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”

Outline of Presentation

• Background

• Rationale

• Hypothesis

• Approach

• Behavioral Dysfunction

• Neuronal Dysfunction

• Dysfunction in Developmental

“MET” Expression

• Model & Conclusions

Benzo(a)Pyrene

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

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)

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.

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

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

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

*

*

Schedule-Controlled Operant Task(Behavioral Learning)

*

*

* *

Correlate: Deficits in Offspring Learning Behavior

*

*

Wormley et al., (2004) Tox. Appl. Pharm. 197 (1) 49-65.

*

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.

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

B(a)P-exposed offspring mice exhibit robust deficits in executive function

Hood et al., (2006) NeuroToxicology

Learning and Memory Correlate (Whisker-to-S1 Cortex)

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

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

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.

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.

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

Prenatal insult during cortical neurogenesis…Prenatal insult during cortical neurogenesis…

MET GluR1subunit

NR2Bsubunit

XRE

B(a)P Metabolites

E14-17 Synapse in S1 Cortex

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…

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.

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

Acknowledgements

Alliance for Research Training in Neuroscience

Gestational Toxicant Exposure

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)

Bench Bedside

Epidemiology (Impacted

Communities)

Bench

Translational Research

Translational Research

Environmental Toxicology

Use epidemiology studies to informdesign of molecular studies of neurological dysfunction

Therapeutics

Recording from the Hippocampus

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

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

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

Schedules of Reinforcement

Schedules of Reinforcement, Las Vegas Style

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 ”