Marc Turiault

2. Stress and Addiction : Glucocorticoid receptor in dopaminoceptive neurons facilitates cocaine seeking Marc Turiault

3. Negative feed back CRH Behavioral modulation + Hypothalamus Pituitary + ACTH Adrenals Glucocorticoïds Gluconeogenesis Lipolysis Tissue repair Immune response Stress response and glucocorticoïds

4. Enhanced vulnerability to drug of abuse in stressed animals Injection GC/stress : response to psychostimulants Adrenalectomy :  response to psychostimulants MR GR Glucocorticoïds and psychostimulants

5. Glucocorticoids hsp G R hsp transcription transcription AP1 NFkB MR GRE AR transcription transcription Stat5 nGRE Action mecanism of the glucocorticoïd receptor

6. Suppression of behavioral sensitization Flattened dose response function for cocaine self administration GR conditionnelle mutation in mice brain (GRNesCre)

7. Prefrontal cortex VTA Nucleus accumbens mesocorticolimbic system Drugs  DA transmission Glucocorticoïds  impulse activity of midbrain DA neurons Which cells are responsible for the GR effets on vulnerability to cocaine ? Do DA neurons show a different activity without GR expression ?

8. GRNesCre Controls 1.8 Hz 4.4 Hz Computer Audio mon Oscilloscop. 4.6 Hz 2.4 Hz Amplifier 1 sec Hz Hz 0,25 4 µ manipulator 0,2  3 0,15  2 0,1 1 0,05 0 0 Cont GRNesCre Cont GRNesCre VTA DA neuron 1 Burst frequency (Hz) Firing rate (Hz) GR is involved in the control of the impulse activity of VTA DA neurons Frédéric Ambroggi

9. NO GR NO GR NO GR Which cells are responsible for the GR effets on DA activity and cocaïne vulnerability ? Prefrontal cortex VTA Nucleus accumbens mesocorticolimbic system

10. DAT BAC 210kb Cre DAT Gene 5’ Homology Cre 3’ Homology GRloxP loxP loxP NO GR NO GR NO GR GRnull Specific GR inactivation in dopaminergic neurons

11. DATiCre mice DATiCre – R26R mice TH Cre TH X-gal Cre STOP LacZ loxP loxP Specific recombination in DA neurons

12. DATiCre – R26R mice β-Gal DAT Ov GR TH Ov GRloxp/loxp GRDATiCre mice GRDAT-Cre Recombination in VTA DA neurons DATiCre mice iCre TH iCre TH iCre DAT

13. Acquisition Dose response Progressive ratio Noses pokes Injections Break point Active hole - GRDATCre Cocaine (mg/kg/inj) Active hole - Controls GRDATCre Controls Inactive hole - GRDATCre Inactive hole - Controls GR inactivation in DA neurons has noeffect on motivational properties of cocaine

14. 4.4 Hz, 7% Bursts 4.6 Hz 7% Burst GRDATCre GRloxP Burst Frequency (Hz) Firing rate (Hz) NO GR NO GR NO GR GR in DA neurons is not involved in the control of DA impulse activity. ? Prefrontal cortex VTA Nucleus accumbens

15. Cre U r a GRloxP loxP loxP NO GR NO GR NO GR GRnull loxP GR inactivation in post-dopaminergic neurons D1A YAC 140 kb D1 Gene T r p C E N 4 5’ Homology Cre 3’ Homology

16. V et VI Cre striatum GR GR NAc olf. tub. GRloxP/loxP GRD1Cre GR - TH TH GR Striatum VTA Hippocampus GR GR Inactivation in post-synaptic structures of the DA system

17. Acquisition Dose response Progressive ratio Injections Nose-Pokes Days Cocaine (mg/kg/inj) Active hole - GRD1Cre GRD1Cre Active hole - Controls Controls Inactive hole - GRD1Cre Inactive hole - Controls GR inactivation in dopaminoceptive neurons reduces motivational properties of cocaine.

18. 30 days Locomotor activity Locomotor activity GRD1Cre Controls Behavioral sensitization is abolished in absence of GR in dopaminoceptive neurons Test 9 injections (intervals:2 days) Group 1 (control) Saline Saline Group 2 (acute) Saline Cocaine (7,5mg/kg) Group 3 (chronic) CocaineCocaine (15mg/kg) (7,5mg/kg)

19. Locomotor Activity Jour Nuit Elevated Plus Maze Morris Maze Open Field Motor Coordination Jours Day 1 Day 3 Day 2 General behavior of GRD1Cre mice

20. GR in dopaminoceptive neurons control the impulse activity of VTA DA neurons 4,5 4 3,5  3 2,5 2 1,5 1 0,5 0 GRloxP GRD1Cre Burst frequency (Hz) Firing rate (Hz) GRD1Cre GRloxP 3.6 Hz, 20% Bursts 2.4 Hz, 0% Bursts 0,18 0,16 NO GR 0,14  NO GR 0,12 0,10 NO GR 0,08 0,06  1 sec 0,04 0,02 0 GRloxP GRD1Cre Prefrontal cortex VTA Nucleus accumbens

21. -8nA…128 -4nA -2nA -1nA Effect of GR inactivation in dopaminoceptive neurons on the NAcc spiny neurons response to glutamate ? VTA Nucleus accumbens

22. Decreased reactivity to glutamate in a subpopulation of NAc neurons in GRD1Cre mice Controls GRD1Cre

23. Kynurenate GR inactivation in dopaminoceptive cells blocks the excitatory tonus of the NAc on VTA DA neurons. VTA Nucleus accumbens

24. Conclusion Response to cocaine We identified the cells responsible for effects of GR on DA activity Le GR is involved in NAc spiny neurons response to glutamate Le GR controls indirectly DA activity Cellular dissociation of GR effects in anxiety and addiction

25. Publications Turiault M, Ambroggi F, Milet A, Marinelli M, Lemberger T, Deroche-Gammonet V, Piazza PV and Tronche F. Stress and addiction: glucocorticoid receptor facilitates cocaine seeking (Nature Neuroscience, 2009). Turiault M, Parnaudeau S, Milet A, Rouzeau JD, Lazar M, Tronche F. Analysis of dopamine transporter gene expression pattern – Generation of DAT-iCre transgenic mice (FEBS journal, 2007). Lemberger T, Parlato R, Dassesse D, Westphal M, Casanova E, Turiault M, Tronche F, Schiffmann SN, Schutz G. Expression of Cre recombinase in dopaminoceptive neurons. BMC Neurosci. 2007 Jan 3;8:4. Parlato R, Rieker C, Turiault M, Tronche F, Schutz G. Survival of DA neurons is independent of CREM upregulation in absence of CREB.Genesis. 2006 Oct;44(10):454-64. Guijarro C, Rutz S, Rothmaier K, Turiault M, Zhi O, Naumann T, Frotscher M, Tronche F, Jackisch R, Kretz O. Maturation and maintenance of cholinergic medial septum neurons require glucocorticoid receptor signaling. J Neurochem. 2006. May; 97(3):747-58. Karst H, Berger S, Turiault M, Tronche F, Schutz G, Joels M Minéralcorticoïd receptors are indispensable for non genomic modulation of hippocampal glutamate transmission by corticosterone. PNAS. 2005. 102:19204 – 19207. Revest JM, Di Blasi F, Kitchener P, Rouge-Pont F, Desmedt A, Turiault M, Tronche F, Piazza PV .2005. The MAPK pathway and Egr-1 mediate stress-related behavorial effects of glucocorticoïds. Nat Neurosci 8:664-672. Tronche F, Casanova E, Turiault M, Sahly I, Kellendonk C .2002. When reverse genetics meets physiology : the use of site-specific recombinases in mice. FEBS Lett 529:116-121.

26. INSERM, Bordeaux Frédéric Ambroggi Véronique DerocheـGamonet Pier Vincenzo Piazza CNRS, Collège de France Sébastien Parnaudeau Aude Milet Dominique Lazar Hélène Cambier JeanـDenis Rouzeau François Tronche DKFZ, Heidelberg Thomas Lemberger Céline Diaz

28. Etude électrophysiologique pour déterminer le mécanisme impliqué dans l’altération de la réponse au glutamate des neurones épineux du NAc Perspectives Déterminer les gènes différentiellement exprimés : ARNm, protéines (voies de signalisation…)

29. ? ? Cortex préfrontal ATV Noyau accumbens Pallidum ventral Par quel mécanisme le GR influence l’activité dopaminergique ? Quelles afférences de l’ATV sont invalidées en GR chez les souris GRD1Cre ? Dans le cortex préfrontal ? Voie directe Dans le noyau accumbens ? Voie indirecte

30. Contrôles GRD1Cre Fluorogold Cre ? Fluorogold GR 100 100 80 80 % FG+/Cre+ GRD1Cre GRD1Cre 60 % FG+/GR+ 60 40 40 20 20 FluoroGold 0 0 La majorite des neurones D1 qui projettent à l’ATV expriment le GR Invalidation dans le cortex préfrontal ? Cortex préfrontal ATV Noyau accumbens Pallidum ventral

31. Contrôles GRD1Cre Fluorogold Cre Fluorogold GR 100 100 ? 80 % FG+/Cre+ 80 60 % FG+/GR+ 60 40 40 * 20 FluoroGold 20 0 GRD1Cre GRD1Cre 0 Cortex préfrontal ATV Noyau accumbens Pallidum ventral La majorité des neurones du NAc qui projettent directement à l’ATV n’expriment plus le GR Invalidation dans les neurones de la voie directe NAc-ATV ?

32. Contrôles GRD1Cre Fluorogold Cre 100 100 80 80 Fluorogold GR % FG+/Cre+ % FG+/GR+ 60 60 40 40 20 20 ? 0 0 GRD1Cre GRD1Cre FluoroGold Cortex préfrontal ATV Noyau accumbens Pallidum ventral Les neurones de la voie indirecte NAc-PV-ATV présentent un faible taux d’invalidation du GR Invalidation dans les neurones de la voie indirecte NAc-PV-ATV ?