Serotonin Signaling in Hippocampus during Initial Cocaine Abstinence Drives Persistent Drug Seeking.

The initiation of abstinence after chronic drug self-administration is stressful. Cocaine-seeking behavior on the first day of the absence of the expected drug (Extinction Day 1, ED1) is reduced by blocking 5-HT signaling in dorsal hippocampal cornu ammonis 1 (CA1) in both male and female rats. We hypothesized that the experience of ED1 can substantially influence later relapse behavior and that dorsal raphe (DR) serotonin (5-HT) input to CA1 may be involved. We inhibited 5-HT1A/1B receptors (WAY-100635 plus GR-127935), or DR input (chemogenetics), in CA1 on ED1 to test the role of this pathway on cocaine-seeking persistence 2 weeks later. We also inhibited 5-HT1A or 5-HT1B receptors in CA1 during conditioned place preference (CPP) for cocaine, to examine mechanisms involved in the persistent effects of ED1 manipulations. Inhibition of DR inputs, or 5-HT1A/1B signaling, in CA1 decreased drug seeking on ED1 and decreased cocaine seeking 2 weeks later revealing that 5-HT signaling in CA1 during ED1 contributes to persistent drug seeking during abstinence. In addition, 5-HT1B antagonism alone transiently decreased drug-associated memory performance when given prior to a CPP test, whereas similar antagonism of 5-HT1A alone had no such effect but blocked CPP retrieval on a test 24 h later. These CPP findings are consistent with prior work showing that DR inputs to CA1 augment recall of the drug-associated context and drug seeking via 5-HT1B receptors and prevent consolidation of the updated nondrug context via 5-HT1A receptors. Thus, treatments that modulate 5-HT-dependent memory mechanisms in CA1 during initial abstinence may facilitate later maintenance of abstinence.

Locus coeruleus and dorsal cingulate morphology contributions to slowed processing speed.

Slowed information processing speed is a defining feature of cognitive aging. Nucleus locus coeruleus (LC) and medial prefrontal regions are targets for understanding slowed processing speed because these brain regions influence neural and behavioral response latencies through their roles in optimizing task performance. Although structural measures of medial prefrontal cortex have been consistently related to processing speed, it is unclear if 1) declines in LC structure underlie this association because of reciprocal connections between LC and medial prefrontal cortex, or 2) if LC declines provide a separate explanation for age-related changes in processing speed. LC and medial prefrontal structural measures were predicted to explain age-dependent individual differences in processing speed in a cross-sectional sample of 43 adults (19-79 years; 63% female). Higher turbo-spin echo LC contrast, based on a persistent homology measure, and greater dorsal cingulate cortical thickness were significantly and each uniquely related to faster processing speed. However, only dorsal cingulate cortical thickness appeared to statistically mediate age-related differences in processing speed. The results suggest that individual differences in cognitive processing speed can be attributed, in part, to structural variation in nucleus LC and medial prefrontal cortex, with the latter key to understanding why older adults exhibit slowed processing speed.

Prelimbic and infralimbic medial prefrontal cortex neuron activity signals cocaine seeking variables across multiple timescales.

RATIONALE AND OBJECTIVES: The prefrontal cortex is critical for execution and inhibition of reward seeking. Neural manipulation of rodent medial prefrontal cortex (mPFC) subregions differentially impacts execution and inhibition of cocaine seeking. Dorsal, or prelimbic (PL), and ventral, or infralimbic (IL) mPFC are implicated in cocaine seeking or extinction of cocaine seeking, respectively. This differentiation is not seen across all studies, indicating that further research is needed to understand specific mPFC contributions to drug seeking. METHODS: We recorded neuronal activity in mPFC subregions during cocaine self-administration, extinction, and cue- and cocaine-induced reinstatement of cocaine seeking. RESULTS: Both PL and IL neurons were phasically responsive around lever presses during cocaine self-administration, and activity in both areas was reduced during extinction. During both cue- and, to a greater extent, cocaine-induced reinstatement, PL neurons exhibited significantly elevated responses, in line with previous studies demonstrating a role for the region in relapse. The enhanced PL signaling in cocaine-induced reinstatement was driven by strong excitation and inhibition in different groups of neurons. Both of these response types were stronger in PL vs. IL neurons. Finally, we observed tonic changes in activity in all tasks phases, reflecting both session-long contextual modulation as well as minute-to-minute activity changes that were highly correlated with brain cocaine levels and motivation associated with cocaine seeking. CONCLUSIONS: Although some differences were observed between PL and IL neuron activity across sessions, we found no evidence of a go/stop dichotomy in PL/IL function. Instead, our results demonstrate temporally heterogeneous prefrontal signaling during cocaine seeking and extinction in both PL and IL, revealing novel and complex functions for both regions during these behaviors. This combination of findings argues that mPFC neurons, in both PL and IL, provide multifaceted contributions to the regulation of drug seeking and addiction.

Orexin Reserve: A Mechanistic Framework for the Role of Orexins (Hypocretins) in Addiction.

In 2014, we proposed that orexin signaling transformed motivationally relevant states into adaptive behavior directed toward exploiting an opportunity or managing a threat, a process we referred to as motivational activation. Advancements in animal models since then have permitted higher-resolution measurements of motivational states; in particular, the behavioral economics approach for studying drug demand characterizes conditions that lead to the enhanced motivation that underlies addiction. This motivational plasticity is paralleled by persistently increased orexin expression in a topographically specific manner-a finding confirmed across species, including in humans. Normalization of orexin levels also reduces drug motivation in addiction models. These new advancements lead us to update our proposed framework for the orexin function. We now propose that the capacity of orexin neurons to exhibit dynamic shifts in peptide production contributes to their role in adaptive motivational regulation and that this is achieved via a pool of reserve orexin neurons. This reserve is normally bidirectionally recruited to permit motivational plasticity that promotes flexible, adaptive behavior. In pathological states such as addiction, however, we propose that the orexin system loses capacity to adaptively adjust peptide production, resulting in focused hypermotivation for drug, driven by aberrantly and persistently high expression in the orexin reserve pool. This mechanistic framework has implications for the understanding and treatment of several psychiatric disorders beyond addiction, particularly those characterized by motivational dysfunction.

Rat Anterior Cingulate Cortex Continuously Signals Decision Variables in a Patch Foraging Task.

In patch foraging tasks, animals must decide whether to remain with a depleting resource or to leave it in search of a potentially better source of reward. In such tasks, animals consistently follow the general predictions of optimal foraging theory (the marginal value theorem; MVT): to leave a patch when the reward rate in the current patch depletes to the average reward rate across patches. Prior studies implicate an important role for the anterior cingulate cortex (ACC) in foraging decisions based on MVT: within single trials, ACC activity increases immediately preceding foraging decisions, and across trials, these dynamics are modulated as the value of staying in the patch depletes to the average reward rate. Here, we test whether these activity patterns reflect dynamic encoding of decision-variables and whether these signals are directly involved in decision-making. We developed a leaky accumulator model based on the MVT that generates estimates of decision variables within and across trials, and tested model predictions against ACC activity recorded from male rats performing a patch foraging task. Model predicted changes in MVT decision variables closely matched rat ACC activity. Next, we pharmacologically inactivated ACC in male rats to test the contribution of these signals to decision-making. ACC inactivation had a profound effect on rats' foraging decisions and response times (RTs) yet rats still followed the MVT decision rule. These findings indicate that the ACC encodes foraging-related variables for reasons unrelated to patch-leaving decisions.SIGNIFICANCE STATEMENT The ability to make adaptive patch-foraging decisions, to remain with a depleting resource or search for better alternatives, is critical to animal well-being. Previous studies have found that anterior cingulate cortex (ACC) activity is modulated at different points in the foraging decision process, raising questions about whether the ACC guides ongoing decisions or serves a more general purpose of regulating cognitive control. To investigate the function of the ACC in foraging, the present study developed a dynamic model of behavior and neural activity, and tested model predictions using recordings and inactivation of ACC. Findings revealed that ACC continuously signals decision variables but that these signals are more likely used to monitor and regulate ongoing processes than to guide foraging decisions.

Novelty preference does not predict trait cocaine behaviors in male rats.

Heightened novelty seeking is a risk factor for the initiation of drug use and development of substance use disorders. In rats, novelty seeking can be examined by assessing preference for a novel environment. Some evidence indicates that high novelty preferring (HNP) rats have higher drug intake compared to low novelty preferring (LNP) rats, although these data are mixed. Moreover, the extent to which the HNP phenotype can predict other initial drug behaviors, including economic demand for cocaine, has not been tested. Here, we screened a cohort (n=60) of male rats for novelty preference and several subsequent cocaine behaviors, including locomotor reactivity to a cocaine priming injection, acquisition of cocaine self-administration, as well as cocaine demand using a within-session behavioral economics procedure. Novelty preference did not correlate with cocaine behaviors, nor were there any differences between HNP and LNP rats identified using a median split strategy. Moreover, regression analyses indicated that novelty preference did not have predictive utility for any of the cocaine behaviors tested. Thus, the extent to which the novelty preference trait can predict initial cocaine-related behaviors in male rats may be limited. This is in contrast to the novel locomotor reactivity phenotype, which is strongly linked with initial cocaine intake, indicating that these traits are distinct and differentially predict cocaine behaviors in rats.

Hormonal milieu drives economic demand for cocaine in female rats.

There are substantial sex differences in drug abuse, and a key feature of cocaine addiction is pathologically high motivation for drug. We investigated the role of ovarian hormones on cocaine demand in female rats using a within-session threshold behavioral economics (BE) procedure, which allows us to compare motivation for drug across hormonal states and sex while controlling for differences in dose and intake. This approach quantifies demand elasticity (α) and free consumption (Q0, consumption at null effort) to determine motivation for cocaine. Overall, female rats showed greater motivation for cocaine compared to males. However, this difference was cycle phase-dependent - motivation for cocaine when females were in proestrus was lower compared to the same animals across cycle phases, and overall similar to that of males. Hormonal cycle phase accounted for 70% of the within-subject variance in demand elasticity, obscuring other individual differences in female demand. High serum progesterone (P4; e.g., in proestrus) predicted decreased cocaine motivation (high demand elasticity), whereas serum estradiol (E2) correlated to greater intake at null effort (Q0). However, individual differences were revealed across OVX females, who displayed a range of demand elasticity, as seen in males. E2 replacement in OVX females increased motivation for cocaine, whereas P4 replacement decreased motivation. We also found that as few as 4 weeks of cocaine self-administration accelerated estropause in female rats as young as 12 weeks old. By 13 weeks of self-administration, proestrus epochs were no longer observed, and cocaine demand was potentiated by persistent estrus in all females. Thus, P4 signaling is a key modulator of cocaine demand in females that may underlie previously observed sex differences in addiction phenotypes.

Orexin-1 receptor signaling in ventral tegmental area mediates cue-driven demand for cocaine.

Drug-associated sensory cues increase motivation for drug and the orexin system is importantly involved in this stimulus-enhanced motivation. Ventral tegmental area (VTA) is a major target by which orexin signaling modulates reward behaviors, but it is unknown whether this circuit is necessary for cue-driven motivation for cocaine. Here, we investigated the role of VTA orexin signaling in cue-driven motivation for cocaine using a behavioral economics (BE) paradigm. We found that infusion of the orexin-1 receptor (Ox1R) antagonist SB-334867 (SB) into VTA prior to BE testing reduced motivation when animals were trained to self-administer cocaine with discrete cues and tested on BE with those cues. SB had no effect when animals were trained to self-administer cocaine without cues or tested on BE without cues, indicating that learning to associate cues with drug delivery during self-administration training was necessary for cues to recruit orexin signaling in VTA. These effects were specific to VTA, as injections of SB immediately dorsal had no effect. Moreover, intra-VTA SB did not have an impact on locomotor activity, or low- or high-effort consumption of sucrose. Finally, we microinjected a novel retrograde adeno-associated virus (AAVretro) containing an orexin-specific short hairpin RNA (OxshRNA) into VTA to knock down orexin in the hypothalamus-VTA circuit. These injections significantly reduced orexin expression in lateral hypothalamus (LH) and decreased cue-driven motivation. These studies demonstrate a role for orexin signaling in VTA, specifically when cues predict drug reward.

Chemogenetic inhibition of trigeminal ganglion neurons attenuates behavioural and neural pain responses in a model of trigeminal neuropathic pain.

BACKGROUND: Nerve injury can lead to ectopic activation of injured nociceptorsand central sensitization characterized by allodynia and hyperalgesia. Reduction in the activity of primary afferent neurons has been shown to be sufficient in alleviating peripherally generated pain. The cell bodies of such trigeminal nociceptors are located in the trigeminal ganglia (TG) with central processes that terminate in the brainstem trigeminal nucleus caudalis (TNC). The TG is therefore a strategic locus where afferent input can be manipulated. We hypothesized that chemogenetic inhibition of TG would suppress TNC neuronal activity and attenuate pain behaviour in a rat model of painful traumatic trigeminal neuropathy (PTTN). METHODS: Trigeminal neuropathic pain was induced in adult male Sprague-Dawley rats (n = 24) via chronic constriction injury to the infraorbital nerve (ION-CCI). Naïve and sham rats were used as controls (n = 20/group). Rats within each group received TG-directed microinjections of AAV virus containing either the inhibitory hM4Di-DREADD construct or EGFP. RESULTS: In the ION-CCI group, systemic administration of the DREADD agonist clozapine N-oxide (CNO) reversed the hypersensitivity phenotype in animals expressing hM4Di but not EGFP. CNO-mediated activation of hM4Di DREADD in ION-CCI animals was also associated with reduced Fos expression in the TNC elicited by repeated mechanical stimulation of the dermatome ipsilateral to the injury. There was no effect of CNO on pain behaviour or TNC Fos expression in eGFP animals. CONCLUSION: Our results indicate that DREADDs may offer an effective therapeutic approach for treatment of trigeminal neuropathic pain. SIGNIFICANCE: Trigeminal neuropathic pain is highly resistant to therapy and we are in dire need of novel approaches. This study provides further evidence for the successful application of DREADDs as an effective tool for modulating central nervous system function. CNO mediated activation of hM4Di-DREADDs in the trigeminal ganglion (TG) attenuates nerve injury induced neuropathic pain by acting on hyperactive TG cells. It also establishes the TG as an effective target to manage pain in the face and head. Accessing the TG in clinical populations is a relatively simple and safe procedure, making this approach highly significant. Moreover, the methodology described here has applications in trigeminal neuropathic pain from traumatic other etiologies and in spinal neuropathic pain. Chronic pain syndromes are characterized by a progressive failure of brain centers to adequately inhibit pain and as these are identified, we may be able to target them for therapy. Therefore, our findings might have wide application in chronic pain syndromes.

A common limiter circuit for opioid choice and relapse identified in a rodent addiction model.

Activity in numerous brain regions drives heroin seeking, but no circuits that limit heroin seeking have been identified. Furthermore, the neural circuits controlling opioid choice are unknown. In this study, we examined the role of the infralimbic cortex (IL) to nucleus accumbens shell (NAshell) pathway during heroin choice and relapse. This model yielded subpopulations of heroin versus food preferring rats during choice, and choice was unrelated to subsequent relapse rates to heroin versus food cues, suggesting that choice and relapse are distinct behavioral constructs. Supporting this, inactivation of the IL with muscimol produced differential effects on opioid choice versus relapse. A pathway-specific chemogenetic approach revealed, however, that the IL-NAshell pathway acts as a common limiter of opioid choice and relapse. Furthermore, dendritic spines in IL-NAshell neurons encode distinct aspects of heroin versus food reinforcement. Thus, opioid choice and relapse share a common addiction-limiting circuit in the IL-NAshell pathway.

The Insomnia-Addiction Positive Feedback Loop: Role of the Orexin System.

Significant sleep impairments often accompany substance use disorders (SUDs). Sleep disturbances in SUD patients are associated with poor clinical outcomes and treatment adherence, emphasizing the importance of normalizing sleep when treating SUDs. Orexins (hypocretins) are neuropeptides exclusively produced by neurons in the posterior hypothalamus that regulate various behavioral and physiological processes, including sleep-wakefulness and motivated drug taking. Given its dual role in sleep and addiction, the orexin system represents a promising therapeutic target for treating SUDs and their comorbid sleep deficits. Here, we review the literature on the role of the orexin system in sleep and drug addiction and discuss the therapeutic potential of orexin receptor antagonists for SUDs. We argue that orexin receptor antagonists may be effective therapeutics for treating addiction because they target orexin's regulation of sleep (top-down) and motivation (bottom-up) pathways.

The sensation seeking trait confers a dormant susceptibility to addiction that is revealed by intermittent cocaine self-administration in rats.

Heightened sensation seeking is associated with an increased risk of substance use disorder in clinical populations. In rats, sensation seeking is often examined by measuring locomotor reactivity to a novel environment. So-called high responders (HR) acquire self-administration of psychostimulants more quickly and consume higher amounts of drug compared to low responder (LR) rats, indicating that the HR trait might confer a stronger addiction propensity. However, studies of addiction-like behaviors in HR vs LR rats have typically utilized self-administration paradigms that do not dissociate individual differences in the hedonic/reinforcing and motivational properties of a drug. Moreover, little attention has been given to whether HR rats are more susceptible to drug-access conditions that promote a state-dependent addiction phenotype. We report that on a behavioral economics task, HR rats have higher preferred brain-cocaine levels compared to LR rats but do not differ with respect to their demand elasticity for cocaine. In contrast, when tested on an intermittent access schedule of cocaine self-administration, which has been shown to promote several addiction-related endophenotypes, HR rats exhibit greater escalation of intake and more drastic reductions in cocaine demand elasticity. Together, these data indicate that the HR trait does not confer higher extant addiction behavior, but rather that this phenotype is associated with a propensity for addiction that remains dormant until it is actuated by intermittent drug intake. These findings reveal a 'trait' (HR) by 'state' (intermittent drug intake) interaction that produces a strong addiction-like phenotype. This article is part of the special issue on 'Vulnerabilities to Substance Abuse'.

Intermittent self-administration of fentanyl induces a multifaceted addiction state associated with persistent changes in the orexin system.

The orexin (hypocretin) system plays a critical role in motivated drug taking. Cocaine self-administration with the intermittent access (IntA) procedure produces a robust addiction-like state that is orexin-dependent. Here, we sought to determine the role of the orexin system in opioid addiction using IntA self-administration of fentanyl. Different groups of male rats were either given continuous access in 1-h period (short access [ShA]), 6-h period (long access [LgA]), or IntA (5 min of access separated by 25 min of no access for 6 h) to fentanyl for 14 days. IntA produced a greater escalation of fentanyl intake, increased motivation for fentanyl on a behavioral economics task, persistent drug seeking during abstinence, and stronger cue-induced reinstatement compared with rats given ShA or LgA. We found that addiction behaviors induced by IntA to fentanyl were reversed by the orexin-1 receptor antagonist SB-334867. IntA to fentanyl was also associated with a persistent increase in the number of orexin neurons. Together, these results indicate that the IntA model is a useful tool in the study of opioid addiction and that the orexin system is critical for the maintenance of addiction behaviors induced by IntA self-administration of fentanyl.

The orexin (hypocretin) neuropeptide system is a target for novel therapeutics to treat cocaine use disorder with alcohol coabuse.

An estimated 50-90% of individuals with cocaine use disorder (CUD) also report using alcohol. Cocaine users report coabusing alcohol to 'self-medicate' against the negative emotional side effects of the cocaine 'crash', including the onset of anxiety. Thus, pharmaceutical strategies to treat CUD would ideally reduce the motivational properties of cocaine, alcohol, and their combination, as well as reduce the onset of anxiety during drug withdrawal. The hypothalamic orexin (hypocretin) neuropeptide system offers a promising target, as orexin neurons are critically involved in activating behavioral and physiological states to respond to both positive and negative motivators. Here, we seek to describe studies demonstrating efficacy of orexin receptor antagonists in reducing cocaine, alcohol- and stress-related behaviors, but note that these studies have largely focused on each of these phenomena in isolation. For orexin-based compounds to be viable in the clinical setting, we argue that it is imperative that their efficacy be tested in animal models that account for polysubstance use patterns. To begin to examine this, we present new data showing that rats' preferred level of cocaine intake is significantly increased following chronic homecage access to alcohol. We also report that cocaine intake and motivation are reduced by a selective orexin-1 receptor antagonist when rats have a history of cocaine + alcohol, but not a limited history of cocaine alone. In light of these proof-of-principle data, we outline what we believe to be the key priorities going forward with respect to further examining the orexin system in models of polysubstance use. This article is part of the special issue on Neurocircuitry Modulating Drug and Alcohol Abuse.

Sex Differences in Demand for Highly Palatable Foods: Role of the Orexin System.

BACKGROUND: The prevalence of eating disorders, including binge eating disorder, is significantly higher in women. These findings are mirrored by preclinical studies, which indicate that female rats have a higher preference for palatable food and show greater binge-like eating compared with male rats. METHODS: Here, we describe a novel within-session behavioral-economic paradigm that allows for the simultaneous measurement of the intake at null cost (Q0) and normalized demand elasticity (α) of 3 types of palatable food (low fat, high fat, and chocolate sucrose pellets) via demand curve analysis. In light of evidence that the orexin (hypocretin) system is critically involved in reward and feeding behaviors, we also examined the role of orexin function in sex differences of economic demand for palatable foods. RESULTS: The novel within-session behavioral-economic approach revealed that female rats have higher intake (demand) than males for all palatable foods at low cost (normalized to body weight) but no difference in intake at higher prices, indicating sex-dependent differences in the hedonic, but not motivational, aspects of palatable food. Immediately following behavioral-economic testing, we observed more orexin-expressing neurons and Fos expression (measure of recent neural activation) in these neurons in female rats compared with male rats. Moreover, the orexin-1 receptor antagonist SB334867 reduced both low- and high-cost intake for palatable food in both male and female rats. CONCLUSIONS: These findings provide evidence of higher demand at low prices for palatable food in females and indicate that these behavioral differences may be associated with sexual dimorphism in orexin system function.

Locus coeruleus: a new look at the blue spot.

The locus coeruleus (LC), or 'blue spot', is a small nucleus located deep in the brainstem that provides the far-reaching noradrenergic neurotransmitter system of the brain. This phylogenetically conserved nucleus has proved relatively intractable to full characterization, despite more than 60 years of concerted efforts by investigators. Recently, an array of powerful new neuroscience tools have provided unprecedented access to this elusive nucleus, revealing new levels of organization and function. We are currently at the threshold of major discoveries regarding how this tiny brainstem structure exerts such varied and significant influences over brain function and behaviour. All LC neurons receive inputs related to autonomic arousal, but distinct subpopulations of those neurons can encode specific cognitive processes, presumably through more specific inputs from the forebrain areas. This ability, combined with specific patterns of innervation of target areas and heterogeneity in receptor distributions, suggests that activation of the LC has more specific influences on target networks than had initially been imagined.

Activation of medial hypothalamic orexin neurons during a Go/No-Go task.

Orexin neurons (Orx; also referred to as hypocretin) are found exclusively in the hypothalamus, and release the neuropeptides orexin A and orexin B (also referred to as hypocretin 1 and 2) throughout the CNS. With its widespread targets, the orexin system is involved in a number of functions including, but not limited to stress, reward, wakefulness, and food seeking. Our laboratory has previously proposed that the dorsomedial hypothalamus (DMH) and perifornical (PFA) orexin neurons function in stress and arousal whereas those in lateral hypothalamus (LH) participate in reward processes (Harris and Aston-Jones, 2006). In the current study, we compared Fos activation in orexin neurons located in medial hypothalamus (DMH and PFA) to those in LH during a Go/No-Go task for a highly palatable food reward, a task that would likely activate regions for arousal/attention as well as reward. The Go/No-Go paradigm is a useful behavioral tool to measure behavioral inhibition, impulsivity, learning, and reaction time. Our results revealed increased activation of medial hypothalamic orexin neurons correlated with greater accuracy on the Go/No-Go task. No correlation was found between Go/No-Go accuracy and activation of lateral hypothalamic orexin neurons. This study supports a functional dichotomy of medial vs lateral orexin neurons, and indicates a role for medial orexin neurons in behavioral performance that requires response inhibition.

Lateral septum inhibition reduces motivation for cocaine: Reversal by diazepam.

The lateral septum (LS) is a brain region implicated in motivation, addiction, anxiety, and affect. We recently found that LS is necessary for cocaine-seeking behaviors including conditioned place preference and reinstatement of extinguished drug seeking, which involve LS input to limbic regions including ventral tegmental area (VTA) and orexin neurons in hypothalamus. Here, we microinjected baclofen-muscimol (B-M) in LS prior to testing in a behavioral economics (BE) paradigm. We found that intra-LS B-M decreased motivation (increased demand elasticity; α) for cocaine, but did not change consumption at low effort (Q0 ). We also compared the effects of LS inhibition with the effects of treatment with the benzodiazepine diazepam, which has been shown to facilitate reward pathways and disinhibit VTA dopamine neurons. Pretreatment with diazepam blocked the effects of LS inhibition and restored cocaine demand to that following vehicle treatment. These changes in cocaine demand after LS inhibition or diazepam were not due to effects on anxiety, as both manipulations produced similar effects on anxiety measures but opposing effects on drug taking. Collectively, these studies point to LS as a critical region driving motivation for cocaine, likely through its interactions with the mesolimbic dopamine system.