Endocannabinoids shape accumbal encoding of cue-motivated behavior via CB1 receptor activation in the ventral tegmentum
SUMMARY
Transient increases in nucleus accumbens (NAc) dopamine concentration are observed when animals are presented with motivationally salient stimuli and are theorized to energize reward seeking. They arise from high frequency firing of dopamine neurons in the ventral tegmental area (VTA), which also results in the release of endocannabinoids from dopamine cell bodies. In this context, endocannabinoids are thought to regulate reward seeking by modulating dopamine signaling, although a direct link has never been demonstrated. To test this, we pharmacologically manipulated endocannabinoid neurotransmission in the VTA while measuring transient changes in dopamine concentration in the NAc during reward seeking. Disrupting endocannabinoid signaling dramatically reduced, whereas augmenting levels of the endocannabinoid 2-arachidonoylglycerol (2AG) increased, cue-evoked dopamine concentrations and reward seeking. These data suggest that 2AG in the VTA regulates reward seeking by sculpting ethologically relevant patterns of dopamine release during reward-directed behavior.
INTRODUCTION
The neural mechanisms responsible for the pursuit of rewards in the environment are essential for the survival of the organism (Nesse and Berridge, 1997; Schultz et al., 1997). Environmental stimuli that predict the availability of reward develop incentive-motivational properties that energize the seeking of future rewards (Bindra, 1968). The NAc is a neural substrate that is critically involved in integrating interoceptive and environmental information with emotional information to initiate reward seeking (Kelley, 1999; Mogenson et al., 1980). When reward seeking is maintained in a controlled experimental setting in which environmental stimuli predict reward-availability, transient dopamine surges in the NAc begin to occur in response to the predictive stimuli (i.e., conditioned cues) following the attribution of incentive salience (Berridge and Robinson, 1998; Flagel et al., 2011). These transient increases in dopamine have been detected in the NAc when animals are exposed to cues predicting various rewards – including drugs of abuse (Phillips et al., 2003), food (Roitman et al., 2004), and brain stimulation reward (Cheer et al., 2007a) – and are required to promote reward-directed behavior (Nicola, 2010).
The brain endocannabinoid system, formed by metabotropic cannabinoid receptors (CB1 and CB2) and their endogenous ligands (e.g. anandamide and 2AG), is important for the regulation of dopamine signaling during reinforcement processing (Lupica and Riegel, 2005; Solinas et al., 2008). When dopamine neurons in the VTA exhibit brief high frequency firing episodes they release endocannabinoids that act as retrograde messengers by binding to pre-synaptic CB1 receptors, thereby indirectly modulating the excitability of dopamine neurons by reducing pre-synaptic neurotransmitter release (Melis et al., 2004). Rather than being released through a vesicular mechanism, endocannabinoids are distinct from other neurotransmitters in that they are formed and released “on demand” during specific neural events (Freund et al., 2003). It is likely, therefore, that endocannabinoids regulate dopamine signaling during reward seeking exclusively in situations in which dopamine neurons fire at high frequencies – like when animals are presented with environmental cues predicting reward (Schultz et al., 1997).
To investigate whether endocannabinoids modulate the neural mechanisms of reward seeking, we measured changes in the concentration of cue-evoked dopamine transients in the NAc shell while pharmacologically altering endocannabinoid signaling during operant behavior. A pharmacological approach was chosen because we previously demonstrated that blocking CB1 receptors using rimonabant (a CB1 receptor antagonist) reduced drug-induced transient dopamine release into the NAc (Cheer et al., 2007b). Operant behavior was maintained by either brain stimulation reward or food reinforcement while an environmental cue signaled the availability of reward. We found that disrupting endocannabinoid signaling uniformly decreased the concentration of cue-evoked dopamine transients and reward seeking. These findings prompted us to investigate whether increasing endocannabinoid levels would facilitate reward seeking, and if so, which endocannabinoid is responsible. Using recently developed pharmacological tools designed to manipulate specific components of the endocannabinoid system, we found that augmenting 2AG, but not anandamide, levels by disrupting metabolic enzyme activity increased dopamine signaling during reward seeking – suggesting that 2AG sculpts ethologically relevant patterns of dopamine release during reward-directed behavior.
RESULTS
Transient dopamine concentrations time-locked to cue presentation develop across trials
Dopamine was measured in the NAc shell using fast-scan cyclic voltammetry (FSCV) while responding was maintained in a previously described intra-cranial self-stimulation (ICSS) task (Cheer et al., 2007a). As in our previous report (Cheer et al., 2007a), a compound cue predicted reward availability. This occurred across multiple sensory modalities; specifically, a house light turned off, an ongoing tone ceased, and then 1-s later a white stimulus light mounted above the lever was presented simultaneously with lever extension. A 10-s timeout followed each lever response. Under these conditions, electrically-evoked dopamine release occurred following a lever response and was temporally dissociable from cue-evoked dopamine release events, allowing for changes in the concentration of cue-evoked dopamine to be measured across trials. In agreement with previous studies (Day et al., 2007; Owesson-White et al., 2008), the concentration of dopamine occurring in response to the cue during this acquisition session increased across trials (Figure 1A, 1B). While the concentration of cue-evoked dopamine rapidly increased (Figure 1C; R2=0.85; n=5), the latency to respond from lever extension (a metric of reward seeking) decreased in a linear fashion (Figure 1D; R2=0.80; n=5; mean values: 7.18, 7.16, 6.91, 6.81-s), demonstrating that the strengthening of Pavlovian associations between the cue and unconditioned stimulus is accompanied by increased and cue-related dopamine signaling (Day et al., 2007). Importantly, increased recruitment of endocannabinoids in the VTA should develop in association with an increasing concentration of cue-evoked dopamine release. As dopamine neurons fire in high frequency bursts, voltage gated CA2+ ion channels open and the resulting CA2+ influx activates the enzymes responsible for the synthesis of endocannabinoids (Wilson and Nicoll, 2002). Thus, endocannabinoid levels should be highest in the VTA after periods of phasic dopamine neural activity. If endocannabinoids are indeed involved in modulating dopamine signaling during reward seeking, pharmacological disruption of endocannabinoids should decrease cue-evoked dopamine concentrations and cue-motivated responding in unison….
…..DISCUSSION
It is well documented that transient dopamine concentrations in the NAc encode information regarding motivationally salient stimuli that predict reward availability (Day et al., 2007; Flagel et al., 2011; Phillips et al., 2003). Little is known however, regarding how these transient increases are modulated at dopamine cell bodies within the VTA. In the present study, we used a cutting-edge electrochemical monitoring technique to investigate how endocannabinoids in the VTA modulate transient dopamine release into the NAc shell during reward seeking. We found that disrupting endocannabinoid modulation of dopamine neurons reduced cue-evoked dopamine concentrations and reward seeking. Moreover, we identified that 2AG, rather than anandamide, is the primary endocannabinoid responsible for facilitating the neural mechanisms of reward seeking. Thus, our findings reveal that the VTA endocannabinoid system is critical for the fine-tuned regulation of dopamine signaling that mediates reward-directed behavior.
Our data demonstrate the existence of a single neural signaling mechanism through which CB1 antagonists can effectively diminish the influence that environmental cues exert over motivated behavior. A number of studies have shown that the endocannabinoid system is involved in the appetitive-motivational aspects of reward-directed behavior. For example, motivation for both palatable foods (Ward and Dykstra, 2005) and drugs of abuse (Solinas et al., 2003; Xi et al., 2007) is decreased by pharmacological disruption of endocannabinoid signaling as assessed by break points under a progressive ratio schedule. A current theory holds that endocannabinoids are specifically involved in modulating the secondary/environmental influences on motivated behavior (Le Foll and Goldberg, 2004; Vries and Schoffelmeer, 2005). In support of this, when responding is maintained by conditioned cues (i.e. under a second order schedule), pharmacological disruption of endocannabinoid signaling decreases responding (Justinova et al., 2008). Moreover, endocannabinoid disruption is particularly effective at reducing cue-induced reinstatement, a model of relapse in humans that incorporates the influence of conditioned environmental stimuli on reward seeking (Epstein et al., 2006). In this model, CB1 receptor antagonists decrease the propensity for conditioned cues to reinstate responding for appetitive food (Ward et al., 2007) and various drugs of abuse (Justinova et al., 2008; Vries and Schoffelmeer, 2005). Importantly, the finding that disrupting endocannabinoid signaling decreases reward seeking regardless of the reinforcer paired with the cue (Vries and Schoffelmeer, 2005) implies that a common neural mechanism is involved through which endocannabinoids regulate cue-motivated behavior. Our data suggest that this common neural mechanism involves endocannabinoid disinhibition of cue-evoked dopamine cell firing in the VTA, as pharmacological disruption of endocannabinoid signaling within this brain region was sufficient to decrease cue-evoked dopamine concentrations and reward seeking behavior in unison. It is likely that following systemic administration of CB1 receptor antagonists; however, diminished surges in dopamine concentration interact with altered accumbal glutamate concentrations (Xi et al., 2007), possibly arising from the prefrontal cortex (Alvarez-Jaimes et al., 2008), to decrease reward seeking. Such an interaction would be consistent with the theory that accumbal dopamine affects reward seeking by modulating convergent cortical, hippocampal and amygdalar input (Brady and O’Donnell, 2004;Floresco et al., 2001). Furthermore, CB1 receptors within the NAc likely contribute to decreased reward seeking following systemic administration of CB1 receptor antagonists (Alvarez-Jaimes et al., 2008). Nevertheless, our findings that intrategmental disruption of endocannabinoid signaling alone simultaneously decreased cue-evoked dopamine concentrations and reward seeking suggests that the VTA endocannabinoid system is critically involved in mediating cue-motivated reward directed behavior.
We therefore predicted that increasing endocannabinoid levels would facilitate the neural mechanisms of reward seeking. VDM11 however, dose-dependently decreased cue-evoked dopamine signaling and reward seeking in a manner that is more consistent with VDM11 reducing presynaptic CB1 receptor activation. These findings are in agreement with recent reports demonstrating that endocannabinoid uptake inhibitors can decrease cue-induced reinstatement of drug-seeking behavior in a manner similar to rimonabant when assessed using self-administration (Gamaleddin et al., 2011) or conditioned place preference paradigms (Scherma et al., 2011). One possible mechanism explaining these findings is that VDM11 decreases CB1 receptor activation by interfering with the bi-directional release of endocannabinoids through a putative transport mechanism (Hillard et al., 1997; Melis et al., 2004;Ronesi et al., 2004). Another mechanistic explanation is that VDM11 might selectively increase anandamide (Van der Stelt et al 2006), which could function as a competitive antagonist at CB1 receptors in the presence of 2AG because, in contrast to 2AG, anandamide is a partial agonist at CB1 receptors (Howlett and Mukhopadhyay, 2000). These findings led us to investigate the respective contributions of 2AG and anandamide. 2AG, but not anandamide, increased motivation, reward seeking and cue-evoked dopamine concentrations. These data demonstrate that 2AG is the primary endocannabinoid that enhances the neural mechanisms of cue-motivated reward seeking and agree with reports demonstrating that 2AG is the principal endocannabinoid for multiple forms of synaptic plasticity across several brain regions (Melis et al., 2004; Tanimura et al., 2010).
Based on our data, we speculate that 2AG might modulate cue-evoked dopamine release through disinhibition of dopamine neurons in the VTA. When dopamine neurons fire at high frequencies they release 2AG (Melis et al, 2004), which then retrogradely binds to CB1 receptors on presynaptic terminals within the VTA (Lupica and Riegel, 2005). Although 2AG would affect both GABAergic and glutamatergic synaptic input through CB1 receptor activation (Matyas et al., 2008) – cue-encoding VTA dopamine neurons are theorized to form discrete neural assemblies with GABAergic synapses, thereby allowing for the fine-tuned regulation of dopamine neural activity during reward seeking (Lupica and Riegel, 2005; Matyas et al., 2008). According to this conceptualization, 2AG activation of CB1 receptors located on GABAergic terminals might decrease GABA release onto VTA dopamine neurons. The reduced GABA tone theoretically would decrease activation of GABA receptors on VTA dopamine neurons, thus resulting in a disinhibition of dopamine neural activity (Lupica and Riegel, 2005). The resulting disinhibition of dopamine neural activity is theorized to facilitate the neural mechanisms of reward seeking. It is important to clarify that using this freely moving recording approach, other mechanisms within the VTA may account for the observed findings.
We further speculate that endocannabinoid modulation of dopamine release from the VTA might affect NAc neural activity through a D1 receptor dependent mechanism. While recent evidence indicates that dopamine does not directly change postsynaptic excitability in the NAc (Stuber et al., 2010; Tecuapetla et al., 2010), it remains well accepted that dopamine can modulate input into the striatum, as occurs during reward seeking, to affect neural responses in a D1 receptor dependent manner (Cheer et al., 2007a; Goto and Grace, 2005; Reynolds et al., 2001). It is possible therefore, that the VTA endocannabinoid system might affect NAc neural activity by increasing D1 receptor occupancy. Recently developed computational models of dopamine signaling offer insight into how dopamine transients might influence NAc neural activity specifically through a D1 receptor-mediated mechanism (Dreyer et al., 2010). When dopamine neurons exhibit regular pacemaker firing, low concentrations (i.e. tonic) of dopamine are released throughout the NAc (Floresco et al., 2003). The computational model predicts that during tonic dopamine signaling, D2 receptors approach maximal occupancy whereas D1 receptors remain relatively unaffected (Dreyer et al., 2010). By contrast, when dopamine neurons fire at high frequency, transient bursts of dopamine are heterogeneously released into discrete microcircuits of the NAc (Dreyer et al., 2010; Wightman et al., 2007). When these higher concentration transients occur – D1 receptor occupancy theoretically increases precipitously whereas D2 receptors, which are already approaching maximal occupancy, remain relatively unaffected (Dreyer et al., 2010). Thus, we hypothesize that endocannabinoid disruption in the VTA might decrease NAc neural activity by preventing sufficient D1 receptor occupancy.
The present study offers previously unseen insights regarding the neural mechanisms underlying reward seeking motivated by conditioned cues. Our data demonstrate for the first time that 2AG within the VTA can modulate cue-evoked dopamine transients, which are theorized to promote reward seeking (Nicola, 2010; Phillips et al., 2003). While we (Cheer et al., 2007b) and others (Perra et al., 2005) have demonstrated that disrupting the VTA endocannabinoid system decreases drug-induced dopamine release, this is the first demonstration that the endocannabinoid system modulates cue-evoked dopamine transients during the pursuit of reward. Furthermore, our data suggest that drugs designed to specifically manipulate 2AG levels may prove to be effective pharmacotherapies for the treatment of neuropsychiatric disorders involving a maladaptive motivational state.
