Revised Model of Endocannabinoid Signaling

Cannabinoids

• Medically and traditionally used for thousands of years

• Active compound of marijuana (D9-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 D9-THC are absent in CB1-/- mice

• “CBX” receptor may exist

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)

Depolarization induced suppression of inhibition (DSI)

The First Problem

CannabinoidsEndocannabinoids

Synthetic cannabinoids

Direct modulation of ligand-gated ion channels:

Acetylcholine receptor, Serotonin 5HT3 receptor(frog oocyte experiments)

Receptors

IC50

Reference

AchR - -7

118 nM 2-AG 163 nM Anandamide 1.03 µM CP 55,940 3.2 µM WIN 5521,2

Oz et al., 2004

5-HT3-R 3.7 µM Anandamide Oz et al., 2002

h5-HT3-R >1 µM SR141617A 129.6 nM Anandamide 646,7 nM CP 55,940 103.5 nM WIN 5521,2

Barann et al., 2002

AMPAR GluR-1 GluR-3 GluR-1/3 GluR-2/3

161 µM 143 µM Anandamide 148 µM 241 µM

Akinshola et al., 1999

NMDAR NR1-NR2A

Potentiation

20% 1µM Anandamide max 50% with 10 µM

Hampson et al., 1998

Direct modulation of ligand-gated ion channels:

Acetylcholine receptor, Serotonin 5HT3 receptor(frog oocyte experiments)

Research questions

Do cannabinoids modulate GABAergic synaptic transmission by a direct action on

ionotropic GABAA receptors?

If yes, what does this modulation mean for local neuronal circuits

CB1R agonists modulate currents through recombinant GABAAR

CB1R agonists modulate currents through recombinant GABAAR

Cannabinoids show CB1 receptor independent reduction of GABAA mediated response

Experimental Procedure

Paired recordings from a FS interneuron innervating a pyramidal neuron

Cannabinoids show CB1 receptor independent reduction of GABAA mediated response

Cannabinoids show CB1 receptor independent reduction of GABAA mediated response

Postsynaptic depolarization causes a CB1R-independent suppression of inhibitory synaptic transmission.

Endocannabinoid synthesis and degradation

Diacylglycerol Lipase (DAGL)Monoacylglycerol Lipase (MAGL)Cyclooxygenase-2 (COX-2)Fatty acid amide hydrolase (FAAH)

Anandamide synthesis and degradation

Postsynaptic depolarization causes a CB1R-independent suppression of inhibitory synaptic transmission.

RHC80267 & THL: Diacylglycerol Lipase inhibitorsURB602 Monoacylglycerol Lipase inhibitorNimesulide: Cyclooxygenase-2 inhibitor

Modulation of microcircuit coupling by cannabinoids

The effect of CP on hippocampal CCK-positive interneuron to CA1 pyramidal neuron connections in wild-type, CB1R-/- and GABAAR a2

-/- mice.

Potentiation of extrasynaptic GABAARs by endocannabinoids

Dual effect of cannabinoids on GABAARs

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 GABAA receptors.

2. Suppression of inhibition by a direct modulation of postsynaptic GABAA receptors has a high impact on a neuronal network activity providing a new dimension in cannabinoid signaling.

Golovko Tatiana Heidelberg University Falconer Caroline Dundee University

Min Rogier Lozovaya Natalia Vrije Universiteit AmsterdamBurnashev Nail Institut de Neurobiologie de la Méditerranée

Depolarization induced suppression of inhibition (DSI)

R I Wilson, R A Nicoll Science 2002;296:678-682

Chronic Suppression of Inhibition (CSI)

WT

CB1 KO

Control AM 251

• Chronic suppression of inhibition (CSI) ?

• Pre- or postsynaptic origin?

• Suitable candidate(s) for mediating the effect ?

• Presynaptic calcium regulation?

CCK (CB1R +) interneurons

100 msPre

Post100 pA50 mV

CCK

PYR

CCK

Location Firing pattern

Asynchronous release Recording protocol

20 mV200 ms

Vh=-70 mVPost CA1 pyr

20 pA

CCK

PYR

50 ms

Pre CB1 interneuroneVm=-65 mV

SR/AM

Blocking CB1Rs relieves CSI

IPSCs potentiated in WT but not CB1 KO mice

THL

Blocking 2-AG synthesis has no effect on CSI

2AG

THL

Chelation of presynaptic calcium potentiates IPSCs

CCK CCKCCKPYR PYR PYR

AM251

A presynaptic calcium-dependent process suppresses IPSCs

AM AMEGTA EGTA

Half maximal IPSC potentiation with 0.1 mM BAPTA

Brief extracellular BAPTA application potentiates IPSCs

CCK

BAPTA

10 mM

PYR

CSI time course

AEA/2-AG/CP+

AEA a potential candidate mediating CSI

Presynaptic calcium regulation: frequency dependence

CCKPYR

CB1R

GABAbR

7 s 12 s 30 s 7 s

What : CSIWhere : PresynapticWho : Anandamide

Why:

Perisomatic inhibition

Freund T and Katona, Neuron 2007; 56:33-42

Synaptic inputs

Freund T, TINS 2003; 489-495

.When: In vivo firing patterns

Bolshakov Alex Falconer CarolineCarl Holmgren Dundee University