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.
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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 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
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possible due to either cell-type specific expression of their respective receptor to the drug (as with opioids, cannabinoids, benzodiazepines), or higher affinity of the drug for the receptor located on GABA neurons (GHB). GHB is a GABAB receptor agonist. However, the sensitivity of GABA neurons GABAB
receptors is much higher than those located on DA neurons. Therefore, a low concentration of GHB will preferentially hyperpolarize GABA cells and disinhibit DA cells, whereas a higher concentration overtakes the disinhibition by directly shutting down DA neurons activity (Labouèbe et al., 2007). This bidirectional effect of GHB is believed to underlie its addictive and aversive properties. Nicotine constitutes group II and directly activates DA neurons (Maskos et al., 2005), whereas group III drugs (including psychostimulants like cocaine and amphetamines) target and perturb the DA transporter (DAT) either by blocking it (cocaine) or reversing its activity (amphetamines, Sulzer et al., 2005).
Drug-evoked synaptic plasticity
Extracellular DA increase occurs within minutes after drug intake and is believed to be at the source of all forthcoming induction of synaptic plasticity and changes in network activity that happen either few hours after a single injection or after days of chronic drug use. Initially, it has been shown that after one shot of cocaine, the glutamatergic connection onto VTA DA neurons is strengthened (Ungless et al., 2001). GABA transmission is also altered by a single injection of opioids (Nugent et al., 2007). The glutamate strengthening is blocked when the drug is co-administered with a DA D1-like receptor antagonist (Argilli et al., 2008), or when given to a genetically modified mouse carrying a DA transporter insensitive to cocaine, with the result that the drug is unable to induce extracellular DA increase (Brown et al., 2010; Bellone et al., 2011). Since then, these observations have been repeated for all three classes of drugs, showing that drug-evoked synaptic plasticity is DA-dependent and common to all addictive drugs (Saal et al., 2003; Brown et al., 2010). This drug-evoked synaptic plasticity switches the mesolimbic system towards a permissive state that may on the long-term lead to the development
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Figure 7. Drugs of abuse target the VTA to increase DA release. Class I drugs decrease the activity of local GABA interneurons, leading to a disinhibition of DA neurons. Class II drug Nicotine directly activates DA neurons and Class III drugs block or reverse DA reuptake at the level of the DA transporter.
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of addiction in vulnerable subjects. If the drug is given only once, the glutamatergic strengthening can go back to baseline after 7 days (Ungless et al., 2001). However, if the animal is repetitively exposed to the drug, the synaptic plasticity extends to other regions of the brain (Mameli et al., 2009) and other neurotransmitters, as GABAergic transmission onto DA neuron is decreased after 5 days of cocaine (Liu et al., 2005). Withdrawal from chronic drug treatment also triggers synaptic adaptations in the NAc, believed to mediate incubation of craving, a behavioral paradigm to measure how much an animal searches for the drug upon withdrawal (Conrad et al., 2008).
If synaptic plasticity of glutamate transmission has motivated a lot of research since the discovery of long-term potentiation (LTP, Bliss and Lomo, 1973), basal and drug-evoked synaptic plasticity of GABAB receptors has not been put forward until recently and its relevance is still not fully understood.
Modulation of GABAB receptor signaling by addictive drugs is another crucial element to identify the cellular correlate of baclofen therapeutic potential, which can be explained by two different hypothesis: either baclofen enhances basal signaling to counteract other synaptic adaptations promoting addiction, either it restores a basal signaling that is altered by drugs of abuse.
GABAB receptor plasticity
Few papers have reported synaptic plasticity of GABAB receptors. It is not always easy to identify which of the receptor or the effector is responsible for modified signaling. For example, a decrease in IBaclofen might be explained by either an internalization of the GABAB receptors or the GIRK channel (in a simplified vision where participation of G protein and regulatory factors are left out). Additionally, activation ratio of GIRK channels by GABAB receptors is not well understood, which makes it difficult to predict what would happen following the upregulation of one or the other. Here we review studies investigating activity and drug-induced plasticity of the GABAB receptor-GIRK complex, among which only few were able to identify the actual players.
Activity-dependent plasticity
The first electrophysiological study to report activity-dependent synaptic plasticity of GABAB receptors in cell cultures and acute hippocampal
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slices was published in 2005 by Huang and colleagues. More than providing evidence for plastic adaptations of GABAB receptors, they showed that the sIPSC amplitude is increased following a classical 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid (AMPA) receptor LTP stimulation protocol in the CA1. The pairing protocol, consisting of 3Hz stimulation paired with a depolarization to 0mV of the postsynaptic cell, induces sIPSC and AMPA EPSC LTP in a NMDA receptor-, intracellular Ca2+-, and CaMKII-dependent fashion, revealing a shared induction molecular pathway. In mice lacking Nova-2, a protein shown to interact with GABAB receptor and GIRK mRNA, pairing protocol did not induce LTP of the sIPSC whereas LTP of AMPA receptors was present (Huang et al., 2005). To date this GABAB receptor LTP has not been further investigated, and its expression mechanism remains elusive. GIRK channels have been shown to be upregulated at the surface of cultured hippocampal neurons following NMDA receptor activation by ending four days of antagonist application. This increase in surface expression is mediated by dephosphorylation of S9 on GIRK2 subunit by PP1 phosphatase, promoting recycling of channels from endosomes to surface. It results in increased basal GIRK currents and A1R-activated currents allowing depotentiation of AMPA receptor LTP, a form of excitatory synaptic plasticity.
However, GABAB receptor-mediated currents were not larger after GIRK surface expression increased, suggesting specific GPCR signaling modulation or coupling to GIRK channels (Chung et al., 2009b; Chung et al., 2009a).
Vargas and collegues showed that glutamate triggers endocytosis and degradation of GABAB receptors in cortical neuron culture (Vargas et al., 2008). The same group further characterized this result and showed that short or prolonged activation of NMDA receptors differentially modulate GABAB
receptors surface expression in cortical and hippocampal neuron cultures. A 5 minutes application of glutamate induced an initial increase of surface receptors due to AMPK-mediated phosphorylation at S783 on GABAB2
subunit, whereas prolonged glutamate exposure decreased the phosphorylation levels of the same S783 through PP2A and redirected GABAB receptors to lysosomes for degradation. Therefore it seems that dephosphorylation of GABAB2 does not directly trigger the internalization of the receptor, but rather modifies its basal recycling at the level of
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postendocytic sorting. As a functional consequence, IBaclofen were significantly smaller. Internalization of GABAB receptors was blocked by NMDA receptor antagonist or baclofen, indicating that GABAB receptor activation may play a neuroprotective role in excitotoxicity situation (Terunuma et al., 2010b). This idea is also supported by a study where NMDA- or oxygen and glucose deprivation-induced excitotoxicity led to a decrease in total GABAB2 protein and cell death, an effect that was blocked by baclofen (Cimarosti et al., 2009).
At the same time, another group also reported a similar NMDA receptor-dependent internalization of GABAB receptors through a different phosphorylation pathway. Within 30 minutes, short application (1 minute) of NMDA decreased the surface expression of GABAB receptors and IBaclofen in cultured hippocampal cells. Internalization of the receptor is mediated by CaMKII activation and phosphorylation of specific S867 on GABAB1b (Guetg et al., 2010). The opposite directions of plasticity despite common molecular mechanisms between these results and those by Huang and colleagues (Table 1) will be discussed in the discussion section. Interestingly, the results by Guetg and colleagues are reminiscent of the decreased NMDA receptor-mediated Ca2+ currents in spines following GABAB receptor activation (Chalifoux and Carter, 2010), suggesting reciprocal interplay between excitatory and inhibitory transmission to modulate input excitability.
Plasticity Protocol Induction Expression Preparation
sIPSC LTP
NMDAR CaMKII activation hippocampal slice cultures and acute
Table 1. Summary of activity-induced plasticity of GABAB receptor signaling
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Drug-evoked synaptic plasticity
Presynaptic auto and hetero GABAB receptor signaling has been investigated in the dorsolateral septum, a region implicated in pleasure and reward (Olds and Milner, 1954). After two or four weeks, but not one, of cocaine treatment, the frequencies of spontaneous EPSPs and IPSPs are increased. In control animals, GABAB receptor antagonist application increased the frequencies to levels similar than the one reported after chronic cocaine treatment, but did not further increase them in cocaine treated animals. In parallel, sensitivity of GABAB auto and heteroreceptors to baclofen was decreased, suggesting the GABAB receptor signaling impairment leads to increased neurotransmitter release (Shoji et al., 1997). Postsynaptic receptor signaling, however, was not altered. These results correlate with early work by Nestler and collaborators who reported a decrease in G!i/o protein in the VTA, NAc and locus coerulus following chronic but not acute cocaine treatment in rats (Nestler et al., 1990). Partial loss of G! may impair G protein complex formation and its coupling to GABAB receptors. However, it remains difficult to predict the consequence on increased release of opposite neurotransmitter on the activity of dorsolateral septum neurons. Manzoni and Williams investigated presynaptic inhibition of glutamate release onto VTA DA neurons in rats undergoing morphine treatment for one week followed by 0 to 24h withdrawal. To do so they recorded AMPA receptor-mediated EPSCs from DA cells while applying GABAB receptor specific pharmacological compounds.
GABAB receptor antagonist had little effect on the EPSC amplitude in both morphine-treated and control conditions. However, baclofen potency was increased after morphine, suggesting an increase in basal sensitivity of the receptor. Additionally, application of a GABA uptake blocker had no effect on the EPSCs amplitude in control animals, whereas it significantly reduced it in morphine treated rats, supporting the increased sensitivity of the presynaptic GABAB receptors on glutamatergic terminals in the VTA (Manzoni and Williams, 1999). Still, some of these effects could also be explained through an increase in basal GABA tone, as previously reported following morphine treatment (Bonci and Williams, 1997).
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Labouèbe and colleagues investigated postsynaptic GABAB receptor signaling in VTA DA and reported that chronic exposure to GHB and morphine (2 daily injections at increasing concentrations for 5 days, no withdrawal) increases the coupling efficiency of GABAB receptors to GIRK channels in VTA DA neurons. They show further that this effect is mediated by a downregulation of RGS2, a protein acting as a brake on the receptor to the effector coupling. Similar results were obtained by incubating slices from drug-naïve animals in morphine for five hours before performing recordings, and were occluded when assessed on RGS2 KO mice (Labouèbe et al., 2007). It was shown more recently that cocaine injection for one or five days triggers a decrease of IBaclofen in VTA DA cells (Arora et al., 2011). This result is underlain by a decrease in GIRK channels density, as seen with electron
Labouèbe and colleagues investigated postsynaptic GABAB receptor signaling in VTA DA and reported that chronic exposure to GHB and morphine (2 daily injections at increasing concentrations for 5 days, no withdrawal) increases the coupling efficiency of GABAB receptors to GIRK channels in VTA DA neurons. They show further that this effect is mediated by a downregulation of RGS2, a protein acting as a brake on the receptor to the effector coupling. Similar results were obtained by incubating slices from drug-naïve animals in morphine for five hours before performing recordings, and were occluded when assessed on RGS2 KO mice (Labouèbe et al., 2007). It was shown more recently that cocaine injection for one or five days triggers a decrease of IBaclofen in VTA DA cells (Arora et al., 2011). This result is underlain by a decrease in GIRK channels density, as seen with electron