1. Revised Model of Endocannabinoid Signaling

2. Cannabinoids Medically and traditionally used for thousands of years Active compound of marijuana (D 9 -THC) identified in 1964 Brain cannabinoid receptor (CB1) identified in 1990. CB1 is one of the most abundant G-protein coupled receptors in the brain Another receptor (CB2) is absent in brain, but enriched in immune tissues Most (but not all) effects of D 9 -THC are absent in CB1 -/- mice “CBX” receptor may exist

3. Cannabinoids At central synapses (hippocampus, cerebellum, neocortex), cannabinoids are released in an activity dependent way, and inhibit presynaptic neurotransmitter release (Depolarization induced Supression of Inhibition/Excitation)

4. Depolarization induced suppression of inhibition (DSI)

5. The First Problem

6. Cannabinoids Endocannabinoids Synthetic cannabinoids

7. Direct modulation of ligand-gated ion channels: Acetylcholine receptor, Serotonin 5HT 3 receptor (frog oocyte experiments)

8. Direct modulation of ligand-gated ion channels: Acetylcholine receptor, Serotonin 5HT 3 receptor (frog oocyte experiments)

9. Research questions Do cannabinoids modulate GABAergic synaptic transmission by a direct action on ionotropic GABA A receptors? If yes, what does this modulation mean for local neuronal circuits

10. CB 1 R agonists modulate currents through recombinant GABA A R

12. Cannabinoids show CB1 receptor independent reduction of GABA A mediated response

13. Experimental Procedure Paired recordings from a FS interneuron innervating a pyramidal neuron

14. Cannabinoids show CB1 receptor independent reduction of GABA A mediated response

16. Postsynaptic depolarization causes a CB 1 R-independent suppression of inhibitory synaptic transmission.

17. Endocannabinoid synthesis and degradation Diacylglycerol Lipase (DAGL) Monoacylglycerol Lipase (MAGL) Cyclooxygenase-2 (COX-2) Fatty acid amide hydrolase (FAAH)

18. Anandamide synthesis and degradation

19. Postsynaptic depolarization causes a CB 1 R-independent suppression of inhibitory synaptic transmission. RHC80267 & THL: Diacylglycerol Lipase inhibitors URB602 Monoacylglycerol Lipase inhibitor Nimesulide: Cyclooxygenase-2 inhibitor

20. Modulation of microcircuit coupling by cannabinoids

21. The effect of CP on hippocampal CCK-positive interneuron to CA1 pyramidal neuron connections in wild-type, CB 1 R -/- and GABA A R a 2 -/- mice.

22. Potentiation of extrasynaptic GABA A Rs by endocannabinoids

23. Dual effect of cannabinoids on GABA A Rs

24. Conclusions These results indicate that: 1.Cannabinoids in addition to the presynaptic (retrograde) mode of action, can suppress inhibition by a direct modulation of postsynaptic GABA A receptors. 2. Suppression of inhibition by a direct modulation of postsynaptic GABA A receptors has a high impact on a neuronal network activity providing a new dimension in cannabinoid signaling.

25. Golovko Tatiana Heidelberg University Falconer Caroline Dundee University Min Rogier Lozovaya NataliaVrije Universiteit Amsterdam Burnashev NailInstitut de Neurobiologie de la Méditerranée

26. Depolarization induced suppression of inhibition (DSI) R I Wilson, R A Nicoll Science 2002;296:678-682

27. Chronic Suppression of Inhibition (CSI) WT CB1 KO Control AM 251

28. Chronic suppression of inhibition (CSI) ? Pre- or postsynaptic origin? Suitable candidate(s) for mediating the effect ? Presynaptic calcium regulation?

29. CCK (CB1R +) interneurons CCK Location Firing pattern Asynchronous release Recording protocol 20 mV 200 ms

30. SR/AM Blocking CB1Rs relieves CSI

31. IPSCs potentiated in WT but not CB1 KO mice

32. THL Blocking 2-AG synthesis has no effect on CSI 2AG THL

33. Chelation of presynaptic calcium potentiates IPSCs CCK PYR AM251

34. A presynaptic calcium-dependent process suppresses IPSCs AM EGTA

35. Half maximal IPSC potentiation with 0.1 mM BAPTA

36. Brief extracellular BAPTA application potentiates IPSCs CCK BAPTA 10 mM PYR

37. CSI time course AEA/2-AG/CP +

38. AEA a potential candidate mediating CSI

39. Presynaptic calcium regulation: frequency dependence CCK PYR CB1R GABAbR 7 s12 s30 s7 s

40. What : CSI Where : Presynaptic Who : Anandamide Why:

41. Perisomatic inhibition Freund T and Katona, Neuron 2007; 56:33-42

42. Synaptic inputs Freund T, TINS 2003; 489-495

43. . When: In vivo firing patterns

44. Bolshakov Alex Falconer Caroline Carl HolmgrenDundee University