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.

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.

Attenuated cocaine-seeking after oxytocin administration in male and female rats.

RATIONALE: Initial drug abstinence (modeled here as Extinction Day 1, ED1) is a critical time point in the progression of addiction that is strongly influenced by stress and sex. ED1 induces corticosterone release in both sexes, and cocaine-seeking during ED1 can be mitigated by corticotrophin-releasing factor (CRF) antagonists more effectively in female rats. Oxytocin (OXT) is a neuropeptide that has several biological functions, including regulation of stress pathways. METHODS: To investigate a relationship between OXT, sex, and cocaine-seeking, we examined Fos on ED1 in OXT neurons of paraventricular (PVN) and supraoptic nuclei (SON) compared to homecage (cocaine experienced) or naïve male and female rats. We also administered OXT 30 min prior to ED1 testing or cued reinstatement testing. RESULTS: OXT neurons had decreased activity (as reflected by Fos protein) in PVN and SON on withdrawal day 1 (homecage) compared to naïve rats. Fos in OXT neurons was further decreased on ED1, compared to homecage controls, in both males and females even though in SON, cocaine exposure increased the number of OXT-expressing neurons. In addition, systemically administered OXT reduced cocaine-seeking during ED1 and cue-induced reinstatement of cocaine-seeking but delayed extinction, similarly among male and female rats. CONCLUSIONS: These data indicate that OXT neurons in PVN and SON may be involved in cocaine-seeking during ED1 and support OXT as a possible therapeutic to decrease cocaine-seeking during initial abstinence and in response to cocaine-associated cues.

Dorsal Hippocampus Drives Context-Induced Cocaine Seeking via Inputs to Lateral Septum.

Lateral septum (LS) has re-emerged as an important structure in reward and addiction; however, LS afferents that drive addiction behaviors are unknown. Here, we used a modified self-administration/reinstatement procedure combined with anatomical, pharmacological, and chemogenetic techniques to characterize LS, and hippocampal inputs to LS, in two established triggers of drug relapse-context- and cue-induced reinstatement of cocaine seeking. We found that inactivation of LS neurons attenuated both context- and cue-induced reinstatement of cocaine seeking. However, dorsal hippocampus inputs to LS showed enhanced neuronal activation (as measured by Fos expression) during context-induced, but not cue-induced reinstatement. Additionally, chemogenetic inhibition of dorsal, but not ventral, hippocampal inputs to LS specifically attenuated context-induced reinstatement. Together these findings elucidate the importance of LS in reinstatement of cocaine seeking, and indicate that dorsal hippocampal inputs to LS mediate context-, but not cue-induced, reinstatement of cocaine seeking.

Increased locus coeruleus tonic activity causes disengagement from a patch-foraging task.

High levels of locus coeruleus (LC) tonic activity are associated with distraction and poor performance within a task. Adaptive gain theory (AGT; Aston-Jones & Cohen, 2005) suggests that this may reflect an adaptive function of the LC, encouraging search for more remunerative opportunities in times of low utility. Here, we examine whether stimulating LC tonic activity using designer receptors (DREADDs) promotes searching for better opportunities in a patch-foraging task as the value of a patch diminishes. The task required rats to decide repeatedly whether to exploit an immediate but depleting reward within a patch or to incur the cost of a time delay to travel to a new, fuller patch. Similar to behavior associated with high LC tonic activity in other tasks, we found that stimulating LC tonic activity impaired task performance, resulting in reduced task participation and increased response times and omission rates. However, this was accompanied by a more specific, predicted effect: a significant tendency to leave patches earlier, which was best explained by an increase in decision noise rather than a systematic bias to leave earlier (i.e., at higher values). This effect is consistent with the hypothesis that high LC tonic activity favors disengagement from current behavior, and the pursuit of alternatives, by augmenting processing noise. These results provide direct causal evidence for the relationship between LC tonic activity and flexible task switching proposed by AGT.

Cocaine Seeking During Initial Abstinence Is Driven by Noradrenergic and Serotonergic Signaling in Hippocampus in a Sex-Dependent Manner.

There is evidence for sex differences in cocaine addiction from both clinical and preclinical studies. In particular, preclinical studies indicate that females may be more sensitive than males to stress-induced drug seeking. The dorsal hippocampus (DH) is prominently involved in the stress response, as are the locus coeruleus norepinephrine (LC-NE) and dorsal raphe serotonin (DR 5-HT) systems. Moreover, DH receives strong inputs from LC-NE and DR 5-HT neurons. We hypothesized that the stress associated with non-reinforced drug seeking during early abstinence (on extinction day 1 (ED1)) may contribute to drug seeking via ß-adrenergic and 5-HT neurotransmission in DH. We observed decreased drug-seeking behavior on ED1 following 10 mg/kg S-propranolol (ß-adrenergic and 5-HT1A/1B receptor antagonist), R-propranolol (5-HT1A/1B receptor antagonist), or racemic propranolol in both male and female rats. ED1 increased Fos expression in DH, LC, and DR, and DH Fos was decreased by systemic S-propranolol. Based on these results, we investigated the effects of blocking 5-HT and ß-adrenoceptor transmission in DH on drug seeking during ED1 by infusing a cocktail of WAY100635 plus GR127935 (5-HT1A/1B receptor antagonists), betaxolol plus ICI-118 551 (ß1 and ß2 antagonists), or S-propranolol alone. In males, WAY100635/GR127935 was most effective in reducing drug-seeking on ED1, whereas betaxolol/ICI-118 551 was ineffective. In contrast, S-propranolol was most effective in females in reducing drug seeking on ED1, and WAY100635/GR127935 and betaxolol/ICI-118 551 were each partially effective. Our results indicate that drug seeking during initial abstinence involves 5-HT and ß-adrenergic signaling in female DH, but only 5-HT signaling in male DH.

Prelimbic to Accumbens Core Pathway Is Recruited in a Dopamine-Dependent Manner to Drive Cued Reinstatement of Cocaine Seeking.

UNLABELLED: Glutamate inputs to nucleus accumbens (NAc) facilitate conditioned drug-seeking behavior and primarily originate from medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and ventral subiculum of the hippocampus (vSub). These regions express Fos (a marker of neural activity) during cue-induced reinstatement of cocaine seeking, but only subpopulations of neurons within these regions drive drug seeking. One way to identify and functionally distinguish neural subpopulations activated during drug-seeking is to examine their projection targets. In rats, we examined Fos expression during cue-induced reinstatement of cocaine- and sucrose-seeking in prelimbic cortex (PL), infralimbic cortex (IL), BLA, and vSub neurons that project to NAc core (NAcC) or NAc shell (NAcSh). Neurons in PL, BLA, and vSub that project to NAcC, but not NAcSh, expressed Fos during cue-induced cocaine seeking, but not sucrose seeking. However, only activation of the PL-NAcC pathway positively correlated with cocaine reinstatement behavior, unlike BLA or vSub inputs to NAcC. To confirm a functional role for the PL-NAcC pathway, and to test the hypothesis that this pathway is recruited in a dopamine-dependent manner, we used a pharmacological disconnection approach whereby dopamine signaling was blocked in PL and glutamate signaling was blocked in the contralateral NAcC. This disconnection attenuated cue-induced reinstatement of cocaine seeking but had no effect on reinstatement of sucrose seeking. Our results highlight a role for the PL-NAcC pathway in cocaine seeking and show that these glutamatergic projections are recruited in a dopamine-dependent manner to drive reinstatement. SIGNIFICANCE STATEMENT: Relapse represents a significant barrier to the successful treatment of cocaine addiction. Here, we characterize the relative activation of glutamatergic inputs to nucleus accumbens during cued reinstatement of cocaine seeking versus sucrose seeking. Prelimbic cortex (PL) projections to nucleus accumbens core (NAcC) uniquely expressed Fos in a manner that positively correlated with cocaine-seeking, but not sucrose-seeking, behavior. Additional functional experiments showed that the PL-NAcC pathway was recruited by drug-associated cues in a dopamine-dependent manner to drive cocaine-seeking, but not sucrose-seeking, behavior. These data highlight PL neurons that project to NAcC, and their regulation by dopamine, as potential targets for therapeutics designed to treat cocaine relapse that do not affect natural reward seeking.

Orexin/hypocretin neuron activation is correlated with alcohol seeking and preference in a topographically specific manner.

Orexin (ORX) (also known as hypocretin) neurons are located exclusively in the posterior hypothalamus, and are involved in a wide range of behaviours, including motivation for drugs of abuse such as alcohol. Hypothalamic subregions contain functionally distinct populations of ORX neurons that may play different roles in regulating drug-motivated and alcohol-motivated behaviours. To investigate the role of ORX neurons in ethanol (EtOH) seeking, we measured Fos activation of ORX neurons in rats following three different measures of EtOH seeking and preference: (i) context-induced reinstatement, or ABA renewal; (ii) cue-induced reinstatement of extinguished responding for EtOH; and (iii) a home cage task in which preference for EtOH (vs. water) was measured in the absence of either reinforcer. We found significant activation of ORX neurons in multiple subregions across all three behavioural tests. Notably, ORX neuron activation in the lateral hypothalamus correlated with the degree of seeking in context reinstatement and the degree of preference in home cage preference testing. In addition, Fos activation in ORX neurons in the dorsomedial hypothalamic and perifornical areas was correlated with context and home cage seeking/preference, respectively. Surprisingly, we found no relationship between the degree of cue-induced reinstatement and ORX neuron activation in any region, despite robust activation overall during reinstatement. These results demonstrate a strong relationship between ORX neuron activation and EtOH seeking/preference, but one that is differentially expressed across ORX field subregions, depending on reinstatement modality.

Designer receptors enhance memory in a mouse model of Down syndrome.

Designer receptors exclusively activated by designer drugs (DREADDs) are novel and powerful tools to investigate discrete neuronal populations in the brain. We have used DREADDs to stimulate degenerating neurons in a Down syndrome (DS) model, Ts65Dn mice. Individuals with DS develop Alzheimer's disease (AD) neuropathology and have elevated risk for dementia starting in their 30s and 40s. Individuals with DS often exhibit working memory deficits coupled with degeneration of the locus coeruleus (LC) norepinephrine (NE) neurons. It is thought that LC degeneration precedes other AD-related neuronal loss, and LC noradrenergic integrity is important for executive function, working memory, and attention. Previous studies have shown that LC-enhancing drugs can slow the progression of AD pathology, including amyloid aggregation, oxidative stress, and inflammation. We have shown that LC degeneration in Ts65Dn mice leads to exaggerated memory loss and neuronal degeneration. We used a DREADD, hM3Dq, administered via adeno-associated virus into the LC under a synthetic promoter, PRSx8, to selectively stimulate LC neurons by exogenous administration of the inert DREADD ligand clozapine-N-oxide. DREADD stimulation of LC-NE enhanced performance in a novel object recognition task and reduced hyperactivity in Ts65Dn mice, without significant behavioral effects in controls. To confirm that the noradrenergic transmitter system was responsible for the enhanced memory function, the NE prodrug l-threo-dihydroxyphenylserine was administered in Ts65Dn and normosomic littermate control mice, and produced similar behavioral results. Thus, NE stimulation may prevent memory loss in Ts65Dn mice, and may hold promise for treatment in individuals with DS and dementia.

Modafinil attenuates reinstatement of cocaine seeking: role for cystine-glutamate exchange and metabotropic glutamate receptors.

Modafinil may be useful for treating stimulant abuse, but the mechanisms by which it acts to do so are unknown. Indeed, a primary effect of modafinil is to inhibit dopamine transport, which typically promotes rather than inhibits motivated behavior. Therefore, we examined the role of nucleus accumbens extracellular glutamate and the group II metabotropic glutamate receptor (mGluR2/3) in modafinil effects. One group of rats was trained to self-administer cocaine for 10 days and extinguished, then given priming injections of cocaine to elicit reinstatement. Modafinil (300 mg/kg, intraperitoneal) inhibited reinstated cocaine seeking (but did not alter extinction responding by itself), and this effect was prevented by pre-treatment with bilateral microinjections of the mGluR2/3 antagonist LY-341495 (LY) into nucleus accumbens core. No reversal of modafinil effects was seen after unilateral accumbens core LY, or bilateral LY in the rostral pole of accumbens. Next, we sought to explore effects of modafinil on extracellular glutamate levels in accumbens after chronic cocaine. Separate rats were administered non-contingent cocaine, and after 3 weeks of withdrawal underwent accumbens microdialysis. Modafinil increased extracellular accumbens glutamate in chronic cocaine, but not chronic saline-pre-treated animals. This increase was prevented by reverse dialysis of cystine-glutamate exchange or voltage-dependent calcium channel antagonists. Voltage-dependent sodium channel blockade partly attenuated the increase in glutamate, but mGluR1 blockade did not. We conclude that modafinil increases extracellular glutamate in nucleus accumbens from glial and neuronal sources in cocaine-exposed rats, which may be important for its mGluR2/3-mediated antirelapse properties.

Regulation of the ventral tegmental area by the bed nucleus of the stria terminalis is required for expression of cocaine preference.

Lateral hypothalamus (LH) orexin neurons are essential for the expression of a cocaine place preference. However, the afferents that regulate the activity of these orexin neurons during reward behaviors are not completely understood. Using tract tracing combined with Fos staining, we examined LH afferents for Fos induction during cocaine preference in rats. We found that the ventral bed nucleus of the stria terminalis (vBNST) was a major input to the LH orexin cell field that was significantly Fos-activated during cocaine conditioned place preference (CPP). Inactivation of the vBNST with baclofen plus muscimol blocked expression of cocaine CPP. Surprisingly, such inactivation of the vBNST also increased Fos induction in LH orexin neurons; as activity in these cells is normally associated with increased preference, this result indicates that a vBNST-orexin connection is unlikely to be responsible for CPP that is dependent on vBNST activity. Because previous studies have revealed that vBNST regulates dopamine cells in the ventral tegmental area (VTA), which is known to be involved in CPP and other reward functions, we tested whether vBNST afferents to the VTA are necessary for cocaine CPP. We found that disconnection of the vBNST and VTA (using local microinjections of baclofen plus muscimol unilaterally into the vBNST and contralateral VTA) significantly attenuated expression of cocaine preference. However, blocking ionotropic glutamatergic afferents to the VTA from the vBNST did not significantly reduce cocaine preference. These results indicate that a non-glutamatergic vBNST-VTA projection is involved in expression of cocaine preference.

Fos activation of selective afferents to ventral tegmental area during cue-induced reinstatement of cocaine seeking in rats.

Ventral tegmental area (VTA) dopamine neurons are crucial for appetitive responses to Pavlovian cues, including cue-induced reinstatement of drug seeking. However, it is unknown which VTA inputs help activate these neurons, transducing stimuli into salient cues that drive drug-seeking behavior. Here we examined 56 VTA afferents from forebrain and midbrain that are Fos activated during cue-induced reinstatement. We injected the retrograde tracer cholera toxin ß subunit (CTb) unilaterally into rostral or caudal VTA of male rats. All animals were trained to self-administer cocaine, then extinguished of this behavior. On a final test day, animals were exposed to response-contingent cocaine-associated cues, extinction conditions, a non-cocaine-predictive CS-, or a novel environment, and brains were processed to visualize CTb and Fos immunoreactivity to identify VTA afferents activated in relation to behaviors. VTA-projecting neurons in subregions of medial accumbens shell, ventral pallidum, elements of extended amygdala, and lateral septum (but not prefrontal cortex) were activated specifically during cue-induced cocaine seeking, and some of these were also activated proportionately to the degree of cocaine seeking. Surprisingly, though efferents from the lateral hypothalamic orexin field were also Fos activated during reinstatement, these were largely non-orexinergic. Also, VTA afferents from the rostromedial tegmental nucleus and lateral habenula were specifically activated during extinction and CS- tests, when cocaine was not expected. These findings point to a select set of subcortical nuclei which provide reinstatement-related inputs to VTA, translating conditioned stimuli into cocaine-seeking behavior.

Halothane-induced hypnosis is not accompanied by inactivation of orexinergic output in rodents.

BACKGROUND: One underexploited property of anesthetics is their ability to probe neuronal regulation of arousal. At appropriate doses, anesthetics reversibly obtund conscious perception. However, individual anesthetic agents may accomplish this by altering the function of distinct neuronal populations. Previously the authors showed that isoflurane and sevoflurane inhibit orexinergic neurons, delaying reintegration of sensory perception as denoted by emergence. Here the authors study the effects of halothane. As a halogenated alkane, halothane differs structurally, has a nonoverlapping series of molecular binding partners, and differentially modulates electrophysiologic properties of several ion channels when compared with its halogenated ether relatives. METHODS: c-Fos immunohistochemistry and in vivo electrophysiology were used to assess neuronal activity. Anesthetic induction and emergence were determined behaviorally in narcoleptic orexin/ataxin-3 mice and control siblings exposed to halothane. RESULTS: Halothane-induced hypnosis occurred despite lack of inhibition of orexinergic neurons in mice. In rats, extracellular single-unit recordings within the locus coeruleus showed significantly greater activity during halothane than during a comparable dose of isoflurane. Microinjection of the orexin-1 receptor antagonist SB-334867-A during the active period slowed firing rates of locus coeruleus neurons in halothane-anesthetized rats, but had no effect on isoflurane-anesthetized rats. Surprisingly, orexin/ataxin-3 transgenic mice, which develop narcolepsy with cataplexy because of loss of orexinergic neurons, did not show delayed emergence from halothane. CONCLUSION: Coordinated inhibition of hypothalamic orexinergic and locus coeruleus noradrenergic neurons is not required for anesthetic induction. Normal emergence from halothane-induced hypnosis in orexin-deficient mice suggests that additional wake-promoting systems likely remain active during general anesthesia produced by halothane.

Activation in extended amygdala corresponds to altered hedonic processing during protracted morphine withdrawal.

Previously we reported that during protracted morphine abstinence rats show reduced conditioned place preferences (CPP) for food-associated environments, compared to non-dependent subjects. To determine the brain regions involved in this altered reward behavior, we examined neural activation (as indexed by Fos-like proteins) induced by a preference test for a food-associated environment in 5-week morphine-abstinent versus non-dependent animals. The results indicate that elevated Fos expression in the anterior cingulate cortex (Cg) and basolateral amygdala (BLA) correlated positively with preference behavior in all groups. In contrast, Fos expression in stress-associated brain areas, including the ventral lateral bed nucleus of the stria terminalis (VL-BNST), central nucleus of the amygdala (CE), and noradrenergic (A2) neurons in the nucleus tractus solitarius (NTS) was significantly elevated only in morphine-abstinent animals. Furthermore, the number of Fos positive neurons in these areas was found to correlate negatively with food preference in abstinent animals. These results indicate that the altered hedonic processing during protracted morphine withdrawal leading to decreased preference for cues associated with natural rewards may involve heightened activity in stress-related brain areas of the extended amygdala and their medullary noradrenergic inputs.

A role for lateral hypothalamic orexin neurons in reward seeking.

The lateral hypothalamus is a brain region historically implicated in reward and motivation, but the identity of the neurotransmitters involved are unknown. The orexins (or hypocretins) are neuropeptides recently identified as neurotransmitters in lateral hypothalamus neurons. Although knockout and transgenic overexpression studies have implicated orexin neurons in arousal and sleep, these cells also project to reward-associated brain regions, including the nucleus accumbens and ventral tegmental area. This indicates a possible role for these neurons in reward function and motivation, consistent with previous studies implicating these neurons in feeding. Here we show that activation of lateral hypothalamus orexin neurons is strongly linked to preferences for cues associated with drug and food reward. In addition, we show that chemical activation of lateral hypothalamus orexin neurons reinstates an extinguished drug-seeking behaviour. This reinstatement effect was completely blocked by prior administration of an orexin A antagonist. Moreover, administration of the orexin A peptide directly into the ventral tegmental area also reinstated drug-seeking. These data reveal a new role for lateral hypothalamus orexin neurons in reward-seeking, drug relapse and addiction.

Brain substrates for increased drug seeking during protracted withdrawal.

Studies are reviewed indicating that both increased anxiety and altered hedonic processing accompany protracted withdrawal from opiates. Increased anxiety may be most apparent in response to stress, whereas decreased motivation for natural rewards but increased interest in drugs reveals substantial alterations in hedonic values. Our recent work indicates that increased norepinephrine (NE) release in the bed nucleus of the stria terminalis (BNST) may underlie anxiety associated with protracted withdrawal. Altered plasticity in afferents to the ventral tegmental area (VTA; accumbens, amygdala and lateral hypothalamus), or in the VTA itself, may be involved in the altered hedonic processing that occurs during protracted withdrawal. We hypothesize that conditioned release of NE in the BNST in response to stressors (including drug-associated stimuli) may elevate anxiety which then augments the reward value of drugs by a negative reinforcement mechanism. We also propose that plasticity in VTA neurons and their afferents during chronic drug exposure and protracted withdrawal decreases the valence of natural rewards whereas sensitization occurs to the motivational effects of drugs that increases their motivational valence. The combination of anxiety, decreased valence of natural rewards, and sensitized incentive for drugs make a potent formula for relapse and drug seeking during protracted withdrawal.

Enhanced morphine preference following prolonged abstinence: association with increased Fos expression in the extended amygdala.

We previously found that chronically morphine-pretreated, abstinent rats show stronger preferences for morphine-associated environments than placebo-pretreated rats. Here we show that this increased preference persisted for at least 5 weeks after withdrawal of chronic morphine. To determine brain regions involved in this behavior, we examined neural activation (as indexed by Fos-like proteins) induced by a morphine-conditioned place preference test. Placebo-pretreated (P) morphine-conditioned rats showed significantly elevated Fos in the anterior cingulate cortex (Cg), nucleus accumbens core (Ac-C) and shell (Ac-S), ventral lateral and dorsal lateral bed nucleus of the stria terminialis (BNST-VL and -DL), and central and basolateral amygdala nuclei (ACE, ABL) when compared to nonconditioned P rats. Chronically morphine-pretreated (M) rats that exhibited enhanced morphine preference 5 weeks after morphine withdrawal showed significantly greater Fos in all the same areas except the BNST-DL relative to conditioned P or nonconditioned M rats. Place preference measures and Fos expression were positively correlated in the Cg and ABL, for conditioned P animals, and in the Cg, ABL and BNST-VL for conditioned M animals. These results indicate a relationship between place preference behavior and neural indices of activation in the forebrain in response to morphine-conditioned cues that may be chronically modulated by prior morphine exposure.