Darryl B. Hood, Ph.D. Department of Neurobiology and Neurotoxicology

1. “Molecular Dysfunction Following Environmental Intoxication During Gestation” Darryl B. Hood, Ph.D. Department of Neurobiology and Neurotoxicology Center for Molecular and Behavioral Neuroscience

3. Benzo(a)Pyrene

4. 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

5. 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)

6. 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 forthe first time.

7. B(a)P Exposure Approach: Susceptibility-Exposure Paradigm GD 0 GD 5 GD 10 GD 15 GD 20 GD 21 Insemination Birth-Rat Birth-Mice Entorhinal /Dentate Gyrus, Cortical Neurogenesis Neuronal Differentiation Down-regulation of Early Developmental Glutamatergic Receptor Subunit Expression Hood et al., 2006; Brown et al., 2007; McCallister et al., 2008 Behavioral Deficit Phenotypes Wormley et al., 2004; Sheng et al., 2008 Onset of Hearing Eye Opening PND 0 PND 10 PND 20 PND 30 PND 60 PND 65 PND 70 Weaning Cortical Synapse Consolidation

8. Exposed Cpr Dam and Offspring have a Similar Composition of B(a)P Metabolites Total Metabolites (%) DamPup PND3 (microsomes)(whole brain)

9. Learning and Memory Correlate (LTP) Deficits in Offspring Plasticity Mechanisms- Hippocampal 275 250 225 200 175 150 Population Spike Amplitude (% Pre-Tetanus) 125 100 75 50 (100Hz, 1s, 3x) 25 0 0 10 20 30 40 50 60 70 80 90 100 Time (min) TCDD Control TCDD/B(a)P B(a)P * * * * * Wormley et al., (2004) Tox. Appl. Pharm. 197 (1) 49-65.

10. Schedule-Controlled Operant Task(Behavioral Learning)

11. Correlate: Deficits in Offspring Learning Behavior * * * * * * * Wormley et al., (2004) Tox. Appl. Pharm. 197 (1) 49-65.

12. 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.

13. B(a)P-exposed Cpr offspring exhibit robust deficits in executive function

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

15. Learning and Memory Correlate (Whisker-to-S1 Cortex) Hood et al., (2006) NeuroToxicology

16. Deficits in Offspring Cortical Neuronal Activity [150mg/kg BW B(a)P] S1 Cortex Control Offspring NMDA Dependent Responses Short Latency AMPA Dependent Responses S1 Cortex B(a)P-Exposed Offspring McCallister et al., 2008

17. Reduced Expression of Glutamatergic Markers in Primary Cortical Neuronal Cultures from B(a)P-exposed Cpr Offspring Exposure during Neurogenesis ; Analysis during Synaptogenesis c c c a a a b b e e e Control Primary Mouse Cortical Neuronal Culture (D7) c a b d a b b d c g g e e e f f f h h h “ex vivo” B(a)P-exposed Primary Mouse Cortical Neuronal Culture (D7) e f g h g e f h MAP2 NR2B Co-localization GluR1 Sheng et al., (2008) In Preparation

18. MET is a pleiotropic receptor and contributes to cortical development In mature cortical neurons, MET signaling:: augments NMDA currents, enhances synaptic long-term potentiation and contributes to glutamatergic synapse formation. MET and NMDA Co-localize at Synapses Tyndall & Walikonis. 2006. Cell Cycle

19. HGF induces increased expression of NR2B and GluR1 during cortical development • 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. Tyndall & Walikonis. 2006. Cell Cycle

20. Offspring MET Developmental Protein Expression is Downegulated in B(a)P-exposed Cpr offspring MET b-Actin 1.4 MET/b-Actin WT Cpr150mg/kg Cpr 300mg/kg Cpr

21. Prenatal insult during cortical neurogenesis… GluR1 subunit NR2B subunit MET B(a)P Metabolites XRE E14-17 Synapse in S1 Cortex

22. …leads to postnatal deficits in… in vivo Neuronal Activity and Behavior MET NR2B and GluR11 mRNA and protein NR2B subunit GluR1 subunit Postnatal Synaptogenesis in Layer 3 S1 Cortex MET

23. 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. CONCLUSIONS

24. Acknowledgements Meharry Vanderbilt Tultul Nayyar Ford Ebner Jie Wu Letha Woods Tianxiang Tu Mark Maguire Deanna Wormley Bill Valentine SaLynn Johnson K. Amernath La’Nissa Brown Anthony Archibong Miki Aschner Aramandla Ramesh Dan Campbell Sheng LiuFP Guengerich Habibeh KhoshboueiPat Levitt Lee E. Limbird RRO3032 (NCRR) ES014156 (NIEHS) Clivel G. Charlton NS041071 (NINDS) Center for Molecular and Behavioral Neuroscience

25. Acknowledgements Alliance for Research Training in Neuroscience

26. Gestational Toxicant Exposure

27. Short Latency AMPA Dependent Response Deficits in Offspring Cortical Neuronal Activity (700ng/kg BW TCDD) NMDA Dependent Responses Evoked Activity * * * * Hood et al., (2006) Neurotoxicology

28. Translational Research Bench Bedside Therapeutics Environmental Toxicology Translational Research Epidemiology (Impacted Communities) Bench Use epidemiology studies to inform design of molecular studies of neurological dysfunction

29. Recording from the Hippocampus

30. Mechanistic Model of Prenatal B(a)P Exposure Effects on Offspring Neuronal Activity and Behavior Deficits in in vivo Neuronal Activity and Behavior AMPA Glu Glu Glu NMDA Glu Glu Glu Glu MET NR2B and GluR1 mRNA and protein Glu siRNAMET ARNT Glu Glu Glu B(a)P Metabolites B(a)P AhR Glu XRE WT Cpr + (brain/liver-Cpr-null) Glial Cell E14-17 Synapse

31. Prenatal Exposure Effects on Postnatal (PND15) MET Protein Expression in WT and B(a)P-exposed Cpr offspring MET 1.4 b-Actin 1.2 1.0 MET/b-Actin 1.4 0.7 0.35 0.0 WTCpr150mg/kg Cpr 300mg/kg Cpr COntrol Sheng et al.,(2008) in preparation

32. B(a)P-induced Reductions in the Magnitude of Inward Currents in ex vivo Primary Cortical Neuronal Cultures Control “ex vivo” Primary Mouse Cortical Neuronal Culture (Day 3) a 20 Cortical neurons from in utero B(a)P- exposed offspring (250mg/kg BW) Cortical neurons from in utero Control offspring -100 -80 -60 -40 -20 20 (mV) Maximum Current At -100 mV pA MAP2 -20 -10 -40 (pA) -20 B(a)P-exposed “ex vivo” Primary Mouse Cortical Neuronal Culture (Day 3) e 10 pA -30 250 mSec MAP2 Offspring control cortical neuronal culture B(a)P-exposed offspring cortical neuronalculture Sheng et al.,2008, in preparation

33. Schedules of Reinforcement

34. Schedules of Reinforcement, Las Vegas Style

35. “Prenatal Environmental Exposures and Early Childhood Development ” PAH Exposure + ETS Autism Spectrum Disorder