1. Dopaminergic Animal Models of RLS William Ondo, MD Department of Neurology, Baylor College of Medicine Houston, TX 77030

2. Animal models of restless legs syndrome 6-OHDA lesioned rat model 6-OHDA lesioned and iron deficient mice Dopamine D3 receptor knockout mice (D3 KO) model An animal model of PLMS

3. Restless Legs Syndrome A common neurological disorder with a prevalence up to 10% in general population A sensor motor disorder characterized by an urge to move the limbs especially the legs Symptoms aggravation at rest; improved with movement, and worse at night About 80% RLS patients also suffer from period limb movements of sleep

4. Hypothesis Diencephalic-spinal dopaminergic tracts (A11) are the anatomic substrate for RLS

5. Why? –Sensation: A11 implicated in anti-nociception –Anatomy: spinal tracts would explain prominent leg involvement, but lack of face involvement –Circadian pattern: A11 adjacent to hypothalamic suprachiasmic nucleus –Dopamine response: RLS responds to dopamine but no clinical evidence of other dopaminergic tracts Does not develop parkinsonism (A9) No change in olfaction (A16) No change in vision (A17) No baseline endocrinology abnormalities (A12)

6. Lesser Known Dopamine Systems

7. A11-A14 Cell Bodies in Mice A11A: A12, B: A13, C:A14

8. Dopaminergic circuits originating from the A11 diencephalic cell cluster

9. A11 Spinal Projections A 11 D3 Dorsal Root Ganglion Motor Neuron D1 D1/D2 Intermedial- lateral tract (autonomic)

10. RLS Rat Model Female Sprague-Dawley rats Diencephalic-spinal dopamine tracts (A 11) –28 lesion, 14 sham 6-H-DOPA lesions (50% cell loss) –No loss in A9, A10, A12-A14 Observed in 6 hour segments by blinded rater Ondo et al. Mov Disord 2000;15:154-158. Ondo et al. Mov Disord 2000;15:154-158.

11. A11 Lesioning Sham Lesion 6-OH-Dopa Lesion

12. A11 Lesioned Animals IP pramipexole normalized the activity

13. Iron Deprived and A11 Lesioned Mice 80 C57BL/6 Mice 4 groups: –Lesioned - iron deprived –Lesioned – normal iron –Sham – iron deprived –Sham – normal iron Assessed activity in laser cage apparatus Assessed D2/3 agonists, D1 agonists, D2 antagonist

14. Groups Lesion-IDLesion-conSham-IDSham-con N20 Day 28Iron deprivationRegular diet Iron deprivationRegular diet Day 58-64Locomotors activities test Serum iron measurement Weight measurement 6-OH DA Saline Day 88-94Locomotors activities test Serum iron measurement Weight measurement Behavior observation Day 94-100Drug intervention ( ropinirole, SKF-38393, haloperidol) After Day 100Mice sacrificed and TH immunohistochemstry of diencephalons neurons Brain and spinal cord iron measurement Time course of 6-OHDA lesion and iron deprivation in mice

15. ** P<0.01 vs. shamed-con, ## P<0.01 vs. lesioned-con Total THi neuron number in different regions

16. Number of TH (+) Neurons

17. Serum iron measurement in mice with iron-deprivation and normal diet ** P<0.01 VS ID groups

18. ** P<0.01 vs. shamed-con, ## P<0.01 vs. lesioned-con, && P<0.01 shamed-ID Iron measurement in serum and CNS

19. No Overt Health Issues in Iron Deprived Mice

20. Fe ++ The metabolic pathway of dopamine

21. Projections of Diencephalic Dopamine Neurons into the Spinal Cord in Mice

22. THi diencephalic neurons (400×). A: A11 THi neurons. B: A13 THi neurons. C: A12 THi neurons. D A14 THi neurons. E: A10 THi neurons.

23. A11 THi neurons after 6-OHDA+iron deprivation; B: 6-OHDA lesion alone; C: Iron deprivation; D: Shem control. E. A12 THi neurons. F. A13 THi neurons. G: A8-10 THi neurons. H. A14 THi neurons. THi neurons in diencephalic and mesencephalic regions

24. Locomotor activity in two groups after one month iron deprivation

25. Locomotor activity one month after 6-0HDA lesioning

26. Locomotor activity after one month 6-0H DA lesioning (cont.)

27. “Hornory” Mice

28. Aggressive behaviors in lesioned-ID mice

29. Changes in Total Movements with: A: Ropinirole, B: SKF, C: Haloperidol

30. ** P<0.01 vs. shamed-con, ## P<0.01 vs. lesioned-con, && P<0.01 shamed-ID Locomotor activities changed after ropinirole D2/3 agonist) intervention

31. ** P<0.01 vs. shamed-con, ## P<0.01 vs. lesioned-con, && P<0.01 shamed-ID Locomotor activities changed after SKF (D1 agonist) intervention

32. ** P<0.01 vs. shamed-con, ## P<0.01 vs. lesioned-con &&, P<0.01 shamed-ID Locomotor activities changes after haloperidole (D2 antagonist) intervention

33. Effects of A11 Lesioning and Iron Deprivation on Spinal Dopamine Receptors (Clinical Results)

34. Effects on mRNA and Protein Levels mRNAProtein Levels

36. Acknowledgement Dr. Shen Qu Dr. Xiong Zhang Dr. Hongru Zhao Dr. Wei Dong Le Shelly Rajaseharan –Ondo WG, He Y, Rajasekaran S, Le WD. Clinical Correlates of 6-Hydroxydopamine Injections into A11 Dopaminergic Neurons in Rats: A Possible Model for Restless Legs Syndrome? Mov Disord 2000;15:154-158. –Qu S, Ondo WG, Zheng X, Le W. Projections of Diencephalic Dopamine Neurons into the Spinal Cord in Mice. Exp Brain Res 2006;168:152-156. –Zhao H, Pan T, Xie W, Zhang A, Ondo WG, Le W. Spinal cord dopamine receptor expression and function in mice with 6-OHDA lesion of the A11 nucleus and dietary iron deprivation. J Neurosci Res 2007;85:1065-1076. –Qu S, Le W, Zhang X, Xie W, Zhang A, Ondo WG. Locomotion is increased in A11 lesioned mice with iron deprivation: A possible animal model for restless legs syndrome. J Neuropath Exp Neurology 2007;66(5):383-8. This work was partially supported by GlaxoSmithKline Inc.

37. Bibliography Ondo WG, He Y, Rajasekaran S, Le WD. Clinical Correlates of 6- Hydroxydopamine Injections into A11 Dopaminergic Neurons in Rats: A Possible Model for Restless Legs Syndrome? Mov Disord 2000;15:154-158. Qu S, Ondo WG, Zheng X, Le W. Projections of Diencephalic Dopamine Neurons into the Spinal Cord in Mice. Exp Brain Res 2006;168:152-156. Zhao H, Pan T, Xie W, Zhang A, Ondo WG, Le W. Spinal cord dopamine receptor expression and function in mice with 6-OHDA lesion of the A11 nucleus and dietary iron deprivation. J Neurosci Res 2007;85:1065-1076. Qu S, Le W, Zhang X, Xie W, Zhang A, Ondo WG. Locomotion is increased in A11 lesioned mice with iron deprivation: A possible animal model for restless legs syndrome. J Neuropath Exp Neurology 2007;66(5):383-8.

38. D3 Knockout Mice D3 receptors –Limbic system –Striatum –Dorsal horn of spinal cord A11 D3 knockouts –Increased locomotor activity –Increased spinal reflexes –hypertension Accili D. Proc Nat Acad Sci 1996

39. D3 Antagonism Nafadotride (D3 anataonist) increased locomotion activity and sleep latency SB-2777011 resulted in increased sleep latency but not increased locomotor activity compared to sulpiride –Some change in exploratory behavior Sautel F. J Pharm Exp Ther 1995, Austin N. Xenobiotica 2001

40. Dopamine Antagonist Medicines and PLM Young Rats (1.4-1.6 months) –No PHLM Older rats (16-21 months) –4/10 had some PHLM –2/10 had > 5/hour Haloperidol did not effect PHLM Baier PC. J Neuro Sci 2002, Manconi Nuero Sci 2007

41. Thank You William Ondo, MD