rat
PND 70
Entorhinal /Dentate Gyrus, Cortical Neurogenesis
GD 0 GD 5 GD 10 GD 15 GD 20 GD 21
Neuronal Differentiation
PND 0 PND 10 PND 20 PND 30 PND 60 PND 65
Eye OpeningOnset of Hearing
Weaning
Down-regulation of Early Developmental Glutamatergic Receptor Subunit Expression
Hood et al., 2006; Brown et al., 2007; McCallister et al., 2008
B(a)P Exposure
Behavioral Deficit Phenotypes
Wormley et al., 2004; Sheng et al., 2008
Birth-Mice
Cortical Synapse Consolidation
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0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
3(OH)Bap
9(OH)BaP
7,8-diol
4,5-diol
9,10-diol
Dam Pup PND3(microsomes) (whole brain)
To
tal
Met
abo
lite
s (%
)
Exposed Cpr Dam and Offspring have a Similar Composition of B(a)P Metabolites
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Control
B(a)P
TCDD
TCDD/B(a)P
Time (min)
0 10 20 30 40 50 60 70 80 90 100
0
25
50
75
100
125
150
175
200
225
250
275
Po
pu
lati
on
Sp
ike
Am
plit
ud
e(%
Pre
-Tet
anu
s)
(100Hz, 1s, 3x)
*
**
Wormley et al., (2004) Tox. Appl. Pharm. 197 (1) 49-65.
Learning and Memory Correlate (LTP)Deficits in Offspring Plasticity Mechanisms- Hippocampal
*
*
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Schedule-Controlled Operant Task(Behavioral Learning)
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*
*
* *
Correlate: Deficits in Offspring Learning Behavior
*
*
Wormley et al., (2004) Tox. Appl. Pharm. 197 (1) 49-65.
*
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Behavioral Correlate2-Choice Novel Object Recognition Task
•A test of executive function and requires more cognitive skills from the animal.
•Task measures exploration of novel environments or a single novel object.
•In order to discriminate between a novel and a familiar object, the animal must first attend to two identical objects and keep the two objects in working memory.
• Upon replacement of one of the familiar objects with a novel object, the animal should display differential behavior directed towards the novel object.
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Control 150 mg/kg 250 mg/kg-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5N
ovel
ty In
dex
B(a)P-exposed Cpr offspring exhibit robust deficits in executive function
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B(a)P-exposed offspring mice exhibit robust deficits in executive function
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Hood et al., (2006) NeuroToxicology
Learning and Memory Correlate (Whisker-to-S1 Cortex)
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Deficits in Offspring Cortical Neuronal Activity
[150mg/kg BW B(a)P]
0
5
10
15
20
25
30
1 to 10 11 to 20 21 to 30 31 to 40
Time Post-Stimulus (ms)
Sp
ikes
Evo
ked
/50
Sti
mu
li
Control
B(a)P exposed
S1 Cortex Control Offspring
S1 Cortex B(a)P-ExposedOffspring
Short Latency AMPA Dependent Responses
NMDA Dependent Responses
McCallister et al., 2008
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Reduced Expression of Glutamatergic Markers in Primary Cortical Neuronal Cultures from B(a)P-exposed
Cpr Offspring
a b c e
e f g h
a b c e
e f g h
a
b
c e
e f h
a b
c
d
e f g h
Control PrimaryMouseCorticalNeuronalCulture (D7)
“ex vivo” B(a)P-exposed Primary Mouse Cortical Neuronal Culture (D7)
MAP2 GluR1 NR2B Co-localization
a b c d
e f g h
Sheng et al., (2008) In Preparation
Exposure during Neurogenesis ; Analysis during Synaptogenesis
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MET is a pleiotropic receptor and contributes to cortical development
MET and NMDA Co-localize at Synapses
Tyndall & Walikonis. 2006. Cell Cycle
In mature cortical neurons, MET signaling::augments NMDA currents, enhances synaptic long-term potentiation and contributes to glutamatergic synapse formation.
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HGF induces increased expression of NR2B and GluR1 during cortical development
Tyndall & Walikonis. 2006. Cell Cycle
In mature cortical neurons, MET signaling
• augments NMDA currents, •enhances synaptic LTP and •contributes to glutamatergic synapse formation.
Therefore, the regulation of MET expression in development is critical.
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Offspring MET Developmental Protein Expression is Downegulated in B(a)P-exposed Cpr offspring
1.4
ME
T/
-Act
inMET
-Actin
00.20.40.60.8
11.21.41.61.8
2
Control 150 300
PND0
PND5
PND10
PND15
WT Cpr 150mg/kg Cpr 300mg/kg Cpr
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Prenatal insult during cortical neurogenesis…Prenatal insult during cortical neurogenesis…
MET GluR1subunit
NR2Bsubunit
XRE
B(a)P Metabolites
E14-17 Synapse in S1 Cortex
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MET
GluR1subunit
NR2Bsubunit
MET NR2B and GluR11mRNA and protein
in vivo Neuronal Activity and Behavior
Postnatal Synaptogenesis in Layer 3 S1 Cortex
……leads to postnatal deficits in…leads to postnatal deficits in…
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CONCLUSIONS
• Prenatal exposure to environmental contaminants causes modulation of developmental glutamate receptor subunit expression.
• Prenatal exposure to these environmental contaminants causes decrements neuronal activity.
• Prenatal exposure to environmental contaminants causes behavioral deficits.
• Current studies are directed at selective knockdown of upstream targets to produce offspring with phenotypes that exhibit robust behavioral deficits.
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AcknowledgementsMeharry Vanderbilt
Tultul Nayyar Ford EbnerJie Wu Letha WoodsTianxiang Tu Mark MaguireDeanna Wormley Bill ValentineSaLynn Johnson K. AmernathLa’Nissa BrownAnthony Archibong Miki AschnerAramandla Ramesh Dan CampbellSheng Liu FP
GuengerichHabibeh Khoshbouei Pat LevittLee E. Limbird
RRO3032 (NCRR)ES014156 (NIEHS)
Clivel G. Charlton NS041071 (NINDS)
Center for Molecular and BehavioralNeuroscience
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Acknowledgements
Alliance for Research Training in Neuroscience
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Gestational Toxicant Exposure
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0
2
4
6
8
10
12
14
16
3-10ms 10-20ms 20-30ms 30-40ms 40-50ms 50-60ms 60-70ms 70-80ms 80-90ms 90-100ms
Time Post-Stimulus(ms)
Sp
ikes
Evo
ked
/50
Sti
mu
li
*
Short Latency AMPA Dependent Response
NMDA Dependent Responses
Evoked Activity
Hood et al., (2006) Neurotoxicology
** *
Deficits in Offspring Cortical Neuronal Activity(700ng/kg BW TCDD)
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Bench Bedside
Epidemiology (Impacted
Communities)
Bench
Translational Research
Translational Research
Environmental Toxicology
Use epidemiology studies to informdesign of molecular studies of neurological dysfunction
Therapeutics
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Recording from the Hippocampus
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Glu
Glial Cell
GluGlu
Glu
Glu
Glu
Glu
NMDA
AMPA
AhR
Glu
Glu
Glu
Glu
Glu
Deficits in in vivo Neuronal Activity and
Behavior
siRNAMETARNT
XRE
B(a)P MetabolitesB(a)P
WT Cpr + (brain/liver-Cpr-null)
MET NR2B and GluR1mRNA and protein
E14-17 Synapse
Mechanistic Model of Prenatal B(a)P Exposure Mechanistic Model of Prenatal B(a)P Exposure Effects on Offspring Neuronal Activity and BehaviorEffects on Offspring Neuronal Activity and Behavior
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Prenatal Exposure Effects on Postnatal (PND15) MET Protein Expression
in WT and B(a)P-exposed Cpr offspring
Sheng et al.,(2008) in preparation
COntrol WTCpr 150mg/kg Cpr 300mg/kg Cpr
1.4
1.4
0.7
1.2
0.35
1.0
0.0
ME
T/
-Act
in
MET
-Actin
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Maximum Current At -100 mV
-30
-20
-10
pA
Offspring control cortical neuronal culture
(mV)
-100 -80 -60 -40 -20 20
(pA
)
-40
-20
20Cortical neurons from in utero Control offspring
B(a)P-exposed offspring cortical neuronalculture
Cortical neurons from in utero B(a)P- exposed offspring (250mg/kg BW)
250 mSec
10 p
A
B(a)P-induced ReductionsB(a)P-induced Reductions in the Magnitude of in the Magnitude of Inward Inward CurrentsCurrents in in ex vivoex vivo Primary Cortical Neuronal Cultures Primary Cortical Neuronal Cultures
Control“ex vivo” Primary
MouseCortical
NeuronalCulture (Day 3)
B(a)P-exposed“ex vivo” Primary MouseCorticalNeuronal Culture (Day 3)
a
e
MAP2
MAP2
Sheng et al., 2008, in preparation
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Schedules of Reinforcement
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Schedules of Reinforcement, Las Vegas Style
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ARCHMeharry Medical College-Vanderbilt University
Advanced Research Cooperation in Environmental Health (ARCH) ConsortiumA Research Program in the Center for Molecular and Behavioral Neuroscience
Department of Neurobiology and Neurotoxicology
Autism Spectrum DisorderPAH Exposure + ETS
““Prenatal Environmental Exposures and Early Childhood Prenatal Environmental Exposures and Early Childhood Development ”Development ”