Modulation of ventromedial orbitofrontal cortical glutamatergic activity affects the explore-exploit balance and influences value-based decision-making.

The balance between exploration and exploitation is essential for decision-making. The present study investigated the role of ventromedial orbitofrontal cortex (vmOFC) glutamate neurons in mediating value-based decision-making by first using optogenetics to manipulate vmOFC glutamate activity in rats during a probabilistic reversal learning (PRL) task. Rats that received vmOFC activation during informative feedback completed fewer reversals and exhibited reduced reward sensitivity relative to rats. Analysis with a Q-learning computational model revealed that increased vmOFC activity did not affect the learning rate but instead promoted maladaptive exploration. By contrast, vmOFC inhibition increased the number of completed reversals and increased exploitative behavior. In a separate group of animals, calcium activity of vmOFC glutamate neurons was recorded using fiber photometry. Complementing our results above, we found that suppression of vmOFC activity during the latter part of rewarded trials was associated with improved PRL performance, greater win-stay responding and selecting the correct choice on the next trial. These data demonstrate that excessive vmOFC activity during reward feedback disrupted value-based decision-making by increasing the maladaptive exploration of lower-valued options. Our findings support the premise that pharmacological interventions that normalize aberrant vmOFC glutamate activity during reward feedback processing may attenuate deficits in value-based decision-making.

Ventral Pallidum GABA Neurons Mediate Motivation Underlying Risky Choice.

Pursuing rewards while avoiding danger is an essential function of any nervous system. Here, we examine a new mechanism helping rats negotiate the balance between risk and reward when making high-stakes decisions. Specifically, we focus on GABA neurons within an emerging mesolimbic circuit nexus: the ventral pallidum (VP). These neurons play a distinct role from other VP neurons in simple motivated behaviors in mice, but their role in more complex motivated behaviors is unknown. Here, we interrogate the behavioral functions of VPGABA neurons in male and female transgenic GAD1:Cre rats (and WT littermates), using a reversible chemogenetic inhibition approach. Using a behavioral assay of risky decision-making, and of the food-seeking and shock-avoidance components of this task, we show that engaging inhibitory Gi/o signaling specifically in VPGABA neurons suppresses motivation to pursue highly salient palatable foods, and possibly also motivation to avoid being shocked. In contrast, inhibiting these neurons did not affect seeking of low-value food, free consumption of palatable food, or unconditioned affective responses to shock. Accordingly, when rats considered whether to pursue food despite potential for shock in a risky decision-making task, inhibiting VPGABA neurons caused them to more readily select a small but safe reward over a large but dangerous one, an effect not seen in the absence of shock threat. Together, results indicate that VPGABA neurons are critical for high-stakes adaptive responding that is necessary for survival, but which may also malfunction in psychiatric disorders.SIGNIFICANCE STATEMENT In a dynamic world, it is essential to implement appropriate behaviors under circumstances involving rewards, threats, or both. Here, we demonstrate a crucial role for VPGABA neurons in high-stakes motivated behavior of several types. We show that this VPGABA role in motivation impacts decision-making, as inhibiting these neurons yields a conservative, risk-averse strategy not seen when the task is performed without threat of shock. These new roles for VPGABA neurons in behavior may inform future strategies for treating addiction, and other disorders of maladaptive decision-making.

Activation of Pedunculopontine Glutamate Neurons Is Reinforcing.

Dopamine transmission from midbrain ventral tegmental area (VTA) neurons underlies behavioral processes related to motivation and drug addiction. The pedunculopontine tegmental nucleus (PPTg) is a brainstem nucleus containing glutamate-, acetylcholine-, and GABA-releasing neurons with connections to basal ganglia and limbic brain regions. Here we investigated the role of PPTg glutamate neurons in reinforcement, with an emphasis on their projections to VTA dopamine neurons. We used cell-type-specific anterograde tracing and optogenetic methods to selectively label and manipulate glutamate projections from PPTg neurons in mice. We used anatomical, electrophysiological, and behavioral assays to determine their patterns of connectivity and ascribe functional roles in reinforcement. We found that photoactivation of PPTg glutamate cell bodies could serve as a direct positive reinforcer on intracranial self-photostimulation assays. Further, PPTg glutamate neurons directly innervate VTA; photostimulation of this pathway preferentially excites VTA dopamine neurons and is sufficient to induce behavioral reinforcement. These results demonstrate that ascending PPTg glutamate projections can drive motivated behavior, and PPTg to VTA synapses may represent an important target relevant to drug addiction and other mental health disorders. SIGNIFICANCE STATEMENT: Uncovering brain circuits underlying reward-seeking is an important step toward understanding the circuit bases of drug addiction and other psychiatric disorders. The dopaminergic system emanating from the ventral tegmental area (VTA) plays a key role in regulating reward-seeking behaviors. We used optogenetics to demonstrate that the pedunculopontine tegmental nucleus sends glutamatergic projections to VTA dopamine neurons, and that stimulation of this circuit promotes behavioral reinforcement. The findings support a critical role for pedunculopontine tegmental nucleus glutamate neurotransmission in modulating VTA dopamine neuron activity and behavioral reinforcement.

Proportion and distribution of neurotransmitter-defined cell types in the ventral tegmental area and substantia nigra pars compacta.

Most studies on the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) have focused on dopamine neurons and their role in processes such as motivation, learning, movement, and associated disorders. However there has been increasing attention on other VTA and SNc cell types that release GABA, glutamate, or a combination of these neurotransmitters. Yet the relative distributions and proportions of neurotransmitter-defined cell types across VTA and SNc has remained unclear. Here, we used fluorescent in situ hybridization in male and female mice to label VTA and SNc neurons that expressed mRNA encoding the canonical vesicular transporters for dopamine, GABA, or glutamate: vesicular monoamine transporter VMAT2, vesicular GABA transporter (VGAT), and vesicular glutamate transporter (VGLUT2). Within VTA, we found that no one type was particularly more abundant, instead we observed similar numbers of VMAT2+ (44%), VGAT+ (37%) and VGLUT2+ (41%) neurons. In SNc we found that a slight majority of neurons expressed VMAT2 (54%), fewer were VGAT+ (42%), and VGLUT2+ neurons were least abundant (16%). Moreover, 20% of VTA neurons and 10% of SNc neurons expressed more than one vesicular transporter, including 45% of VGLUT2 neurons. We also assessed within VTA and SNc subregions and found remarkable heterogeneity in cell-type composition. And by quantifying density across both anterior-posterior and medial-lateral axes we generated heatmaps to visualize the distribution of each cell type. Our data complement recent single-cell RNAseq studies and support a more diverse landscape of neurotransmitter-defined cell types in VTA and SNc than is typically appreciated.

Postnatal Phencyclidine-Induced Deficits in Decision Making Are Ameliorated by Optogenetic Inhibition of Ventromedial Orbitofrontal Cortical Glutamate Neurons.

Background: The orbitofrontal cortex (OFC) is essential for decision making, and functional disruptions within the OFC are evident in schizophrenia. Postnatal phencyclidine (PCP) administration in rats is a neurodevelopmental manipulation that induces schizophrenia-relevant cognitive impairments. We aimed to determine whether manipulating OFC glutamate cell activity could ameliorate postnatal PCP-induced deficits in decision making. Methods: Male and female Wistar rats (n = 110) were administered saline or PCP on postnatal days 7, 9, and 11. In adulthood, we expressed YFP (yellow fluorescent protein) (control), ChR2 (channelrhodopsin-2) (activation), or eNpHR 3.0 (enhanced halorhodopsin) (inhibition) in glutamate neurons within the ventromedial OFC (vmOFC). Rats were tested on the probabilistic reversal learning task once daily for 20 days while we manipulated the activity of vmOFC glutamate cells. Behavioral performance was analyzed using a Q-learning computational model of reinforcement learning. Results: Compared with saline-treated rats expressing YFP, PCP-treated rats expressing YFP completed fewer reversals, made fewer win-stay responses, and had lower learning rates. We induced similar performance impairments in saline-treated rats by activating vmOFC glutamate cells (ChR2). Strikingly, PCP-induced performance deficits were ameliorated when the activity of vmOFC glutamate cells was inhibited (halorhodopsin). Conclusions: Postnatal PCP-induced deficits in decision making are associated with hyperactivity of vmOFC glutamate cells. Thus, normalizing vmOFC activity may represent a potential therapeutic target for decision-making deficits in patients with schizophrenia.

Mesoaccumbal glutamate neurons drive reward via glutamate release but aversion via dopamine co-release.

Ventral tegmental area (VTA) projections to the nucleus accumbens (NAc) drive reward-related motivation. Although dopamine neurons are predominant, a substantial glutamatergic projection is also present, and a subset of these co-release both dopamine and glutamate. Optogenetic stimulation of VTA glutamate neurons not only supports self-stimulation but can also induce avoidance behavior, even in the same assay. Here, we parsed the selective contribution of glutamate or dopamine co-release from VTA glutamate neurons to reinforcement and avoidance. We expressed channelrhodopsin-2 (ChR2) in mouse VTA glutamate neurons in combination with CRISPR-Cas9 to disrupt either the gene encoding vesicular glutamate transporter 2 (VGLUT2) or tyrosine hydroxylase (Th). Selective disruption of VGLUT2 abolished optogenetic self-stimulation but left real-time place avoidance intact, whereas CRISPR-Cas9 deletion of Th preserved self-stimulation but abolished place avoidance. Our results demonstrate that glutamate release from VTA glutamate neurons is positively reinforcing but that dopamine release from VTA glutamate neurons can induce avoidance behavior.

Ventral pallidum GABA and glutamate neurons drive approach and avoidance through distinct modulation of VTA cell types.

The ventral pallidum (VP) contains GABA and glutamate neurons projecting to ventral tegmental area (VTA) whose stimulation drives approach and avoidance, respectively. Yet little is known about the mechanisms by which VP cell types shape VTA activity and drive behavior. Here, we found that both VP GABA and glutamate neurons were activated during approach to reward or by delivery of an aversive stimulus. Stimulation of VP GABA neurons inhibited VTA GABA, but activated dopamine and glutamate neurons. Remarkably, stimulation-evoked activation was behavior-contingent such that VTA recruitment was inhibited when evoked by the subject's own action. Conversely, VP glutamate neurons activated VTA GABA, as well as dopamine and glutamate neurons, despite driving aversion. However, VP glutamate neurons evoked dopamine in aversion-associated ventromedial nucleus accumbens (NAc), but reduced dopamine release in reward-associated dorsomedial NAc. These findings show how heterogeneous VP projections to VTA can be engaged to shape approach and avoidance behaviors.

In vivo visualization of delta opioid receptors upon physiological activation uncovers a distinct internalization profile.

G-protein-coupled receptors (GPCRs) mediate numerous physiological functions and represent prime therapeutic targets. Receptor trafficking upon agonist stimulation is critical for GPCR function, but examining this process in vivo remains a true challenge. Using knock-in mice expressing functional fluorescent delta opioid receptors under the control of the endogenous promoter, we visualized in vivo internalization of this native GPCR upon physiological stimulation. We developed a paradigm in which animals were made dependent on morphine in a drug-paired context. When re-exposed to this context in a drug-free state, mice showed context-dependent withdrawal signs and activation of the hippocampus. Receptor internalization was transiently detected in a subset of CA1 neurons, uncovering regionally restricted opioid peptide release. Importantly, a pool of surface receptors always remained, which contrasts with the in vivo profile previously established for exogenous drug-induced internalization. Therefore, a distinct response is observed at the receptor level upon a physiological or pharmacological stimulation. Altogether, direct in vivo GPCR visualization enables mapping receptor stimulation promoted by a behavioral challenge and represents a powerful approach to study endogenous GPCR physiology.

Ventral pallidum GABA and glutamate neurons drive approach and avoidance through distinct modulation of VTA cell types.

The ventral pallidum (VP) contains GABA and glutamate (Glut) neurons projecting to ventral tegmental area (VTA) whose stimulation drives approach and avoidance, respectively. Yet little is known about the cell-type-specific mechanisms by which VP projections to VTA drive behavior. Here, we found that both VP GABA and Glut neurons were activated during approach to reward or delivery of an aversive stimulus. Stimulation of VP GABA neurons inhibited VTA GABA, but activated dopamine (DA) and glutamate neurons. Remarkably, this cell-type-specific recruitment was behavior-contingent such that VTA recruitment was inhibited when evoked by the subject's own action. Conversely, VP Glut neurons activated VTA GABA, as well as DA and Glut neurons, despite driving aversion. However, VP Glut neurons evoked DA in reward-associated ventromedial nucleus accumbens (NAc), but reduced DA in aversion-associated dorsomedial NAc. These findings show how heterogeneous VP cell types can engage VTA cell types to shape approach and avoidance behaviors.

Neurotensin receptor 1-biased ligand attenuates neurotensin-mediated excitation of ventral tegmental area dopamine neurons and dopamine release in the nucleus accumbens.

Strong expression of the G protein-coupled receptor (GPCR) neurotensin receptor 1 (NTR1) in ventral tegmental area (VTA) dopamine (DA) neurons and terminals makes it an attractive target to modulate DA neuron activity and normalize DA-related pathologies. Recent studies have identified a novel class of NTR1 ligand that shows promising effects in preclinical models of addiction. A lead molecule, SBI-0654553 (SBI-553), can act as a positive allosteric modulator of NTR1 ß-arrestin recruitment while simultaneously antagonizing NTR1 Gq protein signaling. Using cell-attached recordings from mouse VTA DA neurons we discovered that, unlike neurotensin (NT), SBI-553 did not independently increase spontaneous firing. Instead, SBI-553 blocked the NT-mediated increase in firing. SBI-553 also antagonized the effects of NT on dopamine D2 auto-receptor signaling, potentially through its inhibitory effects on G-protein signaling. We also measured DA release directly, using fast-scan cyclic voltammetry in the nucleus accumbens and observed antagonist effects of SBI-553 on an NT-induced increase in DA release. Further, in vivo administration of SBI-553 did not notably change basal or cocaine-evoked DA release measured in NAc using fiber photometry. Overall, these results indicate that SBI-553 blunts NT's effects on spontaneous DA neuron firing, D2 auto-receptor function, and DA release, without independently affecting these measures. In the presence of NT, SBI-553 has an inhibitory effect on mesolimbic DA activity, which could contribute to its efficacy in animal models of psychostimulant use.

Mouse δ opioid receptors are located on presynaptic afferents to hippocampal pyramidal cells.

Delta opioid receptors participate in the control of chronic pain and emotional responses. Recent data have also identified their implication in drug-context associations pointing to a modulatory role on hippocampal activity. We used fluorescent knock-in mice that express a functional delta opioid receptor fused at its carboxy terminus with the green fluorescent protein in place of the native receptor to investigate the receptor neuroanatomical distribution in this structure. Fine mapping of the pyramidal layer was performed in hippocampal acute brain slices and organotypic cultures using fluorescence confocal imaging, co-localization with pre- and postsynaptic markers and correlative light-electron microscopy. The different approaches concurred to identify delta opioid receptors on presynaptic afferents to glutamatergic principal cells. In the latter, only scarce receptors were detected that were confined within the Golgi or vesicular intracellular compartments with no receptor present at the cell surface. In the mouse hippocampus, expression of functional delta opioid receptors is therefore mostly associated with interneurons emphasizing a presynaptic modulatory effect on the pyramidal cell firing rate.

VTA Glutamate Neuron Activity Drives Positive Reinforcement Absent Dopamine Co-release.

Like ventral tegmental area (VTA) dopamine (DA) neurons, VTA glutamate neuron activity can support positive reinforcement. However, a subset of VTA neurons co-release DA and glutamate, and DA release might be responsible for behavioral reinforcement induced by VTA glutamate neuron activity. To test this, we used optogenetics to stimulate VTA glutamate neurons in which tyrosine hydroxylase (TH), and thus DA biosynthesis, was conditionally ablated using either floxed Th mice or viral-based CRISPR/Cas9. Both approaches led to loss of TH expression in VTA glutamate neurons and loss of DA release from their distal terminals in nucleus accumbens (NAc). Despite loss of the DA signal, optogenetic activation of VTA glutamate cell bodies or axon terminals in NAc was sufficient to support reinforcement. These results suggest that glutamate release from VTA is sufficient to promote reinforcement independent of concomitant DA co-release, establishing a non-DA mechanism by which VTA activity can support reward-seeking behaviors.

Ventral pallidum is essential for cocaine relapse after voluntary abstinence in rats.

Addiction is a chronic relapsing disorder, and during recovery many people experience several relapse events as they attempt to voluntarily abstain from drug. New preclinical relapse models have emerged that capture this common human experience, and mounting evidence indicates that resumption of drug seeking after voluntary abstinence recruits neural circuits distinct from those recruited during reinstatement after experimenter-imposed abstinence, or abstinence due to extinction training. Ventral pallidum (VP), a key limbic node involved in drug seeking, has well-established roles in conventional reinstatement models tested following extinction training, but it is unclear whether this region also participates in more translationally relevant models of relapse. Here we show that chemogenetic inhibition of VP neurons decreased cocaine-, context-, and cue-induced relapse tested after voluntary, punishment-induced abstinence. This effect was strongest in the most compulsive, punishment-resistant rats, and reinstatement was associated with neural activity in anatomically defined VP subregions. VP inhibition also attenuated the propensity of rats to display "abortive lever pressing," a species-typical risk assessment behavior seen here during punished drug taking, likely resulting from concurrent approach and avoidance motivations. These results indicate that VP, unlike other connected limbic brain regions, is essential for resumption of drug seeking after voluntary abstinence. Since VP inhibition effects were strongest in the most compulsively cocaine-seeking individuals, this may also indicate that VP plays a particularly important role in the most pathological, addiction-like behavior, making it an attractive target for future therapeutic interventions.

Opponent control of behavioral reinforcement by inhibitory and excitatory projections from the ventral pallidum.

The ventral pallidum (VP) lies at the interface between sensory, motor, and cognitive processing-with a particular role in mounting behavioral responses to rewards. Though the VP is predominantly GABAergic, glutamate neurons were recently identified, though their relative abundances and respective roles are unknown. Here, we show that VP glutamate neurons are concentrated in the rostral ventromedial VP and project to qualitatively similar targets as do VP GABA neurons. At the functional level, we used optogenetics to show that activity in VP GABA neurons can drive positive reinforcement, particularly through projections to the ventral tegmental area (VTA). On the other hand, activation of VP glutamate neurons leads to behavioral avoidance, particularly through projections to the lateral habenula. These findings highlight cell-type and projection-target specific roles for VP neurons in behavioral reinforcement, dysregulation of which could contribute to the emergence of negative symptoms associated with drug addiction and other neuropsychiatric disease.

Ventral tegmental area glutamate neurons co-release GABA and promote positive reinforcement.

In addition to dopamine neurons, the ventral tegmental area (VTA) contains GABA-, glutamate- and co-releasing neurons, and recent reports suggest a complex role for the glutamate neurons in behavioural reinforcement. We report that optogenetic stimulation of VTA glutamate neurons or terminals serves as a positive reinforcer on operant behavioural assays. Mice display marked preference for brief over sustained VTA glutamate neuron stimulation resulting in behavioural responses that are notably distinct from dopamine neuron stimulation and resistant to dopamine receptor antagonists. Whole-cell recordings reveal EPSCs following stimulation of VTA glutamate terminals in the nucleus accumbens or local VTA collaterals; but reveal both excitatory and monosynaptic inhibitory currents in the ventral pallidum and lateral habenula, though the net effects on postsynaptic firing in each region are consistent with the observed rewarding behavioural effects. These data indicate that VTA glutamate neurons co-release GABA in a projection-target-dependent manner and that their transient activation drives positive reinforcement.

Afferent Inputs to Neurotransmitter-Defined Cell Types in the Ventral Tegmental Area.

The ventral tegmental area (VTA) plays a central role in the neural circuit control of behavioral reinforcement. Though considered a dopaminergic nucleus, the VTA contains substantial heterogeneity in neurotransmitter type, containing also GABA and glutamate neurons. Here, we used a combinatorial viral approach to transsynaptically label afferents to defined VTA dopamine, GABA, or glutamate neurons. Surprisingly, we find that these populations received qualitatively similar inputs, with dominant and comparable projections from the lateral hypothalamus, raphe, and ventral pallidum. However, notable differences were observed, with striatal regions and globus pallidus providing a greater share of input to VTA dopamine neurons, cortical input preferentially on to glutamate neurons, and GABA neurons receiving proportionally more input from the lateral habenula and laterodorsal tegmental nucleus. By comparing inputs to each of the transmitter-defined VTA cell types, this study sheds important light on the systems-level organization of diverse inputs to VTA.

Tyramide Signal Amplification for Immunofluorescent Enhancement.

Enzyme-linked signal amplification is a key technique used to enhance the immunohistochemical detection of protein, mRNA, and other molecular species. Tyramide signal amplification (TSA) is based on a catalytic reporter deposit in close vicinity to the epitope of interest. The advantages of this technique are its simplicity, enhanced sensitivity, high specificity, and compatibility with modern multi-label fluorescent microscopy. Here, we describe the use of a TSA kit to increase the signal of enhanced green fluorescent protein (eGFP) expressed under the control of Slc17a6 regulatory elements in the brain of a transgenic mouse. The labeling procedure consists of 6 basic steps: (1) tissue preparation, (2) blocking of nonspecific epitopes, (3) binding with primary antibody, (4) binding with horseradish peroxidase-conjugated secondary antibody, (5) reacting with fluorescent tyramide substrate, and (6) imaging of the signal. The procedures described herein detail these steps and provide additional guidance and background to assist novice users.

A mu-delta opioid receptor brain atlas reveals neuronal co-occurrence in subcortical networks.

Opioid receptors are G protein-coupled receptors (GPCRs) that modulate brain function at all levels of neural integration, including autonomic, sensory, emotional and cognitive processing. Mu (MOR) and delta (DOR) opioid receptors functionally interact in vivo, but whether interactions occur at circuitry, cellular or molecular levels remains unsolved. To challenge the hypothesis of MOR/DOR heteromerization in the brain, we generated redMOR/greenDOR double knock-in mice and report dual receptor mapping throughout the nervous system. Data are organized as an interactive database offering an opioid receptor atlas with concomitant MOR/DOR visualization at subcellular resolution, accessible online. We also provide co-immunoprecipitation-based evidence for receptor heteromerization in these mice. In the forebrain, MOR and DOR are mainly detected in separate neurons, suggesting system-level interactions in high-order processing. In contrast, neuronal co-localization is detected in subcortical networks essential for survival involved in eating and sexual behaviors or perception and response to aversive stimuli. In addition, potential MOR/DOR intracellular interactions within the nociceptive pathway offer novel therapeutic perspectives.

In vivo neuronal co-expression of mu and delta opioid receptors uncovers new therapeutic perspectives.

Opioid receptors belong to the G protein coupled receptor family. They modulate brain function at all levels of neural integration and therefore impact on autonomous, sensory, emotional and cognitive processing. In vivo functional interaction between mu and delta opioid receptors are known to take place though it is still debated whether interactions occur at circuitry, cellular or molecular level. Also, the notion of receptor crosstalk via mu-delta heteromers is well documented in vitro but in vivo evidence remains scarce. To identify neurons in which receptor interactions could take place, we designed a unique double mutant knock-in mouse line that expresses functional red-fluorescent mu receptors and green-fluorescent delta receptors. We mapped mu and delta receptor distribution and co-localization throughout the nervous system and created the first interactive brain atlas with concomitant mu-delta visualization at subcellular resolution (http://mordor.ics-mci.fr/). Mu and delta receptors co-localize in neurons from subcortical networks but are mainly detected in separate neurons in the forebrain. Also, co-immunoprecipitation experiments indicated physical proximity in the hippocampus, a prerequisite to mu-delta heteromerization. Altogether, data suggest that mu-delta functional interactions take place at systems level for high-order emotional and cognitive processing whereas mu-delta may interact at cellular level in brain networks essential for survival, which has potential implications for innovative drug design in pain control, drug addiction and eating disorders.

Cues predicting drug or food reward restore morphine-induced place conditioning in mice lacking delta opioid receptors.

RATIONALE: The exact role of delta opioid receptors in drug-induced conditioned place preference (CPP) remains debated. Under classical experimental conditions, morphine-induced CPP is decreased in mice lacking delta opioid receptors (Oprd1 (-/-)). Morphine self-administration, however, is maintained, suggesting that drug-context association rather than drug reward is deficient in these animals. OBJECTIVES: This study further examined the role of delta opioid receptors in mediating drug-cue associations, which are necessary for the expression of morphine-induced CPP. METHODS: We first identified experimental conditions under which Oprd1 (-/-) mice are able to express CPP to morphine (5, 10 or 20 mg/kg) in a drug-free state and observed that, in this paradigm, CPP was dependent on circadian time conditions. We then took advantage of this particularity to assess the ability of various cues (internal or discrete), predicting either drug or food reward, to restore CPP induced by morphine (10 mg/kg) in Oprd1 (-/-) mice in conditions under which they normally fail to express CPP. RESULTS: We found that presentation of circadian, drug or auditory cues, predicting morphine or food reward, restored morphine CPP in Oprd1 (-/-) mice, which then performed as well as control mice. CONCLUSIONS: This study reveals that, in contrast to spatial cues, internal or discrete morphine-predicting stimuli permit full expression of morphine CPP in Oprd1 (-/-) mice. Delta receptors, therefore, appear to play a crucial role in modulating spatial contextual cue-related responses. This activity may be critical when context gains control over behavior, as is the case for context-induced relapse in drug abuse.