Drug-evoked synaptic plasticity of GABAB receptor signaling in the ventral tegmental area

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Drug-evoked synaptic plasticity of GABAB receptor signaling in the ventral tegmental area

LALIVE D'EPINAY, Arnaud

Abstract

Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABAB receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir3) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABABR-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABABRs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABAB1 and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABAB2, a site implicated in regulating GABABR surface expression.

Inhibition of protein phosphatases recovered GABABR-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABABR signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the [...]

LALIVE D'EPINAY, Arnaud. Drug-evoked synaptic plasticity of GABAB receptor

signaling in the ventral tegmental area. Thèse de doctorat : Univ. Genève et Lausanne, 2012, no. Neur. 85

URN : urn:nbn:ch:unige-219889

DOI : 10.13097/archive-ouverte/unige:21988

Available at:

http://archive-ouverte.unige.ch/unige:21988

Disclaimer: layout of this document may differ from the published version.

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FACULTE DES SCIENCES

DOCTORAT EN NEUROSCIENCES des Universités de Genève

et de Lausanne

UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Professeur Christian Lüscher, directeur de thèse

TITRE DE LA THESE

DRUG-EVOKED SYNAPTIC PLASTICITY OF GABAB RECEPTOR SIGNALING IN THE VENTRAL TEGMENTAL AREA

THESE Présentée à la Faculté des Sciences de l’Université de Genève

pour obtenir le grade de Docteur en Neurosciences

par

Arnaud LALIVE D’EPINAY de Genève

Thèse N° 85 Genève

Editeur ou imprimeur : Université de Genève 2012

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Acknowledgements

I would like to thank my thesis supervisor Christian Lüscher for passing on to me the concepts of objective and constructive scientific thinking and experimentation, for making the lab a stimulating working environment but also a place to meet and share with people, and for all the support regarding my future projects.

I would like to thank Paul Slesinger for his encouragement towards publication and his kindness and support throughout my PhD, especially during the last experiments.

I would like to thank Prof. Bernhard Bettler for accepting to join the jury of my PhD thesis and for useful discussions about my results.

I would like to thank Prof. Anthony Holtmaat for constructive feedback as a member of the jury and Prof. Ivan Rodriguez for accepting (again !) to join the jury.

I am extremely grateful to my past and present labmates for being a constant and inhexaustible source of support (Camilla, Manu, Kelly, Matt, Eoin, Cyril, Gwen, Vincent, TiFei, Marc, Clio, Christina), Françoise for all the help with technical preparations, Ancilla for bringing laughter around everyday and Catherine for making sure I get my refunds!

Finally I would like to thank Christelle, my family and my friends for always encouraging me in my choices, pushing me to question myself, supporting me throughout my PhD, and much more.

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Summary

Addiction is a brain disease defined as compulsive substance intake despite negative consequences, and characterized by the risk of relapse after a prolonged period of abstinence. On average, the disease develops only in 20% of all drug consumers, and symptoms are only observed after repetitive drug use. However, a single drug exposure is sufficient to modify neuronal networks and switch the brain to a permissive state that may allow the development of addiction upon repeated drug intake.

Addictive drugs target the ventral tegmental area (VTA), a core nucleus of the reward system, and evoke synaptic plasticity. This region is composed of two main subpopulations, which are dopamine (DA) and !–Amunobutyric acid (GABA) neurons. GABA neurons form synapses onto DA cells and maintain a brake on their activity. Many studies have revealed changes of glutamate or GABAA receptor mediated transmission onto VTA dopamine (DA) neurons after drug exposure. However, little is known about psychostimulant-evoked plasticity of the GABAB receptor, which mediates slow inhibition and thereby contributes to the control of excitability of both VTA DA and GABA neurons. Here we report that a single ip injection of psychostimulant like methamphetamine (METH) or cocaine depresses GABAB

receptor signaling selectively in GABA cells of the VTA.

GABAB receptors are Gi/o protein-coupled receptors (GPCR) activated by GABA and coupling to different effectors depending on their cellular location. When expressed perisynaptically on dendrites (postsynaptic receptor), they activate G protein coupled inwardly-rectifying K+ channels (GIRK) and hyperpolarize the cell. In contrast, when located at synaptic terminals (presynaptic receptor), they inhibit voltage-gated Ca2+ channels (VGCC) and decrease neurotransmitter release. 24h after a single intraperitoneum drug injection, postsynaptic GABAB receptor currents were depressed, and presynaptic receptor sensitivity was decreased in VTA GABA cells. Quantitative immunogold electron microscopy revealed internalization of GABAB1 subunit and GIRK2, which occurred coincident with dephosphorylation of serine (S) 783 in GABAB2 subunit, a site implicated in regulating GABAB receptor surface expression. Inhibition of protein

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phosphatases recovered GABAB-GIRK currents in VTA GABA neurons of METH-injected mice. This novel psychostimulant-evoked depression of GABAB receptor signaling removes an intrinsic brake on GABA neurons activity and potentially increasing inhibition onto DA neurons. This adaptation may reflect a compensatory mechanism to the strengthening of excitation occurring at the same time in the VTA.

Our results demonstrate that GABAB receptor signaling rapidly adapts to drugs of abuse and opens discussion regarding their contribution to the development of addiction. Understanding how these early changes shape the brain’s response to upcoming drug exposure is crucial to the comprehension of neural bases of addiction.

Résumé en français

L’addiction est une maladie du cerveau définie comme un abus compulsif de substances malgré des conséquences négatives, et caractérisée par le risque de rechute après une période prolongée d’abstinence. En moyenne, l’addiction ne se développe que chez 20% des consommateurs de drogue, toutes substances confondues, et les symptômes n’apparaissent qu’après plusieurs expositions à la drogue. Cependant, une seule prise suffit à modifier le fonctionnement de certains réseaux neuronaux et induire un état permissif qui, si la prise de drogue est réitérée, permet des adaptations cellulaires plus profondes et le développement de l’addiction.

Les drogues addictives agissent sur l’aire ventrale tegmentale (VTA), un noyau central du système de récompense, et induisent une certaine plasticité synaptique. Cette région est composée principalement de deux types cellulaires : les neurones dopaminergiques (DA) et les neurones GABAergiques (!–Amunobutyric acid, GABA). Les neurones GABA forment des synapses sur les neurones DA et maintiennent ainsi un frein sur leur activité. De nombreuses études ont révélé des changements dans la transmission excitatrice (via les récepteurs au glutamate), et inhibitrice rapide (via le récepteur GABAA), après une injection de drogue. Cependant, les adaptations plastiques suite à une prise de drogue addictive du récepteur

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GABAB, responsable de l’inhibition lente et impliqué dans le contrôle de l’activité des neurones de la VTA, demeurent majoritairement méconnues.

Dans ce travail de thèse, nous avons découvert qu’une unique injection de psychostimulant comme la méthamphetamine (METH) ou la cocaïne réduit la transmission du récepteur GABAB de manière sélective dans les neurones GABA de la VTA.

Le récepteur GABAB est couplé par une protéine G de type i/o à différents effecteurs, selon sa localisation cellulaire. Dans les dendrites et compartiments postsynaptiques, il active les canaux potassiques rectifiants entrants dépendants des proteines G (GIRK) qui hyperpolarisent la cellule.

Dans les terminaux des axones, compartiments présynaptiques, il inhibe les canaux calciques dépendants du voltage et diminue la libération de neurotransmetteur. 24 heures après une injection intrapéritoine de drogue, les courants postsynaptiques du récepteur GABAB sont réduits, et la sensibilité des récepteurs présynaptiques est diminuée dans les neurones GABA de la VTA. La quantification d’immunoparticules d’or par microscopie électronique révèle que les sous-unités GABAB1 et GIRK2 sont internalisées, et des analyses biochimiques montrent une diminution du niveau de phosphorylation de la sérine 783 de la sous-unité GABAB2, un site impliqué dans la régulation de l’expression du récepteur GABAB à la surface des neurones. En effet, l’inhibition de protéines phosphatases rétablit la transmission basale GABAB- GIRK dans les neurones GABA de la VTA d’animaux traités à la drogue.

Cette altération de la transmission du récepteur GABAB supprime un frein potentiel à l’activité des neurones GABA et pourrait mener à un renforcement de l’inhibition sur les cellules DA. Il pourrait s’agir d’un mécanisme de compensation face à l’augmentation concomitante de la transmission excitatrice dans la VTA.

Nos résultats d’une part démontrent que dans leur adaptation aux drogues, les réseaux neuronaux modifient de manière spécifique la transmission du récepteur GABAB, et d’autre part fournissent de nouveaux éléments quant à la contribution de ce récepteur au développement de l’addiction. L’étude de ces adaptations synaptiques rapides suite à la première prise de drogue est cruciale pour la mise en place d’un modèle et la compréhension des bases neuronales de l’addiction.

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List of abbreviations

AAV adeno-associated virus

AMPA 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid AMPK 5’adenosine monophosphate-dependent protein kinase CaMKII Ca2+/calmodulin-dependent protein kinase II

cAMP cyclic adenosine monophosphate ChR2 channel rhodopsin 2

CPP conditionned place preference CREB cAMP response element-binding

DA dopamine

DAT dopamine transporter D1R dopamine 1 receptor D2R dopamine 2 receptor

EC50 half maximal effective concentration EM electron microscopy

EPSC excitatory postsynaptic currents ER endoplasmic reticulum

ERK extracellular-signal-regulated kinase GABA !–Ammunobutyric acid

GAD glutamate decarboxylase GAP GTPase-accelerating protein

GEF guanine nucleotide exchange factor GDP guanosine-5'-diphosphate

GFP green fluorescent protein GHB !-hydroxybutyrate

GIRK G protein-dependent inwardly rectifying K+ channel GRK G protein kinase

GTP guanosine-5'-triphosphate GTPase guanine triphosphate hydrolase GPCR G protein-coupled receptor

G protein guanine-nucleotide binding protein HEK human embryonic kidney cell HRP horseradish peroxidase

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IBaclofen baclofen-evoked current

IC50 half maximal inhibitory concentration ICSS intracranial self-stimulation

Ih hyperpolarization-activated current I-O input-output

IPSC inhibitory postsynaptic current IPSP inhibitory postsynaptic potential

KCTD K+ channel tetramerization domain-containing protein KO knockout

LTP long-term potentiation

mEPSC mini excitatory postsynaptic currents METH methamphetamine

mRNA messenger ribonucleic acid NAc nucleus accumbens

NMDA N-methyl-D-aspartate OA okadaic acid

PFC prefrontal cortex Pitx3 pituitary homeobox 3 PKA protein kinase A PKC protein kinase C PP protein phosphatase

RGS regulator of G protein signaling

S serine

SD sushi domain

sIPSC slow inhibitory postsynaptic current sIPSP slow inhibitory postsynaptic potential TH tyrosine hydroxylase

TTX tetrodotoxin

VGCC voltage-gated Ca2+ channel VTA ventral tegmental area YFP yellow fluorescent protein

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Table of Contents

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ACKNOWLEDGEMENTS ...3

SUMMARY ...4

RESUME EN FRANÇAIS...5

LIST OF ABBREVIATIONS ...7

LIST OF FIGURES ...10

LIST OF TABLES...11

INTRODUCTION ...12

GABAB RECEPTORS : STRUCTURE, FUNCTION, LOCALIZATION AND EXPRESSION...12

STRUCTURE OF THE SIGNALING COMPLEX...12

GABAB receptor ...12

G protein: a signal transducer ...14

Effectors ...14

HETEROGENEITY OF GABAB RECEPTOR RESPONSES...19

FUNCTION AND LOCALIZATION OF GABAB RECEPTOR SUBUNITS...22

Spillover for a sIPSC ...22

Unraveling function according to localization ...23

Revealing localization through function ...24

SURFACE EXPRESSION...26

Through endoplasmic reticulum and golgi ...26

Axonal and dendritic transport and targeting ...27

Endocytosis and recycling...27

Phosphorylation and surface expression ...28

GABAB RECEPTORS: DOPAMINE, SYNAPTIC PLASTICITY AND ADDICTION..31

VENTRAL TEGMENTAL AREA, A CORE NUCLEUS OF THE REWARD SYSTEM...31

Cellular composition...31

Electrophysiological and pharmacological characteristics ...33

DRUG-EVOKED SYNAPTIC PLASTICITY AND ADDICTION...34

A brain disease ...34

Targeting GABAB receptors for treating addiction...34

NEURONAL BASES OF ADDICTION...36

Drugs of abuse target the VTA to increase DA ...36

Drug-evoked synaptic plasticity...37

GABAB RECEPTOR PLASTICITY...39

Activity-dependent plasticity...39

Drug-evoked synaptic plasticity...42

MATERIAL AND METHODS...46

RESULTS ...51

DISCUSSION...73

CONCLUSION...82

CONTRIBUTIONS ...83

REFERENCES ...84

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List of Figures

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Figure1. Schematic representation of a GABAB receptor heterodimer composed of GABAB1a and GABAB2, and coupling to effectors.

Figure 2. Activation pathways of the different G" subunits.

Figure 3. Schematic representation of the different cell populations in the VTA.

Figure 4. Subunit composition and localization of GABAB receptors at synapses.

Figure 5. Phosphorylation sites on GABAB receptor subunits.

Figure 6. DA projections from the VTA.

Figure 7. Drugs of abuse target the VTA to increase DA release.

Figure 8. Absence of slow inhibitory postsynaptic currents in VTA GABA neurons 24h and 7d following a single METH exposure.

Figure 9. Reduced GABAB-GIRK currents in VTA GABA neurons 24h and 7d following a single METH exposure.

Figure 10. Methamphetamine does not alter GABAB receptor signaling in hippocampal and prelimbic cortex neurons.

Figure 11. Reduced sensitivity of presynaptic GABAB receptor-mediated inhibition 24h and 7d following a single METH exposure.

Figure 12. Characterization of blue light-evoked currents in VTA of floxed ChR2-injected GAD65-Cre mice.

Figure 13. GABAB receptor-dependent presynaptic inhibition of glutamate onto VTA DA neurons is not altered by METH.

Figure 14. Depression of GABAB-GIRK signaling in VTA GABA neurons 24h following a single cocaine injection.

Figure 15. D1-like receptor antagonist blocks METH-induced depression of GABAB-GIRK signaling.

Figure 16. Reduced surface expression of GABAB1 and GIRK2 subunits in GABA neurons of METH injected mice.

Figure 17. GABAB receptor dephosphorylation-dependent depression of GABAB-GIRK currents in GABA neurons following METH.

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Figure 18. METH-injected mice lack GABAB receptor-dependent inhibition of VTA GABA neuron firing.

Figure 19. Proposed model of phosphosrylation-dependent GABAB receptor constitutive recycling.

Figure 20. Drug-evoked GABAB receptor plasticity in the VTA following different drug treatment protocols.

List of Tables

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Table 1. Summary of activity-induced plasticity of GABAB receptor signaling.

Table 2. Summary drug-evoked modulation of GABAB receptor signaling.

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Introduction

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GABA

B

receptors : structure, function, localization and expression

Structure of the signaling complex

GABAB receptor

The GABAB receptor is a metabotropic or GPCR. Its cloning revealed the existence of two subunits, GABAB1 (Kaupmann et al., 1997) and GABAB2

(Kaupmann et al., 1998; Jones et al., 1998; White et al., 1998; Kuner et al., 1999; Ng et al., 1999), both displaying the classic GPCR structure of an extracellular N-terminal domain, seven transmembrane domains and an internal C-terminal tail. Additionally, GABAB1 comes in two different splice variants, GABAB1a and GABAB1b, where 1a differs by the presence of two protein interacting sushi domains (SD) in the N-terminal tail (Kaupmann et al., 1997). Heterodimerization of GABAB1 and GABAB2 is required for the formation of functional receptors (Figure 1), as expression of individual subunit is unable to activate K+ currents (Kaupmann et al., 1998; Jones et al., 1998; White et al., 1998; Kuner et al., 1999; Ng et al., 1999). It is mediated through coiled-coil domains interactions located in the C-terminal tail of both subunits (Kammerer et al., 1999). Additionnally, assembly of the two subunits allows hiding of an endoplasmic reticulum (ER) retention signal located in the C-terminal tail of GABAB1 (Margeta-Mitrovic et al., 2000) and thus promotes surface expression of the receptor. Each subunit of the dimer specifically participates to activation or signal transduction. Both subunits N-terminal domains arbor a venus flytrap configuration, but ligand efficiently binds to GABAB1 only (Galvez et al., 1999; Malitschek et al., 1999). However, the N- terminal tail of GABAB2 is also needed for proper receptor signaling, as mutated receptors containing two GABAB1 N-terminals show impaired activation (Margeta-Mitrovic et al., 2001a). On the intracellular side, only GABAB2 can bind to the G protein via its three intracellular loops linking the transmembrane domains (Margeta-Mitrovic et al., 2001b; Robbins et al.,

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2001). Similarly, the GABAB1 C-terminal tail contributes to and enhances coupling efficiency, further supporting allosteric interactions and necessary heterodimerization for wild-type (WT) receptor signaling (Galvez et al., 2001).

Figure1. Schematic representation of a GABAB receptor heterodimer composed of GABAB1a and GABAB2, and coupling to effectors. GABAB1a

binds GABA in a Venus flytrap located in the extracellular N-terminal domain.

This domain also contains a pair of sushi domains that allow axonal targeting of the receptor. The intracellular C terminal tail of GABAB1a contains LL and ER retention motifs, crucial to surface expression of the dimer. GABAB1b only differs by the absence of the sushi domains. The two subunits interact through their intracellular coiled-coil domains, represented here by the dashed lines.

GABAB2 can bind auxiliary KCTD proteins that modulate receptor signaling. It also binds to Gi/o type protein via intracellular loops. Upon receptor activation, G" inhibits adenylate cyclase, thus decreasing the production of camp, whereas G#! either activates GIRK channels and depolarize the membrane, or inhibits VGCC and Ca2+ dependent processes.

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G protein: a signal transducer

G proteins are widespread triheteromeric signal transducers composed of one ", one # and one ! subunits, the latter forming the #! complex.

Isoforms exist for each subunit, allowing numerous combinations for the assembly of G proteins, thus impinging on various and distinct molecular signaling pathways. G proteins are called after the name of the ! isoform within the complex, and fall into four groups. Briefly, G!s activates adenylate cyclase, whereas G!i/o inhibits this pathway. G!q activates phospholipase C, and G!12/13 activates Rho guanine nucleotide exchange factor (GEF) proteins (Figure 2). G protein activation by GPCR is not yet fully understood. It has been suggested that ligand binding induces a conformation change that allows the receptor to act as a GEF and switch the guanosine-5'-diphosphate!

(GDP) for a guanosine-5'-triphosphate (GTP) on G!, now activated (Cabrera- Vera et al., 2003). As a result the ! and "# subunits split and travel locally along the membrane to reach their respective targets (Soejima and Noma, 1984). Hydrolysis of GTP determines deactivation by reassembly of the triheteromeric complex (for review see Cabrera-Vera et al., 2003;

Wettschureck and Offermanns, 2005).

Effectors

GABAB receptor couples to pertussis toxin-sensitive Gi/o protein (Andrade et al., 1986), similar to many other receptors for neurotransmitters and neuromodulators like glutamate (metabotropic glutamate (mGlu) group II and III receptors), dopamine (D2-like receptors), adenosine (A1 and A3 receptors), acetylcholine (M2 and M4 receptors), serotonin (5-HT1 and 5-HT5

receptors) and opioids ( µ-Opioid R), among many others (Figure 2).

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Figure 2. Activation pathways of the different G" subunits.

Corresponding receptors are cited underneath.

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Voltage-gated Ca2+ channels

Presynaptic GABAB receptors inhibit VGCCs, thus decreasing neurotransmitter release from terminals. This was the very first action of high affinity GABAB receptor agonists like baclofen to be reported (Pierau and Zimmermann, 1973) before GABAB receptors had even been identified. These early results led to the discovery of the GABA-activated and bicuculine- insensitive GABAB receptor, modulating release of evoked adrenaline and serotonin from sympathetic ganglia terminals (Bowery et al., 1980). It was then shown that baclofen inhibits Ca2+ currents in the dorsal root ganglion (Dunlap and Fischbach, 1981), suggesting Ca2+ channels as potential effectors of GABAB receptors. GABAB receptors inhibit mainly P, Q and N- type VGCCs, through a physical interaction with G"# and not G! (Ikeda, 1996; Herlitze et al., 1996). Presynaptic inhibition of multivesicular release was also reported at the level of a single synapse using two-photon optical quantal analysis (Chalifoux and Carter, 2010). Further characterization established presynaptic inhibition as a control mechanism for synaptic transmission common to most neuronal cell types throughout the brain (for review see Wu and Saggau, 1997). Autoreceptor refers to GABAB receptors located on GABAergic terminals, whereas any other presynaptic GABAB

receptor is called heteroreceptor. Some papers also reported GABAB

receptor-mediated presynaptic inhibition downstream of Ca2+ entrance. For example, baclofen decreased the frequency of action potential-independent mini excitatory postsynaptic currents (mEPSC) on hippocampal CA3 neurons, whereas blocking Ca2+ channels with Cd2+ did not (Scanziani et al., 1992).

Similar results were obtained in the cerebellum with baclofen, suggesting VGCC-dependent inhibition of evoked release, and additional modulation of spontaneous release downstream of Ca2+ entry (Dittman and Regehr, 1996).

Synaptic release is also indirectly modulated by a slow down of vesicle recruitment during recovery from synaptic depression in the giant Calyx of Held synapse, independent of Ca2+ and rather mediated by G! and a decrease in cyclic adenosine monophosphate (cAMP) levels (Sakaba and Neher, 2003).

Postsynaptic GABAB receptors also inhibit VGCC-mediated Ca2+

currents. In the supraoptic nucleus, baclofen modulates firing capabilities of

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the cells (Harayama et al., 1998; Li and Stern, 2004). In the somatosensory cortex, activation of GABAB receptors inhibits dendritic Ca2+-spike propagation (Pérez-Garci et al., 2006). More recently, it has been shown that GABAB

receptors inhibit Ca2+ signals evoked by action potentials in dendrites and spines of pyramidal neurons of the prefrontal cortex. This is mediated by the inhibition of VGCC, potentially including L-type channels (Chalifoux and Carter, 2011).

GIRK channels

The prominent action of postsynaptic GABAB receptors is to hyperpolarize membranes by activating GIRK channels. These channels are part of a large inwardly-rectifying K+ channels family called Kir1-7. Mammals express four different subunits, GIRK 1-4 (Kir3.1-3). Only GIRK1-3 are found in the CNS (Wickman et al., 2000), where they form GIRK1-GIRK2, GIRK1- GIRK3, GIRK2-GIRK3 heterotetramers (Jelacic et al., 1999; Jelacic et al., 2000), and GIRK2 homotetramers (Kofuji et al., 1995; Slesinger et al., 1996).

Initially baclofen was reported to hyperpolarize hippocampal CA1 neurons in a Cl--independent way (Newberry and Nicoll, 1984). The activation of a K+ conductance was then demonstrated (Gähwiler and Brown, 1985; Newberry and Nicoll, 1985), before the suggestion of GIRK channels as the main postsynaptic effector of GABAB receptors (Misgeld et al., 1995).

Demonstration of the GIRK implication in post but not presynaptic effects of GABAB receptor was possible due to generation of GIRK2 knockout (KO) mice. In these animals, baclofen, adenosine and serotonin-evoked postsynaptic currents were gone, whereas presynaptic inhibition of glutamate and GABA release was unaffected, compared to WT mice (Lüscher et al., 1997). Molecular activation of GIRK channels remained controversial for some time before it was finally accepted that G"# activates GIRK channels (Reuveny et al., 1994; Wickman et al., 1994). G"# binding sites have been identified on all GIRK subunits, within the intracellular N- and C-terminal domains (Huang et al., 1995; Slesinger et al., 1995). GIRK1 has a higher affinity for G"# due to a unique amino acid sequence absent in other subunits.

Additionally, the N- and C-terminal domains can interact and enhance G"#

binding (Huang et al., 1995). Investigating the gating of GIRK channel

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activation revealed its complex mechanism. It has been suggested that up to four G"# proteins can bind a GIRK tetramer (Corey and Clapham, 2001).

Building of chimeric GIRK channels containing 0 to 4 subunits devoid of G protein interaction domain revealed that the presence of one WT subunit was sufficient to allow G"# activation of the receptor, and that three WT subunits recovered full activation of the channel (Sadja et al., 2002).

Few papers have described the presence of presynaptic GIRK channels to inhibit glutamate release in the cerebellum (Ladera et al., 2008; Fernández- Alacid et al., 2009). Another study reported that activation of GABAB receptors decreases the amplitude of GABAA-mediated inhibitory postsynaptic current (IPSC) onto VTA neurons, and that this effect is blocked by a GIRK channel inhibitor (Michaeli and Yaka, 2010). Even though knocking out GIRK channels did not alter presynaptic inhibition in the hippocampus (Lüscher et al., 1997), it is possible that GIRK channels participate in this function in a region or cell type-specific manner.

Enzymes

Once activated, G!i/o inhibits adenylate cyclase and the production of cAMP, leading to decreased protein kinase A (PKA) activity (Wojcik and Neff, 1984). However the physiological consequences of this interaction are much less understood than those ensuing from G"# and ion channels. Decreased PKA activity has been implicated in surface stability and desensitization of the GABAB receptor mediated by phosphorylation levels of serine (S) 892 on the GABAB2 subunit (Couve et al., 2002). On the opposite, GABAB receptor- activated G"# can enhance binding affinity of G!s to adenylate cyclase, therefore increasing the production of cAMP triggered by concomitant activation of adrenergic receptors (Robichon et al., 2004). This suggests crosstalk between metabotropic receptors and opens to more complex regulations of cellular responses in a physiological situation. Directly linked to synaptic transmission, Chalifoux and colleagues investigated postsynaptic modulation of N-methyl-D-aspartate (NMDA) receptor signaling by GABAB

receptors, taking advantage of two-photon laser-scanning microscopy and glutamate uncaging. They showed that GABAB receptor activation inhibits NMDA receptor-mediated Ca2+ signals without changing the size of unitary

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EPSC in single spines. This is mediated by antagonization of PKA and independent of VGCCs, but the implication of rather G! than G"# remains elusive (Chalifoux and Carter, 2010). On another level, G"# has also been reported to induce phosphorylation of extracellular-signal-regulated kinase (ERK) in the cerebellum, leading to activation of transcription factor cAMP response element-binding (CREB) activation and potentially gene expression (Tu et al., 2007).

Heterogeneity of GABAB receptor responses

Even if the main functions of pre and postsynaptic GABAB receptors are now better understood, the large heterogeneity among native GABAB

receptor responses is still a question of great interest. The major discovery that only two molecularly distinct and functional GABAB receptor heterodimers are expressed at the membrane, but fail to reproduce in heterologous systems the functional diversity observed with native GABAB receptors, fanned the debate over the mechanisms underlying this discrepancy (for reviews see (Pinard et al., 2010; Raiteri, 2008).

For example, presynaptic receptors are usually more sensitive to agonist than the postsynaptic ones. In the ventral tegmental area (VTA), baclofen is 10 times more efficient at inhibiting IPSC than activating GIRK currents on DA neurons (Cruz et al., 2004). Shown in a different way, higher concentrations of antagonist are needed to block presynaptic versus postsynaptic GABAB receptors (Pozza et al., 1999). Among presynaptic GABAB receptors, it has been shown in the dorsolateral septal nucleus that the GABAB receptor agonist CGP44533 has a higher efficacy and potency at inhibiting hetero than autoreceptors (Yu et al., 1999). Upon continuous baclofen application, GABAB receptor-mediated inhibition of GABA release, but not glutamate release, desensitizes (Tosetti et al., 2004). Similar differences have been reported for postsynaptic receptors in the VTA. DA neurons of the VTA exhibit large and desensitizing baclofen-evoked currents (IBaclofen), whereas neighboring GABA interneurons respond with small and steady state currents to baclofen (Figure 3). Additionally, GABAB receptor- GIRK coupling efficiency is higher in GABA than DA cells, reported by a 10-

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fold decrease in half maximal! effective concentration (EC50, (Cruz et al., 2004).

Some of these differences have been molecularly explained. In the VTA, the lower postsynaptic coupling efficiency in DA versus GABA cells is partially explained by a differential GIRK channel subunit composition (Figure 3). GABA cells express GIRK1-3 subunits, whereas DA neurons only arbor GIRK2-3. Reexpression of GIRK1 in DA cells increased the EC50 of baclofen, potentially underlying higher binding affinity of G protein for GIRK1 containing channels (Labouèbe et al., 2007). Similar partial recovery was obtained by knocking out regulator of G protein signaling (RGS) 2. RGS proteins belong to a guanine triphosphate hydrolase (GTPase)-accelerating proteins (GAP) family that promote GTP hydrolysis, thus reducing the activation of G proteins.

In other words, RGS proteins act as a brake on GABAB receptor coupling to effectors. RGS2 modulates GIRK channels coupling (Doupnik et al., 2004), and RGS2 KO mice VTA DA cells exhibited postsynaptic GABAB receptors with higher sensitivity to baclofen than WT (Labouèbe et al., 2007). G protein kinase (GRK) 4 has been implicated in the desensitization of the receptor, surprisingly through a phosphorylation-independent mechanism (Perroy et al., 2003). In the same way, a study recently reported that GRK2 accelerates GIRK current desensitization by physically interacting with G"#, without involving phosphorylation (Raveh et al., 2010). However, this effect is GPCR activation-dependent and has not been tested with GABAB receptors. Most interestingly, four K+ channel tetramerization domain-containing (KCTD) proteins have been identified as auxiliary subunits of the GABAB receptors. By forming tetramers and binding to the C-terminal tail of GABAB2, KCTDs increase agonist potency, accelerate onset and promote desensitization of the receptor. These proteins are differentially expressed throughout the brain, on both pre and postsynaptic sites, and cluster with GABAB1 subunits (Schwenk et al., 2010; Bartoi et al., 2010; Metz et al., 2011). KCTDs are promising candidates in the elucidation of the heterogeneity of native GABAB receptor responses, and generation of KO mice for these proteins will tell us more about their implication in mediating GABAB receptor signaling.

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Figure 3. Schematic representation of the different cell populations in the VTA. DA neurons (red) express GIRK2,3 subunits and high levels of RGS2, contributing to the largely desensitizing IBaclofen (at saturating concentration, representative traces shown in right panels). GABA neurons (green) synapse onto DA neurons, express GIRK1,2,3 and low levels of RGS2, contributing to the non desensitizing IBaclofen (right panel). The current is blocked by CGP54626, a GABAB receptor antagonist. GABAB receptor dependent currents in glutamate neurons (blue) have not been investigated yet.

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Function and localization of GABAB receptor subunits

Given the heterogeneity in GABAB receptor actions and responses observed since its discovery, it rapidly became evident that localization and subunit composition knowledge would be the key to the comprehension of GABAB receptor physiological roles. In the next paragraphs, we show how functional and structural studies have influenced each other’s progress in this interest, depending on technical limitations and advances.

Spillover for a sIPSC

Early on, functional activation of GABAB receptor-GIRK currents provided insights on the localization of the receptors. It seemed that GABAB

receptors would not be activated by basal GABA release, as in slice preparations, a low intensity stimulus evokes a fast GABAA-mediated, whereas stronger intensity stimulation is needed to generate a slower component, called late or slow IPSC/inhibitory postsynaptic potential (sIPSC/IPSP, Dutar and Nicoll, 1988). This suggested GABAB receptors are extrasynaptically located and require spillover of GABA to reach them.

Blocking GABA transporters increases the amplitude and slows down the kinetics of the sIPSP, further supporting the spillover hypothesis (Isaacson et al., 1993). In the hippocampus, double patch experiments revealed that train stimulation of one interneuron is unable to drive a sIPSC in a connected pyramidal cell, unless GABA uptake is inhibited. This also occurs in non- connected neurons, arguing for recruitment of GABAB receptors following synchronized GABA neuron activity, like during hippocampal rhythmic oscillations (Scanziani, 2000). Still, single cell-generated GABAB receptor sIPSC in pair-connected neurons have been reported in the thalamus and neocortex, but it seems to be cell type-specific (Kim et al., 1997; Tamás et al., 2003). GABA spillover has also been suggested for the activation of presynaptic receptors (Dittman and Regehr, 1997). The model that came out from these functional experiments suggest that GABAB receptors are extrasynaptically located and require intense GABA release and spillover for modulation of neuronal excitability and synaptic transmission. However, identification of in vivo sIPSC as a form synaptic transmission has not been

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yet demonstrated and remains a high potential discovery for synaptic physiologists.

Unraveling function according to localization

Twenty-five years ago, autoradiography studies showed that GABAB

receptors localize in many different regions of the brain (Bowery et al., 1987;

Chu et al., 1990). Over years technical improvement and cloning of the receptor allowed refinement of this visualization by identifying GABAB1a, GABAB1b and GABAB2 expression patterns separately either with in situ hybridization (Kaupmann et al., 1997; Bischoff et al., 1999) or immunohistochemistry (Margeta-Mitrovic et al., 1999; Billinton et al., 2000). It appears that most neuronal cell-types express all GABAB subunits, with some exceptions. GABAB2 messenger ribonucleic acid (mRNA) expression is low in the putamen, medial basal hypothalamus, septum and brainstem whereas GABAB1 mRNA is abundant (Clark et al., 2000). In the striatum, differential immunoreactivity for GABAB1 and GABAB2 was reported (Ng and Yung, 2001).

The distribution of GABAB1a versus GABAB1b also differed within regions.

Cerebellum is a striking example where 1b is restricted to Purkinje cells wheras 1a appears in granule, stellate and basket cells (Bischoff et al., 1999).

Electron microscopy (EM) studies then revealed the cellular compartmentalization of GABAB receptor subunits, showing that GABAB1 and GABAB2 colocalize presynaptically at both GABA and glutamate terminals, and postsynaptically in spines and dendritic shafs. (Gonchar et al., 2001; Kulik et al., 2002). Postsynaptic receptors are mostly perisynaptically located, indicating that they are not the prime target of synaptically released GABA (Kulik et al., 2003), in agreement with the requirement of GABA spillover previously mentioned. These observations also support that heterodimerizartion is needed for the functionality of the receptor and argue in favor of a widespread regulatory function of GABAB receptors.

These observations were supported by electrophysiological data from GABAB1-/- and GABAB2-/- mice. Knocking out one subunit induced a downregulation of the other’s expression. GABAB receptor-mediated inhibition of glutamate and GABA release was absent in both mice (Schuler et al., 2001;

Gassmann et al., 2004). Postsynaptic K+ currents were gone in GABAB1-/-

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mice, however in the GABAB2-/- mouse postsynaptic activation of GABAB

receptors blocked a constitutively active K+ current, instead of opening GIRK channels (Gassmann et al., 2004).

Revealing localization through function

Imaging studies reached their limit when it came to localizing GABAB1a

versus GABAB1b isoforms at axon terminals and dendrites, due to the lack of discriminative antibodies. The only difference between the two isoforms is the pair of sushi domains on the N-terminal tail of GABAB1a. It had been foreseen already that this could be an explanation to the variety of responses following GABAB receptor activation. KO mice were generated for GABAB1a and GABAB1b to formerly identify subunit isoforms functional implication in pre versus postsynaptic actions of GABAB receptor and deduce their subcellular localization.

Expression of green fluorescent protein (GFP)-tagged GABAB1

isoforms in hippocampal slice cultures revealed that GABAB1a preferentially targets axons, whereas GABAB1b is more recurrent in dendritic spines. Patch- clamp recordings provided a functional support to this observation. Several studies then investigated the isoform participation to heteroreceptor function at glutamatergic terminals and all reported that decrease of release is reduced in GABAB1a-/- but not GABAB1b-/- animals, be it onto hippocampal CA1 neurons (Vigot et al., 2006), CA3 neurons (Guetg et al., 2009), thalamus (Ulrich et al., 2007) or amygdala (Shaban et al., 2006). Two of these studies also investigated presynaptic inhibition through autoreceptors, and reported an equal contribution of GABAB1a and GABAB1b to decrease in GABA release, indicating differential distribution of subunits between auto and heteroreceptors (Vigot et al., 2006; Ulrich et al., 2007). GABAB receptor- activated Kir3 postsynaptic currents appeared to be dependent on both GABAB1 isoforms except in CA1 neurons where most of the current relied on GABAB1b (Vigot et al., 2006). It also appeared that only GABAB1b containing receptors enter spine heads. Roughly, GABAB1a and GABAB1b function seem predominant at presynaptic and postsynaptic sites respectively (Figure 4).

This conclusion is supported by the recent discovery that the pair of sushi

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Figure 4. Subunit composition and localization of GABAB receptors at synapses. Presynaptic inhibition at GABA terminals is mediated by autoreceptors containing either GABAB1a or GABAB1b, and GABAB2 that decrease Ca2+ entry through VGCC, whereas it is mostly mediated by GABAB1a containing heteroreceptors at excitatory terminals. On the postsynaptic side, spillover of GABA activates the peri and extrasynaptically located receptors. Both GABAB1a and GABAB1b-containing receptors are present at the membrane, mainly hyperpolarize the neuron by activating GIRK channels but can also inhibit VGCCs. Mostly GABAB1b-containing receptors are found in spines, where they balance input excitation (Inspired from Pinard et al., 2010).

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domains, present on GABAB1a, serves as an axonal targeting signal and promotes GABAB1a containing receptors trafficking towards terminals (Biermann et al., 2010).

Surface expression

GABAB receptor surface expression is a highly regulated process that organizes at the same time vertical transportation from the ER through golgi to the membrane and lateral trafficking towards dendrites and axons. It requires heterodimerisation of the receptor and undergoes at least two checkpoints implicating two different motifs on the GABAB1 subunit.

Through endoplasmic reticulum and golgi

An Arginine-rich ER retention signal is present within the C-terminal tail coiled-coil domain of the GABAB1 subunit (Calver et al., 2001; Margeta- Mitrovic et al., 2000; Pagano et al., 2001). If GABAB1 reaches the cis-golgi, its ER retention signal is spotted by COPI protein, which carries the receptor back to the ER (Brock et al., 2005). Heterodimerization with GABAB2 seems to induce a conformational change that renders the retention signal invisible to COPI. If the retention signal is mutated, GABAB1 accumulates in trans golgi, but is not further transported to the cell surface.

A second motif within the coiled-coil domain of GABAB1, LL motif, inhibits transport from trans golgi to the cell surface. When the LL motif is mutated, GABAB1 alone can be brought to the membrane (Restituito et al., 2005). However the LL motif could be masked by dimerization and its role in surface expression of functional receptors remains to be demonstrated (Figure 1). In the C-terminal tail of GABAB2, an amino acid sequence has been implicated in surface trafficking of GABAB2 and the full receptor at the membrane, actually by promoting its exit from the ER (Pooler et al., 2009). So far no proteins are known to interact with this sequence. Finally, CHOP, a transcription factor activated after ER stress, interacts with the GABAB

receptor and downregulates its surface expression (Sauter et al., 2005).

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Axonal and dendritic transport and targeting

One open question asks whether GABAB receptor subunits assemble before or after transport to dendrites and axons. In cultured hippocampal neurons, immunofluorescence experiments reported that a fraction GABAB1

and GABAB2 subunits do not colocalize in intracellular compartments. This observation argues for the transport of individual subunits in the ER before heterodimerization and surface expression (Ramírez et al., 2009). Along the same line, Marlin-1 interacts with both GABAB1 and the molecular motor kinesin-I, providing subunit specific transport mechanism (Vidal et al., 2007).

Contradicting data indicates that GABAB2 is required for dendritic targeting of GABAB1 (Pooler et al., 2009). It has also been shown that GABAB1a can be transported into axons without GABAB2, but not the opposite, indicating that dimerization is required for presynaptic targeting and transportation (Biermann et al., 2010). So far it is difficult to build a uniform model of whether single subunits or full receptors are transported in the ER, as it may depend on subcellular targeting.

Endocytosis and recycling

Constitutive endocytosis of GABAB receptors has been reported in different cell culture preparations, taking advantage of various visualization methods such as biotinylation (Grampp et al., 2007, Grampp et al., 2008;

Vargas et al., 2008), "-bungarotoxin tag (Wilkins et al., 2008) and human influenza hemagglutinin (Pooler et al., 2009). Intracellular accumulation of recombinant GABAB1a,2 receptors occurrs within 10 minutes. Internalization is clathrin and dynamin-mediated. Clathrin coats the vesicules whereas dynamin is a GTPase implicated in membrane fission. Blocking dynamin inhibits GABAB receptor endocytosis. Interfering with the clathrin pathway inhibits internalization in both human embryonic kidney (HEK) cells and neurons (Grampp et al., 2007; Laffray et al., 2007; Vargas et al., 2008). After internalization, GABAB receptors are transferred to fast and slow recycling endosomes, as well as lysosomes. Receptors that are not degraded can recycle back to the membrane (Grampp et al., 2008). Blocking vesicle fusion to the plasma membrane by monensin induced degradation of 50% of the

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receptors by redirecting them to lysosomes, suggesting a tight equilibrium between internalization and recycling (for review see Benke, 2010).

Prolonged GPCR activation leads to internalization through GRK phosphorylation, implicating arrestin, dynamin and clathrin, providing a mean to downregulate signaling at the membrane. However, GABAB receptors do not seem to abide by this rule: they are not a phosphorylation substrate of GRK1-4, and most of the studies that investigated the consequences of baclofen activation of GABAB receptors never reported an intracellular accumulation, either in ectopic preparations or neurons (Fairfax et al., 2004;

Grampp et al., 2007; Grampp et al., 2008; Perroy et al., 2003; Mutneja et al., 2005; Vargas et al., 2008; but see González-Maeso et al., 2003). One offered explanation is that ligand binding accelerates basal recycling and compensates for internalization of the receptor (Wilkins et al., 2008; Grampp et al., 2008). In the same line, blocking endocytosis while applying baclofen increases the intracellular pool of receptors (Laffray et al., 2007).

Altogether these results indicate that GABAB receptors are not usual GPCRs. The necessity for heterodimerization suggests tight regulatory mechanisms for subcellular and surface expression and will require further high definition visualization like electron microscopy to understand and standardize its functioning. The fact that GABAB receptors do not undergo agonist-induced internalization indicates that complex phenomena like desensitization, observed in cell cultures and slices, can not be explained simply by a decreased number of membrane receptors but rather implicates adaptations at several levels like G protein coupling or effector activation rate.

Therefore, future studies will need to take in account the relationship between GABAB receptor, G protein, effectors and modulators.

Phosphorylation and surface expression

Surface expression of receptors is highly dependent on phosphorylation status, and several kinases are known to interact with GABAB

receptors (Figure 5). Again, GABAB receptors do not follow the usual line of conduct of canonical GPCRs following phosphorylation (Terunuma et al., 2010a).

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Phosphorylation of GPCRs by PKA usually triggers internalization of the receptor (Bouvier et al., 1988; Moffett et al., 1996), and GABAB2 S892 is the only PKA phosphorylation site by PKA on GABAB receptors.

Unexpectedly, short agonist exposure induces PKA-mediated phosphorylation of the receptor, decreases the desensitization rate through potential stabilization of the receptor at the membrane and promotes GABAB receptor signaling (Couve et al., 2002). However, PKA activation mechanism remains elusive and may be GABAB receptor-independent, as G"i/o inhibits adenylate cyclase and decreases PKA activity. Indeed, GABAB2 S892 phosphorylation level is decreased after prolonged exposure to baclofen, leading to degradation of the receptor, which is attenuated by increasing PKA activity (Fairfax et al., 2004).

Protein kinase C (PKC) can phosphorylate GABAB1 upon receptor activation. This results in the dissociation of N-ethylmaleimide-sensitive fusion protein (NSF) from the receptor and reduces G protein activation. Preventing NSF from binding to the receptor blocks PKC-dependent phosphorylation.

This mechanism promotes an internalization-free desensitization of the receptor by decreasing the coupling of the receptor to the G protein (Pontier et al., 2006).

5’AMP-dependent protein kinase (AMPK) is activated by AMP following high metabolic activity, binds to GABAB1 C-terminal domain but phosphorylates both GABAB1 (at S917, S923) and GABAB2 (at S783).

Whereas the consequences of GABAB1 phosphorylation remain elusive, phosphorylation of GABAB2 stabilizes the complex at the membrane and decreases desensitization. This could reflect a protective mechanism following metabolic stress or ischemia to reduce excitotoxicity (Kuramoto et al., 2007).

Two recent studies have identified two independent pathways implicating Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of GABAB1 at S867 (Guetg et al., 2010) and protein phosphatase 2A (PP2A)-mediated dephosphorylation of GABAB2 at S783 (Terunuma et al., 2010b), both of them leading to degradation of the receptor.

The functional modulation of GABAB receptor signaling by changes in phosphorylation levels will be reviewed and discussed in the next chapters.

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Figure 5. Phosphorylation sites on GABAB receptor subunits.

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GABA

B

receptors: dopamine, synaptic plasticity and addiction

Ventral tegmental area, a core nucleus of the reward system

Cellular composition

The mesocorticolimbic system is implicated in reward function, decision-making and goal-directed actions (Figure 6). It is composed of the ventral tegmental area (VTA) and its principal target regions, the Nucleus Accumbens (NAc) and prefrontal cortex (PFC). The VTA comprises mainly three neuronal subtypes (Figure 3): DA neurons, representing the majority of cells in the VTA (~60%, Cameron, 1997), GABA interneurons (~15%, Johnson and North, 1992b), and recently identified glutamate neurons (~25%, Yamaguchi et al., 2007). DA cells release DA within the VTA and in numerous projection regions like the NAc, the striatum, prefrontal cortex and the hippocampus. They are stained for tyrosine hydroxylase (TH), an enzyme implicated in the synthesis of DA (Gupta et al., 1990). GABA interneurons form synapses onto DA neurons, thus controlling their activity by acting as a brake. They also project to the NAc (Xia et al., 2011) and the PFC (Carr and Sesack, 2000). They are stained for glutamate decarboxylase (GAD) 65 and 67, an enzyme responsible for GABA synthesis, and specifically express !1- containing GABAA receptors within the VTA (Tan et al., 2010). Glutamate cells express vesicular glutamate transporter 2 mRNA and send axons to the NAc and the prefrontal cortex, but their function remains elusive (Yamaguchi et al., 2011).

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Figure 6. DA projections from the VTA. Hip: hippocampus; NAcc: Nucleus Accumbens; PFC: prefrontal cortex.

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Electrophysiological and pharmacological characteristics

Unlike in structurally well-organized regions like the hippocampus, neuronal populations of the VTA are intermingled and uneasy to discriminate among. A lot of work has been dedicated to identifying electrophysiological and pharmacological properties of the different cell types. Additionally, generation of genetically modified mice expressing green fluorescent protein (GFP) along GAD (Tamamaki et al., 2003) or pituitary homeobox 3 (Pitx3) protein (Zhao et al., 2004) now allows identification with certainty for GABA and DA cells, respectively.

!

DA neurons

DA neurons membrane potential rests around -55mV. These cells have a bigger capacitance than GABA neurons (~50pF) along with a lower input resistance (~150M$). Hyperpolarization of the membrane reveals a hyperpolarization-activated current (Ih). In vivo, DA neurons display a large action potential width (>1.1ms, Ungless et al., 2004). Pharmacologically, they are hyperpolarized by D2 and GABAB receptors activation (Figure 3), but not by µ-opioid receptors agonists. GABAB receptor-mediated currents desensitize upon constant agonist application (Cruz et al., 2004). Additionally, DA neurons are excited by unexpected natural rewards and reward predicting cues, and inhibited by both absence of predicted reward (Schultz et al., 1997) and aversive stimuli (Ungless et al., 2004). There is however variability in the predictive strength of these individual signatures that seems to depend on which afferences DA neurons receive and where they project. This variability supports the hypothesis of DA cells subpopulations within the VTA that would differentially respond to salient stimuli (Brischoux et al., 2009; Bromberg- Martin et al., 2010; Lammel et al., 2011; Margolis et al., 2006).

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GABA neurons

GABA neurons have a small capacitance (~15pA), a large input resistance (~500M$), a resting membrane potential around -79mV and show no Ih (German et al., 1993; Johnson and North, 1992a). They are hyperpolarized upon GABAB and µ-opioid receptor activation, but not by D2 receptor agonists. Their GABAB receptor-evoked current is not desensitizing

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(Figure 3). Their action potential width is shorter than DA neurons (<1.1ms, (Ungless et al., 2004). They are activated by aversive stimuli and directly mediate the following inhibition of DA cells (Tan et al. in 2012).

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Glutamate neurons

So far VTA glutamatergic neurons have been identified by positive VGluT2 mRNA expression and negative GAD and TH staining (Yamaguchi et al., 2011). They seem to form local synapses onto DA and non-DA cells (Dobi et al., 2010), and may therefore oppose the role of local GABA cells onto DA neurons activity. However electrophysiological studies are still lacking to further characterize this neuronal cell type and understand its functional relevance.

Drug-evoked synaptic plasticity and addiction

A brain disease

Addiction is considered a brain disease defined by the World Health Organization as compulsive substance intake despite negative consequences, and characterized by the risk of relapse after a prolonged period of abstinence. On average, the disease develops only in 20% of all drug consumers, and symptoms are only observed after repetitive drug use.

However, a single injection of drug triggers adaptive changes in synaptic function within the VTA, which persist beyond elimination of the compound from the body. These changes are referred to as drug-evoked synaptic plasticity, and are necessary but not sufficient to cause addiction. Increasing evidence suggests that addiction develops through hijacking of normal experience-dependent synaptic plasticity, reinforcing biased learning and associations that lead to maladaptive behaviors (Lüscher and Malenka, 2011;

Kauer and Malenka, 2007; Wolf and Ferrario, 2010).

Targeting GABAB receptors for treating addiction

Lots of studies have investigated how the modulation of GABAB

receptor signaling in the brain modifies reward processing and the rewarding value of addictive drugs. Positive effects of compounds like baclofen are presumably due to the decrease of drug-evoked DA release in the reward

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system (Fadda 2003, Brebner 2005). We highlight here only the studies where pharmacological intervention was restricted to a specific brain area, allowing better understanding of the implicated neuronal networks.

In drug naïve rats performing intracranial self-stimulation (ICSS) of electrical pulses into the medial forebrain bundle or the ventral pallidum, intra- VTA administration of baclofen increased the ICSS threshold, indicating a decrease of the reward value of the self-stimulation (Willick and Kokkinidis, 1995; Panagis and Kastellakis, 2002). It was also shown that rats would self- administer baclofen in the medial Raphe nucleus, whereas baclofen injections in the dorsal Raphe nucleus would induce conditionned place preference (CPP), a behavioral paradigm used to evaluate association of a specific environment with a positive reward (Shin and Ikemoto, 2010).

Studies have also looked at the ability of baclofen to modulate addiction-related behaviors like self-administration. In rats that have learned to self-administer cocaine under different reinforcement schedules, intra VTA, NAc or striatum injection of baclofen reduces self-administration (Shoaib et al., 1998; Brebner et al., 2000). However, it was not the case for intra PPT baclofen injection (Corrigall et al., 2001). Intra VTA baclofen treatment also decreased nicotine self-administration (Corrigall et al., 2000) and heroin self- administration in rats (Xi and Stein, 2000). Intra VTA injection of baclofen decreased ethanol-induced CPP (Bechtholt and Cunningham, 2005) and morphine-induced CPP (Tsuji et al., 1996), the acquisition of the latter being also altered by intra-dorsal hippocampus injection of baclofen (Zarrindast et al., 2006).

In humans, clinical studies showed the ability of baclofen (15-75 mg/kg/day) to decrease cocaine self-administration in human addicts (Ling et al., 1998), to reduce alcohol consumption and craving and help establishing abstinence (Addolorato et al., 2009). Similar results were obtained with baclofen (10-80mg/kg/day for 9 weeks) to reduce cigarette smoking (Franklin et al., 2009). However severe withdrawal symptoms may appear upon treatment cessation (Leo and Baer, 2005).

These results indicate that GABAB receptors could be a promising target for curing addiction, but some links remain missing. For example, the positive outcome of baclofen intake highly depends on the concentration

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(ranging from 50 to 500ng/kg when locally administered, and from 0.1 to 10 mg/kg when systemically given in rodents, and up to 80mg/kg for humans) and so makes it difficult to conclude from these studies which receptors are affected by baclofen and how subsequent changes in neuronal activity alter the development of addiction. Within the VTA, GABAB receptors on GABA cells are much more sensitive to baclofen than those located on the DA neurons. Therefore, a low concentration of baclofen would preferentially shut down the GABA cells activity and disinhibit DA neurons, whereas a higher concentration would directly inhibit the DA neurons, thus inducing an opposite effect (Cruz et al., 2004). A recent study looked at the effects of oral baclofen administration in humans on a monetary reward gambling task. A low concentration of baclofen (0.3mg/kg) increased the efficiency of reward- associated learning, whereas a higher dose (0.8mg/kg) did not affect learning curves (Terrier et al., 2011), supporting the need for a better cellular understanding of GABAB receptor function in the modulation of the reward system.

Neuronal bases of addiction

Drugs of abuse target the VTA to increase DA

In order to understand how baclofen counteracts addiction-related behaviors in drug-treated animals, we first need to describe how drugs of abuse initially modify the reward system.

A common particularity of addictive drugs is that they all target the VTA (Figure 7) and acutely increase extrasynaptic levels of DA within the VTA and target regions (Di Chiara and Imperato, 1988). Drugs of abuse may therefore be classified according to the three cellular mechanisms of action that exist to cause this acute dopamine increase (Lüscher and Ungless, 2006). Group I includes opioids (Johnson and North, 1992a), cannabinoids (Szabo et al., 2002), !-hydroxybutyrate (GHB, Cruz et al., 2004) and benzodiazepines (Tan et al., 2010) which decrease the release of GABA from VTA interneurons and thereby remove the inhibitory transmission brake onto DA neurons (for a comprehensive review on benzodiazepine addiction, see Lalive et al., 2011).

This indirect increase of DA cells activity is known as disinhibition, and is

Figure

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References

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