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This article presents the proceedings of a symposium presented at the meeting of the Research Society on Alcoholism, held in Vancouver, British Columbia, Canada, in June 2004. The organizers and chairpersons were Subhash C. Pandey and Fulton Crews. The presentations were (1) Ethanol Modulation of CREB: Role in Dependence and Withdrawal, by Fulton Crews; (2) Effects of D1 Dopamine Receptor Activation During Withdrawal From Chronic Morphine: Enhanced CREB Activation and Decreased Conditioned Place Aversion, by Elena H. Chartoff; (3) CREB-Haplodeficient Mice: Role in Anxiety and Alcohol-Drinking Behaviors, by Subhash C. Pandey; and (4) A Role for CREB in Stress and Drug Addiction, by Julie A. Blendy. [ABSTRACT FROM AUTHOR]

Targeted intracranial delivery of molecularly-specific therapies within intricate brain structures poses a formidable challenge due to the heterogeneity of neuronal phenotypes and functions. Here we report the use of an implantable, miniaturized neural drug delivery system permitting dynamic adjustment of pharmacotherapies. Specifically, we exploit the spatial accuracy afforded by this method for targeting modulation of neuronal microstructures. Kappa opioid receptors (KOR) within the dorsal medial nucleus accumbens shell (NASh) are selectively activated through micro infusions of the KOR agonist, U-50488. Remarkably, we demonstrate that micro infusions of U-50488 into the dorsal NASh induces reward-like conditioned place preferences, whereas a mere 1 mm shift ventrally results in conditioned place aversions. The striking precision afforded by this method may prove useful in other neurotherapeutic interventions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 International Brain Research Organization (IBRO). Published by Elsevier Inc. All rights reserved.)

Opioid use disorder is marked by a progressive change in the motivation to administer the drug even in the presence of negative consequences. After long periods of abstinence, the urge to return to taking the drug intensifies over time, known as incubation of craving. Conditioned responses to drug-related stimuli, can acquire motivational properties and exert control over motivated behaviors leading to relapse. Although, preclinical data suggest that the behavioral expression of opioid use is similar between male and female rodents, we do not have conclusive results on sex differences on craving and relapse across abstinence periods. Here, we investigated the effects of abstinence from oxycodone self-administration on neurotransmission in the paraventricular thalamus (PVT) to nucleus accumbens shell (NAcSh) pathway in male and female rats. Using optogenetics and ex vivo electrophysiology, we assessed synaptic strength and glutamate release probability in this pathway, as well as NAcSh medium spiny neurons (MSN) intrinsic excitability, in slices from rats which were subjected to either 1 (acute) or 14 (prolonged) days of forced abstinence after self-administration. Our results revealed no sex differences in oxycodone self-administration or somatic withdrawal symptoms following acute abstinence. However, we found a sex-specific enhancement in cue-induced relapse after prolonged, but not acute, abstinence from oxycodone self-administration, with females exhibiting higher relapse rates. Notably, prolonged abstinence led to similar increases in synaptic strength at PVT-NAcSh inputs compared to saline controls in both sexes, which was not observed after acute abstinence. Thus, prolonged abstinence results in a time-dependent increase in PVT-NAcSh synaptic strength and sex-specific effects on cue-induced relapse rates. These findings suggest that prolonged abstinence leads to significant synaptic changes, contributing to heightened relapse vulnerability, highlighting the need for targeted therapeutic strategies in opioid use disorder., Competing Interests: Conflict of Interest: No conflict of interest to disclose.

Oxycodone is one of the most widely prescribed and misused opioid painkillers in the United States. Evidence suggests that biological sex and hormonal status can impact drug reward in humans and rodents, but the extent to which these factors can influence the rewarding effects of oxycodone is unclear. The purpose of this study was to utilize place conditioning to determine the effects of sex and female hormonal status on the expression of oxycodone conditioned reward in rats. Gonadally intact adult Sprague-Dawley male and female rats were used to test: (1) whether both sexes express conditioned reward to oxycodone at similar doses, (2) the impact of conditioning session length on oxycodone conditioned reward expression in both sexes, and (3) the influence of female estrous cycle stage on oxycodone conditioned reward expression. Both sexes expressed conditioned reward at the same doses of oxycodone. Increasing the length of conditioning sessions did not reveal an effect of sex and resulted in lower magnitude conditioned reward expression. Importantly however, female stage of estrous cycle significantly influenced oxycodone conditioned reward expression. These results suggest that female hormonal status can impact the rewarding effects of opioids and thus have important implications for prescription opioid treatment practices., (© 2023. Springer Nature Limited.)

Introduction: Rates of relapse to drug use during abstinence are among the highest for opioid use disorder (OUD). In preclinical studies, reinstatement to drug-seeking has been extensively studied as a model of relapse-but the work has been primarily in males. We asked whether biological sex contributes to behaviors comprising self-administration of the prescription opioid oxycodone in rats, and we calculated the relative contribution of these behavioral measures to reinstatement in male and female rats., Materials and Methods: Rats were trained to self-administer oxycodone (8 days, training phase), after which we examined oxycodone self-administration behaviors for an additional 14 days under three conditions in male and female rats: short access (ShA, 1 h/d), long access (LgA, 6 h/d), and saline self-administration. All rats were then tested for cue-induced reinstatement of drug-seeking after a 14-d forced abstinence period. We quantified the # of infusions, front-loading of drug intake, non-reinforced lever pressing, inter-infusion intervals, escalation of intake, and reinstatement responding on the active lever., Results: Both male and female rats in LgA and ShA conditions escalated oxycodone intake to a similar extent. However, males had higher levels of non-reinforced responding than females under LgA conditions, and females had greater levels of reinstatement responding than males. We then correlated each addiction-related measure listed above with reinstatement responding in males and females and ranked their respective relative contributions. Although the majority of behavioral measures associated with oxycodone self-administration did not show sex differences on their own, when analyzed together using partial least squares regression, their relative contributions to reinstatement were sex-dependent. Front-loading behavior was calculated to have the highest relative contribution to reinstatement in both sexes, with long and short inter-infusion intervals having the second greatest contribution in females and males, respectively., Discussion: Our results demonstrate sex differences in some oxycodone self-administration measures. More importantly, we demonstrate that a sex- dependent constellation of self-administration behaviors can predict the magnitude of reinstatement, which holds great promise for relapse prevention in people., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Guha, Alonso-Caraballo, Driscoll, Babb, Neal, Constantino, Lintz, Kinard and Chartoff.)

The current opioid epidemic has dramatically increased the number of children who are prenatally exposed to opioids, including oxycodone. A number of social and cognitive abnormalities have been documented in these children as they reach young adulthood. However, little is known about the mechanisms underlying developmental effects of prenatal opioid exposure. Microglia, the resident immune cells of the brain, respond to acute opioid exposure in adulthood. Moreover, microglia are known to sculpt neural circuits during typical development. Indeed, we recently found that microglial phagocytosis of dopamine D1 receptors (D1R) in the nucleus accumbens (NAc) is required for the natural developmental decline in NAc-D1R that occurs between adolescence and adulthood in rats. This microglial pruning occurs only in males, and is required for the normal developmental trajectory of social play behavior. However, virtually nothing is known as to whether this developmental program is altered by prenatal exposure to opioids. Here, we show in rats that maternal oxycodone self-administration during pregnancy leads to reduced adolescent microglial phagocytosis of D1R and subsequently higher D1R density within the NAc in adult male, but not female, offspring. Finally, we show prenatal and adult behavioral deficits in opioid-exposed offspring, including impaired extinction of oxycodone-conditioned place preference in males. This work demonstrates for the first time that microglia play a key role in translating prenatal opioid exposure to changes in neural systems and behavior., (© 2022. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Objectives: Gender differences in the prevalence of opioid misuse continue to evolve and have not been well characterized in recent years. Our objective was to investigate gender differences in the prevalence of opioid misuse and use disorder in the US over the 5-year period from 2015 to 2019., Methods: We used annual survey data from the 2015-2019 National Survey on Drug Use and Health to estimate gender differences in the prevalence of opioid misuse. We examined past-year opioid analgesic misuse initiation, opioid analgesic misuse, heroin use, opioid analgesic use disorder and heroin use disorder. Logistic regression models were used to test gender differences, adjusting for sociodemographic variables., Results: In adjusted analyses, women had higher odds of having initiated opioid analgesic misuse in the past year compared to men. In contrast, men had higher odds of misuse of opioid analgesics, heroin use, and an opioid analgesic or heroin use disorder., Conclusions: Although opioid misuse has historically been more prevalent in men, the gender difference in opioid analgesic misuse continues to narrow, with more women initiating misuse than men including higher rates of misuse in adolescent girls. Heroin use continues to be approximately twice as common in men as women., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Rationale: Methamphetamine (MA) addiction is a major public health issue in the USA, with a poorly understood genetic component. We previously identified heterogeneous nuclear ribonucleoprotein H1 (Hnrnph1; H1) as a quantitative trait gene underlying sensitivity to MA-induced behavioral sensitivity. Mice heterozygous for a frameshift deletion in the first coding exon of H1 (H1 +/- ) showed reduced MA phenotypes including oral self-administration, locomotor activity, dopamine release, and dose-dependent differences in MA conditioned place preference. However, the effects of H1 +/- on innate and MA-modulated reward sensitivity are not known., Objectives: We examined innate reward sensitivity and facilitation by MA in H1 +/- mice via intracranial self-stimulation (ICSS)., Methods: We used intracranial self-stimulation (ICSS) of the medial forebrain bundle to assess shifts in reward sensitivity following acute, ascending doses of MA (0.5-4.0 mg/kg, i.p.) using a within-subjects design. We also assessed video-recorded behaviors during ICSS testing sessions., Results: H1 +/- mice displayed reduced normalized maximum response rates in response to MA. H1 +/- females had lower normalized M50 values compared to wild-type females, suggesting enhanced reward facilitation by MA. Finally, regardless of genotype, there was a dose-dependent reduction in distance to the response wheel following MA administration, providing an additional measure of MA-induced reward-driven behavior., Conclusions: H1 +/- mice displayed a complex ICSS phenotype following MA, displaying indications of both blunted reward magnitude (lower normalized maximum response rates) and enhanced reward sensitivity specific to H1 +/- females (lower normalized M50 values).

Drugs of abuse and highly palatable foods (e.g. high fat or sweet foods) have powerful reinforcing effects, which can lead to compulsive and addictive drives to ingest these substances to the point of psychopathology and self-harm--specifically the development of Substance Use Disorder (SUD) and obesity. Both SUD and binge-like overeating can be defined as disorders in which the salience of the reward (food or drug) becomes exaggerated relative to, and at the expense of, other rewards that promote well-being. A major roadblock in the treatment of these disorders is high rates of relapse after periods of abstinence. It is common, although not universal, for cue-induced craving to increase over time with abstinence, often triggered by cues previously paired with the reinforcing substance. Accumulating evidence suggests that similar neural circuits and cellular mechanisms contribute to abstinence-induced and cue-triggered seeking of drugs and palatable food. Although much research has focused on the important role of corticolimbic circuitry in drug-seeking, our goal is to expand focus to the more recently explored hypothalamic-thalamic-striatal circuitry. Specifically, we review how connections, and neurotransmitters therein, among the lateral hypothalamus, paraventricular nucleus of the thalamus, and the nucleus accumbens contribute to abstinence-induced opioid- and (high fat or sweet) food-seeking. Given that biological sex and gonadal hormones have been implicated in addictive behavior across species, another layer to this review is to compare behaviors and neural circuit-based mechanisms of abstinence-induced opioid- or food-seeking between males and females when such data is available., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Neuropathy is major source of chronic pain that can be caused by mechanically or chemically induced nerve injury. Intraplantar formalin injection produces local necrosis over a two-week period and has been used to model neuropathy in rats. To determine whether neuropathy alters dopamine (DA) receptor responsiveness in mesolimbic brain regions, we examined dopamine D 1 -like and D 2 -like receptor (D 1/2 R) signaling and expression in male rats 14 days after bilateral intraplantar formalin injections into both rear paws. D 2 R-mediated G-protein activation and expression of the D 2 R long, but not short, isoform were reduced in nucleus accumbens (NAc) core, but not in NAc shell, caudate-putamen or ventral tegmental area of formalin- compared to saline-treated rats. In addition, D 1 R-stimulated adenylyl cyclase activity was also reduced in NAc core, but not in NAc shell or prefrontal cortex, of formalin-treated rats, whereas D 1 R expression was unaffected. Other proteins involved in dopamine neurotransmission, including dopamine uptake transporter and tyrosine hydroxylase, were unaffected by formalin treatment. In behavioral tests, the potency of a D 2 R agonist to suppress intracranial self-stimulation (ICSS) was decreased in formalin-treated rats, whereas D 1 R agonist effects were not altered. The combination of reduced D 2 R expression and signaling in NAc core with reduced suppression of ICSS responding by a D 2 R agonist suggest a reduction in D 2 autoreceptor function. Altogether, these results indicate that intraplantar formalin produces attenuation of highly specific DA receptor signaling processes in NAc core of male rats and suggest the development of a neuropathy-induced allostatic state in both pre- and post-synaptic DA receptor function., (Copyright © 2020. Published by Elsevier Ltd.)

Background: New treatments for stress-related disorders including depression, anxiety, and substance use disorder are greatly needed. Kappa opioid receptors are expressed in the central nervous system, including areas implicated in analgesia and affective state. Although kappa opioid receptor agonists share the antinociceptive effects of mu opioid receptor agonists, they also tend to produce negative affective states. In contrast, selective kappa opioid receptor antagonists have antidepressant- and anxiolytic-like effects, stimulating interest in their therapeutic potential. The prototypical kappa opioid receptor antagonists (e.g., norBNI, JDTic) have an exceptionally long duration of action that complicates their use in humans, particularly in tests to establish safety. This study was designed to test dose- and time-course effects of novel kappa opioid receptor antagonists with the goal of identifying short-acting lead compounds for future medication development., Methods: We screened 2 novel, highly selective kappa opioid receptor antagonists (CYM-52220 and CYM-52288) with oral efficacy in the warm water tail flick assay in rats to determine initial dose and time course effects. For comparison, we tested existing kappa opioid receptor antagonists JDTic and LY-2456302 (also known as CERC-501 or JNJ-67953964)., Results: In the tail flick assay, the rank order of duration of action for the antagonists was LY-2456302 < CYM-52288 < CYM-52220 << JDTic. Furthermore, LY-2456302 blocked the depressive (anhedonia-producing) effects of the kappa opioid receptor agonist U50,488 in the intracranial self-stimulation paradigm, albeit at a higher dose than that needed for analgesic blockade in the tail flick assay., Conclusions: These results suggest that structurally diverse kappa opioid receptor antagonists can have short-acting effects and that LY-2456302 reduces anhedonia as measured in the intracranial self-stimulation test., (© The Author(s) 2019. Published by Oxford University Press on behalf of CINP.)

Background: There is an urgent need to identify factors that increase vulnerability to opioid addiction to help stem the opioid epidemic and develop more efficient pharmacotherapeutics. MicroRNAs are small non-coding RNAs that regulate gene expression at a posttranscriptional level and have been implicated in chronic drug-taking in humans and in rodent models. Recent evidence has shown that chronic opioid treatment regulates the microRNA miR-9. The present study was designed to test the hypothesis that miR-9 in the nucleus accumbens potentiates oxycodone addictive-like behavior., Methods: We utilized adeno-associated virus (AAV) to overexpress miR-9 in the nucleus accumbens of male rats and tested the effects on intravenous self-administration of the highly abused prescription opioid, oxycodone, in 1-hour short-access followed by 6-h long-access sessions, the latter of which leads to escalation of drug intake. In separate rats, we assessed the effects of nucleus accumbens miR-9 overexpression on mRNA targets including RE1-silencing transcription factor (REST) and dopamine D2 receptor (DRD2), which have been shown to be regulated by drugs of abuse., Results: Overexpression of miR-9 in the nucleus accumbens significantly increased oxycodone self-administration compared with rats expressing a control, scrambled microRNA. Analysis of the pattern of oxycodone intake revealed that miR-9 overexpression increased "burst" episodes of intake and decreased the inter-infusion interval. Furthermore, miR-9 overexpression decreased the expression of REST and increased DRD2 in the nucleus accumbens at time points that coincided with behavioral effects., Conclusions: These results suggest that nucleus accumbens miR-9 regulates oxycodone addictive-like behavior as well as the expression of genes that are involved in drug addiction., (© The Author(s) 2019. Published by Oxford University Press on behalf of CINP.)

The neuropeptide dynorphin (DYN) activates kappa opioid receptors (KORs) in the brain to produce depressive-like states and decrease motivation. KOR-mediated suppression of dopamine release in the nucleus accumbens (NAc) is considered one underlying mechanism. We previously showed that, regardless of estrous cycle stage, female rats are less sensitive than males to KOR agonist-mediated decreases in motivation to respond for brain stimulation reward, measured with intracranial self-stimulation (ICSS). However, the explicit roles of KORs, circulating gonadal hormones, and their interaction with dopamine signaling in motivated behavior are not known. As such, we measured the effects of the KOR agonist U50,488 on ICSS stimulation thresholds before and after gonadectomy (or sham surgery). We found that ovariectomized females remained less sensitive than sham or castrated males to KOR-mediated decreases in brain stimulation reward, indicating that circulating gonadal hormones do not play a role. We used qRT-PCR to examine whether sex differences in gene expression in limbic brain regions are associated with behavioral sex differences. We found no sex differences in Pdyn or Oprk1 mRNA in the NAc and ventral tegmental area (VTA), but tyrosine hydroxylase (Th) mRNA was significantly higher in female compared to male VTA. To further explore sex-differences in KOR-mediated suppression of dopamine, we used fast scan cyclic voltammetry (FSCV) and demonstrated that U50,488 was less effective in suppressing evoked NAc dopamine release in females compared to males. These data raise the possibility that females are protected from KOR-mediated decreases in motivation by an increased capacity to produce and release dopamine., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Rationale: Mood disorders can be triggered by stress and are characterized by deficits in reward processing, including disrupted reward learning (the ability to modulate behavior according to past rewards). Reward learning is regulated by the anterior cingulate cortex (ACC) and striatal circuits, both of which are implicated in the pathophysiology of mood disorders., Objectives: Here, we assessed in rats the effects of a potent stressor (social defeat) on reward learning and gene expression in the ACC, ventral tegmental area (VTA), and striatum., Methods: Adult male Wistar rats were trained on an operant probabilistic reward task (PRT) and then exposed to 3 days of social defeat before assessment of reward learning. After testing, the ACC, VTA, and striatum were dissected, and expression of genes previously implicated in stress was assessed., Result: Social defeat blunted reward learning (manifested as reduced response bias toward a more frequently rewarded stimulus) and was associated with increased nociceptin/orphanin FQ (N/OFQ) peptide mRNA levels in the striatum and decreased Fos mRNA levels in the VTA. Moreover, N/OFQ peptide and nociceptin receptor mRNA levels in the ACC, VTA and striatum were inversely related to reward learning., Conclusions: The behavioral findings parallel previous data in humans, suggesting that stress similarly disrupts reward learning in both species. Increased striatal N/OFQ mRNA in stressed rats characterized by impaired reward learning is consistent with accumulating evidence that antagonism of nociceptin receptors, which bind N/OFQ, has antidepressant-like effects. These results raise the possibility that nociceptin systems represent a molecular substrate through which stress produces reward learning deficits in mood disorders.

Unlabelled: Dependence is a hallmark feature of opiate addiction and is defined by the emergence of somatic and affective withdrawal signs. The nucleus accumbens (NAc) integrates dopaminergic and glutamatergic inputs to mediate rewarding and aversive properties of opiates. Evidence suggests that AMPA glutamate-receptor-dependent synaptic plasticity within the NAc underlies aspects of addiction. However, the degree to which NAc AMPA receptors (AMPARs) contribute to somatic and affective signs of opiate withdrawal is not fully understood. Here, we show that microinjection of the AMPAR antagonist NBQX into the NAc shell of morphine-dependent rats prevented naloxone-induced conditioned place aversions and decreases in sensitivity to brain stimulation reward, but had no effect on somatic withdrawal signs. Using a protein cross-linking approach, we found that the surface/intracellular ratio of NAc GluA1, but not GluA2, increased with morphine treatment, suggesting postsynaptic insertion of GluA2-lacking AMPARs. Consistent with this, 1-naphthylacetyl spermine trihydrochloride (NASPM), an antagonist of GluA2-lacking AMPARs, attenuated naloxone-induced decreases in sensitivity to brain stimulation reward. Naloxone decreased the surface/intracellular ratio and synaptosomal membrane levels of NAc GluA1 in morphine-dependent rats, suggesting a compensatory removal of AMPARs from synaptic zones. Together, these findings indicate that chronic morphine increases synaptic availability of GluA1-containing AMPARs in the NAc, which is necessary for triggering negative-affective states in response to naloxone. This is broadly consistent with the hypothesis that activation of NAc neurons produces acute aversive states and raises the possibility that inhibiting AMPA transmission selectively in the NAc may have therapeutic value in the treatment of addiction., Significance Statement: Morphine dependence and withdrawal result in profound negative-affective states that play a major role in the maintenance of addiction. However, the underlying neurobiological mechanisms are not fully understood. We use a rat model of morphine dependence to show that GluA1 subunits of AMPA glutamate receptors in the nucleus accumbens (NAc), a brain region critical for modulating affective states, are necessary for aversive effects of morphine withdrawal. Using biochemical methods in NAc tissue, we show that morphine dependence increases cell surface expression of GluA1, suggesting that neurons in this area are primed for increased AMPA receptor activation upon withdrawal. This work is important because it suggests that targeting AMPA receptor trafficking and activation could provide novel targets for addiction treatment., (Copyright © 2016 the authors 0270-6474/16/365748-15$15.00/0.)

Negative affective states can increase the rewarding value of drugs of abuse and promote drug taking. Chronic cocaine exposure increases levels of the neuropeptide dynorphin, an endogenous ligand at kappa opioid receptors (KOR) that suppresses dopamine release in the nucleus accumbens (NAc) and elicits negative affective states upon drug withdrawal. However, there is evidence that the effects of KOR activation on affective state are biphasic: immediate aversive effects are followed by delayed increases in reward. The impact of KOR-induced affective states on reward-related effects of cocaine over time is not known. We hypothesize that the initial aversive effects of KOR activation increase, whereas the delayed rewarding effects decrease, the net effects of cocaine on reward and dopamine release. We treated rats with cocaine at various times (15 min to 48 h) after administration of the selective KOR agonist salvinorin A (salvA). Using intracranial self-stimulation and fast scan cyclic voltammetry, we found that cocaine-induced increases in brain stimulation reward and evoked dopamine release in the NAc core were potentiated when cocaine was administered within 1 h of salvA, but attenuated when administered 24 h after salvA. Quantitative real-time PCR was used to show that KOR and prodynorphin mRNA levels were decreased in the NAc, whereas tyrosine hydroxylase and dopamine transporter mRNA levels and tissue dopamine content were increased in the ventral tegmental area 24 h post-salvA. These findings raise the possibility that KOR activation-as occurs upon withdrawal from chronic cocaine-modulates vulnerability to cocaine in a time-dependent manner.

Unconditioned rewarding stimuli evoke phasic increases in dopamine concentration in the nucleus accumbens (NAc) while discrete aversive stimuli elicit pauses in dopamine neuron firing and reductions in NAc dopamine concentration. The unconditioned effects of more prolonged aversive states on dopamine release dynamics are not well understood and are investigated here using the malaise-inducing agent lithium chloride (LiCl). We used fast-scan cyclic voltammetry to measure phasic increases in NAc dopamine resulting from electrical stimulation of dopamine cell bodies in the ventral tegmental area (VTA). Systemic LiCl injection reduced electrically evoked dopamine release in the NAc of both anesthetized and awake rats. As some behavioral effects of LiCl appear to be mediated through glucagon-like peptide-1 receptor (GLP-1R) activation, we hypothesized that the suppression of phasic dopamine by LiCl is GLP-1R dependent. Indeed, peripheral pretreatment with the GLP-1R antagonist exendin-9 (Ex-9) potently attenuated the LiCl-induced suppression of dopamine. Pretreatment with Ex-9 did not, however, affect the suppression of phasic dopamine release by the kappa-opioid receptor agonist, salvinorin A, supporting a selective effect of GLP-1R stimulation in LiCl-induced dopamine suppression. By delivering Ex-9 to either the lateral or fourth ventricle, we highlight a population of central GLP-1 receptors rostral to the hindbrain that are involved in the LiCl-mediated suppression of NAc dopamine release.

Behavioral, biological, and social sequelae that lead to drug addiction differ between men and women. Our efforts to understand addiction on a mechanistic level must include studies in both males and females. Stress, anxiety, and depression are tightly linked to addiction, and whether they precede or result from compulsive drug use depends on many factors, including biological sex. The neuropeptide dynorphin (DYN), an endogenous ligand at kappa opioid receptors (KORs), is necessary for stress-induced aversive states and is upregulated in the brain after chronic exposure to drugs of abuse. KOR agonists produce signs of anxiety, fear, and depression in laboratory animals and humans, findings that have led to the hypothesis that drug withdrawal-induced DYN release is instrumental in negative reinforcement processes that drive addiction. However, these studies were almost exclusively conducted in males. Only recently is evidence available that there are sex differences in the effects of KOR activation on affective state. This review focuses on sex differences in DYN and KOR systems and how these might contribute to sex differences in addictive behavior. Much of what is known about how biological sex influences KOR systems is from research on pain systems. The basic molecular and genetic mechanisms that have been discovered to underlie sex differences in KOR function in pain systems may apply to sex differences in KOR function in reward systems. Our goals are to discuss the current state of knowledge on how biological sex contributes to KOR function in the context of pain, mood, and addiction and to explore potential mechanisms for sex differences in KOR function. We will highlight evidence that the function of DYN-KOR systems is influenced in a sex-dependent manner by: polymorphisms in the prodynorphin (pDYN) gene, genetic linkage with the melanocortin-1 receptor (MC1R), heterodimerization of KORs and mu opioid receptors (MORs), and gonadal hormones. Finally, we identify several gaps in our understanding of "if" and "how" DYN and KORs modulate addictive behavior in a sex-dependent manner. Future work may address these gaps by building on the mechanistic studies outlined in this review. Ultimately this will enable the development of novel and effective addiction treatments tailored to either males or females.

Rationale: Intermittent social defeat stress can induce neuroadaptations that promote compulsive drug taking. Within the mesocorticolimbic circuit, repeated cocaine administration activates extracellular signal-regulated kinase (ERK)., Objective: The present experiments examine whether changes in ERK phosphorylation are necessary for the behavioral and neural adaptations that occur as a consequence of intermittent defeat stress., Materials and Methods: Rats were exposed to four brief intermittent defeats over the course of 10 days. Ten days after the last defeat, rats were challenged with cocaine (10 mg/kg, i.p.) or saline, and ERK activity was examined in mesocorticolimbic regions. To determine the role of ERK in defeat stress-induced behavioral sensitization, we bilaterally microinjected the MAPK/ERK kinase inhibitor U0126 (1 μg/side) or vehicle (20 % DMSO) into the ventral tegmental area (VTA) prior to each of four defeats. Ten days following the last defeat, locomotor activity was assessed for the expression of behavioral cross-sensitization to cocaine (10 mg/kg, i.p.). Thereafter, rats self-administered cocaine under fixed and progressive ratio schedules of reinforcement, including a 24-h continuous access "binge" (0.3 mg/kg/infusion)., Results: We found that repeated defeat stress increased ERK phosphorylation in the VTA. Inhibition of VTA ERK prior to each social defeat attenuated the development of stress-induced sensitization and prevented stress-induced enhancement of cocaine self-administration during a continuous access binge., Conclusions: These results suggest that enhanced activation of ERK in the VTA due to brief defeats is critical in the induction of sensitization and escalated cocaine taking.

Both the development and relief of stress-related psychiatric conditions such as major depression (MD) and post-traumatic stress disorder (PTSD) have been linked to neuroplastic changes in the brain. One such change involves the birth of new neurons (neurogenesis), which occurs throughout adulthood within discrete areas of the mammalian brain, including the dorsal hippocampus (HIP). Stress can trigger MD and PTSD in humans, and there is considerable evidence that it can decrease HIP neurogenesis in laboratory animals. In contrast, antidepressant treatments increase HIP neurogenesis, and their efficacy is eliminated by ablation of this process. These findings have led to the working hypothesis that HIP neurogenesis serves as a biomarker of neuroplasticity and stress resistance. Here we report that local alterations in the expression of Sprouty2 (SPRY2), an intracellular inhibitor of growth factor function, produces profound effects on both HIP neurogenesis and behaviors that reflect sensitivity to stressors. Viral vector-mediated disruption of endogenous Sprouty2 function (via a dominant negative construct) within the dorsal HIP of adult rats stimulates neurogenesis and produces signs of stress resilience including enhanced extinction of conditioned fear. Conversely, viral vector-mediated elevation of SPRY2 expression intensifies the behavioral consequences of stress. Studies of these manipulations in HIP primary cultures indicate that SPRY2 negatively regulates fibroblast growth factor-2 (FGF2), which has been previously shown to produce antidepressant- and anxiolytic-like effects via actions in the HIP. Our findings strengthen the relationship between HIP plasticity and stress responsiveness, and identify a specific intracellular pathway that could be targeted to study and treat stress-related disorders.

Drug withdrawal is often conceptualized as an aversive state that motivates drug-seeking and drug-taking behaviors in humans. Stress is more difficult to define, but is also frequently associated with aversive states. Here we describe evidence for the simple theory that drug withdrawal is a stress-like state, on the basis of common effects on behavioral, neurochemical, and molecular endpoints. We also describe data suggesting a more complex relationship between drug withdrawal and stress. As one example, we will highlight evidence that, depending on drug class, components of withdrawal can produce effects that have characteristics consistent with mood elevation. In addition, some stressors can act as positive reinforcers, defined as having the ability to increase the probability of a behavior that produces it. As such, accumulating evidence supports the general principles of opponent process theory, whereby processes that have an affective valence are followed in time by an opponent process that has the opposite valence. Throughout, we identify gaps in knowledge and propose future directions for research. A better understanding of the similarities, differences, and overlaps between drug withdrawal and stress will lead to the development of improved treatments for addiction, as well as for a vast array of neuropsychiatric conditions that are triggered or exacerbated by stress.

Background: Dynorphin, an endogenous ligand at kappa opioid receptors (KORs), produces depressive-like effects and contributes to addictive behavior in male nonhuman primates and rodents. Although comorbidity of depression and addiction is greater in women than men, the role of KORs in female motivated behavior is unknown., Methods: In adult Sprague-Dawley rats, we used intracranial self-stimulation to measure effects of the KOR agonist (±)-trans-U-50488 methanesulfonate salt (U-50488) (.0-10.0 mg/kg) on brain stimulation reward in gonadally intact and castrated males and in females at estrous cycle stages associated with low and high estrogen levels. Pharmacokinetic studies of U-50488 in plasma and brain were conducted. Immunohistochemistry was used to identify sex-dependent expression of U-50488-induced c-Fos in brain., Results: U-50488 dose-dependently increased the frequency of stimulation (threshold) required to maintain intracranial self-stimulation responding in male and female rats, a depressive-like effect. However, females were significantly less sensitive than males to the threshold-increasing effects of U-50488, independent of estrous cycle stage in females or gonadectomy in males. Although initial plasma concentrations of U-50488 were higher in females, there were no sex differences in brain concentrations. Sex differences in U-50488-induced c-Fos activation were observed in corticotropin releasing factor-containing neurons of the paraventricular nucleus of the hypothalamus and primarily in non-corticotropin releasing factor-containing neurons of the bed nucleus of the stria terminalis., Conclusions: These data suggest that the role of KORs in motivated behavior of rats is sex-dependent, which has important ramifications for the study and treatment of mood-related disorders, including depression and drug addiction in people., (Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Prescription opioid abuse is an increasing public health concern in the USA. A vaccine comprising a hapten (OXY) conjugated to the carrier protein keyhole limpet hemocyanin (OXY-KLH) has been shown to attenuate the antinociceptive effects of oxycodone. Here, the vaccine's ability to prevent acquisition of intravenous (i.v.) oxycodone self-administration was studied in rats. Effects of vaccination on oxycodone-induced changes in the expression of several genes within the mesolimbic system, which are regulated by chronic opiate use, were also examined. Vaccination with OXY-KLH reduced the proportion of rats acquiring i.v. self-administration of oxycodone under a fixed ratio (FR) 3 schedule of reinforcement compared to control rats immunized with the unconjugated KLH carrier protein. Vaccination significantly reduced the mean number of infusions at FR3, total number of infusions, and total oxycodone intake during the entire protocol. Compared to oxycodone self-administering control rats immunized with the carrier alone, rats vaccinated with the OXY-KLH immunogen showed increased levels of adenylate cyclase 5 (Adcy5) and decreased levels of early growth response protein 2 (Egr2) and the early immediate gene c-Fos in the striatum. These data suggest that vaccination with OXY-KLH can attenuate the reinforcing effects of oxycodone at a clinically-relevant exposure level. Analysis of mRNA expression identified some addiction-relevant markers that may be of interest in understanding oxycodone effects or the protection provided by vaccination.

Opioids selective for the G protein-coupled mu opioid receptor (MOR) produce potent analgesia and euphoria. Heroin, a synthetic opioid, is considered one of the most addictive substances, and the recent exponential rise in opioid addiction and overdose deaths has made treatment development a national public health priority. Existing medications (methadone, buprenorphine, and naltrexone), when combined with psychosocial therapies, have proven efficacy in reducing aspects of opioid addiction. Unfortunately, these medications have critical limitations including those associated with opioid agonist therapies (e.g., sustained physiological dependence and opioid withdrawal leading to high relapse rates upon discontinuation), non-adherence to daily dosing, and non-renewal of monthly injection with extended-release naltrexone. Furthermore, current medications fail to ameliorate key aspects of addiction such as powerful conditioned associations that trigger relapse (e.g., cues, stress, the drug itself). Thus, there is a need for developing novel treatments that target neural processes corrupted with chronic opioid use. This requires a basic understanding of molecular and cellular mechanisms underlying effects of opioids on synaptic transmission and plasticity within reward-related neural circuits. The focus of this review is to discuss how crosstalk between MOR-associated G protein signaling and glutamatergic neurotransmission leads to immediate and long-term effects on emotional states (e.g., euphoria, depression) and motivated behavior (e.g., drug-seeking, relapse). Our goal is to integrate findings on how opioids modulate synaptic release of glutamate and postsynaptic transmission via α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptors in the nucleus accumbens and ventral tegmental area with the clinical (neurobehavioral) progression of opioid dependence, as well as to identify gaps in knowledge that can be addressed in future studies.

Early life stress exposure (ELS) yields risk for psychiatric disorders that might occur though a population-specific mechanism that impacts prefrontal cortical development. Sex differences in ELS effects are largely unknown and are also essential to understand social and cognitive development. ELS can cause dysfunction within parvalbumin (PVB)-containing inhibitory interneurons in the prefrontal cortex and in several prefrontal cortex-mediated behaviors including social interaction. Social behavior deficits are often the earliest observed changes in psychiatric disorders, therefore the time-course and causation of social interaction deficits after ELS are important to determine. PVB interneuron dysfunction can disrupt social behavior, and has been correlated in males with elevated markers of oxidative stress and inflammation, such as cyclooxygenase-2 after ELS. Here, we measured the effects of maternal separation ELS on social interaction behaviors in males and females. Prefrontal cortex PVB and cyclooxygenase-2 were also measured in juveniles and adolescents using Western blots. ELS led to social interaction alterations earlier in females than males. Sexually dimorphic behavioral changes were consistent with prefrontal cortex PVB loss after ELS. PVB levels were decreased in ELS-exposed juvenile females, while males exposed to ELS do not display parvalbumin decreases until adolescence. Early behavioral and PVB changes in females did not appear to be mediated through cyclooxygenase-2, since levels were not affected in ELS females. Therefore, these data suggest that ELS affects males and females differently and with distinct developmental profiles., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Brain kappa-opioid receptors (KORs) are implicated in states of motivation and emotion. Activation of KORs negatively regulates mesolimbic dopamine (DA) neurons, and KOR agonists produce depressive-like behavioral effects. To further evaluate how KOR function affects behavior, we developed mutant mice in which exon 3 of the KOR gene (Oprk1) was flanked with Cre-lox recombination (loxP) sites. By breeding these mice with lines that express Cre-recombinase (Cre) in early embryogenesis (EIIa-Cre) or only in DA neurons (dopamine transporter (DAT)-Cre), we developed constitutive KOR knockouts (KOR(-/-)) and conditional knockouts that lack KORs in DA-containing neurons (DAT-KOR(lox/lox)). Autoradiography demonstrated complete ablation of KOR binding in the KOR(-/-) mutants, and reduced binding in the DAT-KOR(lox/lox) mutants. Quantitative reverse transcription PCR (qPCR) studies confirmed that KOR mRNA is undetectable in the constitutive mutants and reduced in the midbrain DA systems of the conditional mutants. Behavioral characterization demonstrated that these mutant lines do not differ from controls in metrics, including hearing, vision, weight, and locomotor activity. Whereas KOR(-/-) mice appeared normal in the open field and light/dark box tests, DAT-KOR(lox/lox) mice showed reduced anxiety-like behavior, an effect that is broadly consistent with previously reported effects of KOR antagonists. Sensitization to the locomotor-stimulating effects of cocaine appeared normal in KOR(-/-) mutants, but was exaggerated in DAT-KOR(lox/lox) mutants. Increased sensitivity to cocaine in the DAT-KOR(lox/lox) mutants is consistent with a role for KORs in negative regulation of DA function, whereas the lack of differences in the KOR(-/-) mutants suggests compensatory adaptations after constitutive receptor ablation. These mouse lines may be useful in future studies of KOR function.

The development of diet-induced obesity (DIO) can potently alter multiple aspects of dopamine signaling, including dopamine transporter (DAT) expression and dopamine reuptake. However, the time-course of diet-induced changes in DAT expression and function and whether such changes are dependent upon the development of DIO remains unresolved. Here, we fed rats a high (HFD) or low (LFD) fat diet for 2 or 6 weeks. Following diet exposure, rats were anesthetized with urethane and striatal DAT function was assessed by electrically stimulating the dopamine cell bodies in the ventral tegmental area (VTA) and recording resultant changes in dopamine concentration in the ventral striatum using fast-scan cyclic voltammetry. We also quantified the effect of HFD on membrane associated DAT in striatal cell fractions from a separate group of rats following exposure to the same diet protocol. Notably, none of our treatment groups differed in body weight. We found a deficit in the rate of dopamine reuptake in HFD rats relative to LFD rats after 6 but not 2 weeks of diet exposure. Additionally, the increase in evoked dopamine following a pharmacological challenge of cocaine was significantly attenuated in HFD relative to LFD rats. Western blot analysis revealed that there was no effect of diet on total DAT protein. However, 6 weeks of HFD exposure significantly reduced the 50 kDa DAT isoform in a synaptosomal membrane-associated fraction, but not in a fraction associated with recycling endosomes. Our data provide further evidence for diet-induced alterations in dopamine reuptake independent of changes in DAT production and demonstrates that such changes can manifest without the development of DIO.

Background: Repeated exposure to drugs of abuse and stress increase dynorphin, a κ opioid receptor (KOR) ligand, in the nucleus accumbens (NAc). Acute KOR activation produces dysphoria that might contribute to addictive behavior. How repeated KOR activation modulates reward circuitry is not understood., Methods: We used intracranial self-stimulation (ICSS), a method that provides a behavioral index of reward sensitivity, to measure the effects of repeated administration of the KOR agonist salvinorin A (salvA) (2 mg/kg) on the reward-potentiating effects of cocaine (5.0 mg/kg). In separate rats, we measured the effects of salvA on activation of extracellular signal regulated kinase (ERK), cyclic adenosine monophosphate (cAMP) response element binding protein, and c-Fos within the NAc., Results: SalvA had biphasic effects on reward: an immediate effect was to decrease the rewarding impact of ICSS, whereas a delayed effect was to increase the rewarding impact of ICSS. Repeated salvA produced a net decrease in the reward-potentiating effects of cocaine. In the NAc, both acute and repeated salvA administration increased phosphorylated ERK, whereas only acute salvA increased c-Fos and repeated salvA increased phosphorylated cAMP response element binding protein. The KOR antagonist nor-binaltorphimine (20 mg/kg) blocked the immediate and delayed effects of salvA and prolonged the duration of cocaine effects in ICSS., Conclusions: Repeated salvA might trigger opponent processes such that "withdrawal" from the dysphoric effects of KOR activation is rewarding and decreases the net rewarding valence of cocaine. The temporal effects of salvA on ERK signaling suggest KOR-mediated engagement of distinct signaling pathways within the NAc that might contribute to biphasic effects on reward sensitivity., (Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Bone marrow-derived mesenchymal stromal cells (MSCs) hold promise for autologous treatment of neuropathologies. Intranasal delivery is relatively noninvasive and has recently been reported to result in transport of MSCs to the brain. However, the ability of MSCs to migrate from nasal passages to sites of neuropathology and ultimately survive has not been fully examined. In this paper, we harvested MSCs from transgenic mice expressing enhanced green fluorescent protein (cells hereafter referred to as MSC-EGFP) and delivered them intranasally to wild-type mice sustaining mechanical lesions in the striatum. Using fluorescent, colorimetric, and ultrastructural detection methods, GFP-expressing cells were undetectable in the brain from 3 hours to 2 months after MSC delivery. However, bright autofluorescence that strongly resembled emission from GFP was observed in the olfactory bulb and striatum of lesioned control and MSC-EGFP-treated mice. In a control experiment, we directly implanted MSC-EGFPs into the mouse striatum and detected robust GFP expression 1 and 7 days after implantation. These findings suggest that-under our conditions-intranasally delivered MSC-EGFPs do not survive or migrate in the brain. Furthermore, our observations highlight the necessity of including appropriate controls when working with GFP as a cellular marker.

Rationale: Kappa opioid receptors (KORs) have been implicated in depressive-like states associated with chronic administration of drugs of abuse and stress. Although KOR agonists decrease dopamine in the nucleus accumbens (NAc), KOR modulation of phasic dopamine release in the core and shell subregions of the NAc-which have distinct roles in reward processing-remains poorly understood., Objectives: Studies were designed to examine whether the time course of effects of KOR activation on phasic dopamine release in the NAc core or shell are similar to effects on motivated behavior., Methods: The effect of systemic administration of the KOR agonist salvinorin A (salvA)-at a dose (2.0 mg/kg) previously determined to have depressive-like effects-was measured on electrically evoked phasic dopamine release in the NAc core or shell of awake and behaving rats using fast scan cyclic voltammetry. In parallel, the effects of salvA on intracranial self-stimulation (ICSS) and sucrose-reinforced responding were assessed. For comparison, a threshold dose of salvA (0.25 mg/kg) was also tested., Results: The active, but not threshold, dose of salvA significantly decreased phasic dopamine release without affecting dopamine reuptake in the NAc core and shell. SalvA increased ICSS thresholds and significantly lowered breakpoint on the progressive ratio schedule, indicating a decrease in motivation. The time course of the KOR-mediated decrease in dopamine in the core was qualitatively similar to the effects on motivated behavior., Conclusions: These data suggest that the effects of KOR activation on motivation are due, in part, to inhibition of phasic dopamine signaling in the NAc core.

Rationale: Chronic opiate administration induces neuroadaptations within the nucleus accumbens (NAc) and ventral tegmental area (VTA) that can contribute to dependence. We have shown that morphine dependence shifts the behavioral consequences of D1 dopamine (DA) receptor signaling: systemic administration of a D1 receptor agonist is rewarding and blocks naloxone-precipitated withdrawal signs in morphine-dependent rats, but has minimal effects in nondependent rats. These data suggest that D1 receptors acquire the ability to regulate reward and withdrawal in morphine-dependent rats. The brain regions involved in these effects are not known., Objective: Studies were designed to test the hypothesis that the nucleus accumbens shell (NASh) and the ventral tegmental area (VTA) are important sites for mediating the behavioral effects of D1 receptor activation in morphine-dependent rats., Materials and Methods: The effects of microinjecting the D1 receptor agonist SKF 82958 into the NASh or the VTA on place conditioning and somatic withdrawal signs were studied in morphine-dependent and nondependent rats., Results: Intra-NASh microinjection of SKF 82958 (1 microg/side) established conditioned place preferences in morphine-dependent but not nondependent rats, but had no effect on naloxone-induced place aversions or somatic withdrawal signs. Intra-VTA microinjection of SKF 82958 (2 microg) did not establish place preferences under any conditions, but blocked naloxone-induced place aversions without effects on somatic withdrawal signs., Conclusions: There is an anatomical dissociation between D1 receptor-mediated reward and relief of withdrawal in morphine-dependent rats. When combined, the individual effects of D1 receptor activation in the NASh and VTA on the affective signs of precipitated morphine withdrawal resemble those seen with systemic administration.

The nucleus accumbens (NAc) is a critical brain area for reward and motivated behavior. Accumulating evidence suggests that altered function of the transcription factor cAMP response element binding protein (CREB) within the NAc is involved in depressive behavior. In rats, stress activates CREB within the NAc, and elevation of CREB expression in this region produces depressive-like behaviors that are accompanied by activation of CREB-regulated target genes. The depressive-like behaviors seem to be due, at least in part, to CREB-mediated increases in dynorphin function, because they are mimicked by kappa-opioid receptor (KOR) agonists and attenuated by KOR antagonists. We hypothesized that if CREB-mediated dynorphin expression in the NAc contributes to depressive behavior, then antidepressants might reduce dynorphin function in this region. Here, we demonstrate that desipramine (DMI), a norepinephrine reuptake inhibitor that has been used for decades to treat clinical depression, blocks swim stress-induced activation of prodynorphin (encodes dynorphin) in the NAc. In primary cultures of NAc and striatum, DMI decreases basal and stimulated CREB phosphorylation by causing reductions in intracellular calcium (Ca(2+)) availability that are independent of norepinephrine or other monoaminergic inputs, identifying a potential mechanism for alterations in CREB-mediated gene expression. Fluoxetine (FLX), a selective serotonin reuptake inhibitor, has similar effects in culture, suggesting a common intracellular effect of these antidepressants. These findings raise the possibility that a therapeutically relevant mechanism of action of DMI occurs through attenuation of CREB-mediated gene transcription, which is mediated via previously uncharacterized mechanisms that occur directly within the NAc.

Background: Increasing evidence indicates that brain kappa-opioid receptors (KORs) are involved in regulation of mood states. In animal models often used to study psychiatric illness, KOR agonists produce depressive-like effects (e.g., anhedonia), whereas KOR antagonists produce antidepressant- and anxiolytic-like effects. The ability of KOR agonists to produce anhedonia-like signs in laboratory animals raises the possibility that this class of drugs might be useful to ameliorate states characterized by excess reward or motivation, such as mania or stimulant intoxication., Methods: We examined how the selective KOR agonist U69,593 affects cocaine-induced facilitation of intracranial self-stimulation (ICSS), a model of the abnormally increased reward function that characterizes mania and stimulant intoxication. Rats with stimulating electrodes implanted in the medial forebrain bundle (MFB) were tested with intraperitoneal injections of U69,593 (.063-.5 mg/kg) alone, cocaine (1.25-10 mg/kg) alone, and combinations of the drugs., Results: Cocaine dose-dependently decreased ICSS thresholds, indicating that it enhanced the rewarding impact of MFB stimulation. In contrast, U69,593 dose-dependently increased ICSS thresholds, indicating that it decreased the rewarding impact of the stimulation. Pretreatment with U69,593 blocked cocaine-induced decreases in ICSS thresholds at doses that had negligible effects on their own., Conclusions: Activation of KORs reduces the reward-related effects of cocaine. Inasmuch as cocaine-induced behavioral stimulation in rodents may model key aspects of enhanced mood in humans, these findings raise the possibility that KOR agonists might ameliorate symptoms of conditions characterized by increased motivation and hyperfunction of brain reward systems, such as mania and stimulant intoxication.

Stress and chronic exposure to drugs of abuse can trigger addictive and depressive disorders. Both stimuli increase activity of dynorphin, a neuropeptide that acts at kappa-opioid receptors (KORs). In humans, KOR agonists cause dysphoria, raising the possibility that dynorphin modulates the depressive-like effects of stress and chronic drug use. We examined if KOR activation alters sensitivity to stimulant drugs by assessing the effects of the selective KOR agonist, salvinorin A (SalvA), on cocaine-induced locomotor activity and c-Fos expression. Acute administration of SalvA blocked the locomotor-stimulant effects of cocaine, whereas repeated SalvA together with concomitant exposure to activity testing chambers potentiated the locomotor response to a cocaine challenge. In contrast, repeated SalvA administered in home cages rather than the activity chambers failed to potentiate the locomotor response to a cocaine challenge. One potential explanation for these findings is that activation of KORs disrupts context conditioning: acute locomotor responses to SalvA alone did not fully habituate with repeated testing in the activity chambers. The effects of SalvA on locomotor activity paralleled its effects on cocaine-induced c-Fos expression in the dorsal striatum: acute SalvA attenuated cocaine-induced c-Fos, whereas repeated SalvA potentiated it when administered in the activity chambers but not the home cage. Acute SalvA also blocked the locomotor stimulant effects of the D1 receptor agonist SKF 82958, whereas repeated SalvA potentiated these effects when administered in the activity chambers. These findings suggest that SalvA regulates the stimulant effects of cocaine through interactions with D1 receptor-mediated signaling in the dorsal striatum.

It has become increasingly important to assess mood states in laboratory animals. Tests that reflect reward, reduced ability to experience reward (anhedonia) and aversion (dysphoria) are in high demand because many psychiatric conditions that are currently intractable in humans (e.g., major depression, bipolar disorder, addiction) are characterized by dysregulated motivation. Intracranial self-stimulation (ICSS) can be utilized in rodents (rats, mice) to understand how pharmacological or molecular manipulations affect the function of brain reward systems. Although many different methodologies are possible, we will describe in this protocol the use of medial forebrain bundle (MFB) stimulation together with the 'curve-shift' variant of analysis. This combination is particularly powerful because it produces a highly reliable behavioral output that enables clear distinctions between the treatment effects on motivation and the treatment effects on the capability to perform the task.

Background: Calcium (Ca2+) influx within the nucleus accumbens shell (NASh) can influence brain reward processes. We found previously that rats self-administer NMDA receptor antagonists (which block Ca2+ influx through NMDA receptors) into the NASh. We also found that manipulations which increase expression of Ca2+-permeable AMPA receptors within this region make cocaine aversive. Here we examined if Ca2+ influx via L-type Ca2+ channels within the NASh would influence cocaine reward., Methods: Rats received bilateral microinjections of the L-type Ca2+ channel antagonist diltiazem into the ventral NASh prior to place conditioning with systemic cocaine., Results: Microinjections of diltiazem (10 nmol/hemisphere) into the ventral NASh facilitated the ability of a sub-threshold dose of cocaine (5.0 mg/kg) to establish place preferences, but did not affect place conditioning on their own (5.0-40 nmol/hemisphere). Microinjections into more dorsal regions had no effects., Conclusions: Blockade of Ca2+ influx through L-type channels Ca2+ within the ventral NASh increases cocaine reward.

Morphine dependence is characterized by somatic and motivational signs of withdrawal that likely contribute to the maintenance of addictive behavior. The nucleus accumbens (NAc) receives extensive dopaminergic input and is an important substrate for mediating these aversive states. In the NAc, the function of the transcription factor cAMP response element binding protein (CREB) and AMPA glutamate receptor subunit, type 1 (GluR1) can be regulated by dopamine (DA) D1 receptor-mediated phosphorylation (P-CREB, P-GluR1). However, the roles of D1 receptors, CREB, and GluR1 in morphine dependence are not well understood. Here, we show that somatic signs of naloxone-precipitated withdrawal were associated with increased P-CREB, but not P-GluR1, in the NAc of morphine-dependent rats. The D1 receptor agonist chloro-APB hydrobromide (SKF 82958) was rewarding in morphine-dependent rats and blocked naloxone-induced place aversions and somatic signs of withdrawal. Surprisingly, SKF 82958 increased P-GluR1, but not P-CREB, in the NAc, and naloxone reduced SKF 82958-mediated P-GluR1 induction specifically in morphine-dependent rats. Together, these results confirm that aversive treatments can increase CREB function in the NAc. Furthermore, they suggest a dependence-associated shift in the molecular mechanisms that regulate the consequences of D1 receptor stimulation, favoring activation of GluR1 rather than CREB. These data raise the possibility that the rewarding effects of SKF 82958 in morphine-dependent rats involve increased P-GluR1 in the NAc, although the involvement of other brain regions cannot be ruled out. Regardless, these findings suggest for the first time that D1 agonists might be useful for the treatment of withdrawal symptoms that contribute to the maintenance of opiate addiction in humans.

The amygdala plays key roles in several aspects of addiction to drugs of abuse. This brain structure has been implicated in behaviours that reflect drug reward, drug seeking, and the aversive effects of drug withdrawal. Using a model that involves repeated cocaine injections to approximate 'binge' intoxication, we show in rats that during cocaine withdrawal, the impact of rewarding brain stimulation is attenuated, as quantified by alterations in intracranial self-stimulation (ICSS) behaviour. These behavioural signs of withdrawal are accompanied by enhancements of glutamatergic synaptic transmission within the lateral amygdala (LA) that occlude electrically induced long-term potentiation (LTP) in tissue slices. Synaptic enhancements during periods of cocaine withdrawal are mechanistically similar to LTP induced with electrical stimulation in control slices, as both forms of synaptic plasticity involve an increase in glutamate release. These results suggest that mechanisms of LTP within the amygdala are recruited during withdrawal from repeated exposure to cocaine. As such, they raise the possibility that the development and maintenance of addictive behaviours may involve, at least in part, mechanisms of synaptic plasticity within specific amygdala circuits.

N-methyl-D-aspartate (NMDA) receptor antagonists can elicit symptoms in humans that resemble those seen in schizophrenic patients. Rodents manifest locomotor and stereotypic behaviors when treated with NMDA receptor antagonists such as phencyclidine (PCP) or dizocilpine maleate (MK-801); these behaviors are usually associated with an activated dopamine system. However, recent evidence suggests that increased glutamatergic transmission mediates the effects of these NMDA receptor antagonists. The role of dopamine in PCP- and MK-801-induced behavior (eg hyperlocomotion) remains unclear. We used dopamine-deficient (DD) mice in which tyrosine hydroxylase is selectively inactivated in dopaminergic neurons to determine whether dopamine is required for the locomotor and molecular effects of PCP and MK-801. DD mice showed a similar increase in locomotor activity and c-fos mRNA induction in the striatum in response to these NMDA receptor antagonists as control mice. Restoration of dopamine signaling in DD mice enhanced their locomotor response to PCP and MK-801. Administration of LY379268, a group II metabotropic glutamate receptor agonist that inhibits glutamate release, blocked PCP- and MK-801-induced hyperlocomotion in both DD and control mice. These results suggest that glutamate, rather than dopamine, is required for the locomotor and molecular effects of NMDA receptor antagonists, but that glutamate and dopamine can act cooperatively.

The neuropeptide neurotensin (NT) is highly sensitive to changes in dopaminergic signaling in the striatum, and is thought to modulate dopamine-mediated behaviors. To explore the interaction of NT with the dopamine system, we utilized mice with a targeted deletion of dopamine synthesis specifically in dopaminergic neurons. Dopamine levels in dopamine-deficient (DD) mice are less than 1% of control mice, and they require daily administration of the dopamine precursor L-dihydroxyphenylalanine (L-DOPA) for survival. DD mice are supersensitive to the effects of dopamine, becoming hyperactive relative to control mice in the presence of L-DOPA. We show that 24 h after L-DOPA treatment, when DD mice are in a "dopamine-depleted" state, Nt mRNA levels in the striatum of DD mice are similar to those in control mice. Administration of L-DOPA or L-DOPA plus the L-amino acid decarboxylase inhibitor, carbidopa, (C/L-DOPA) induced Nt expression in the striatum of DD mice. The dopamine D1 receptor antagonist, SCH23390, blocked C/L-DOPA-induced Nt. To test the hypothesis that this striatal Nt expression modulated dopamine-mediated behavior in DD mice, we administered SR 48692, an antagonist of the high affinity NT receptor, together with L-DOPA or C/L-DOPA. L-DOPA-induced hyperlocomotion and C/L-DOPA-induced stereotypy were potentiated by peripheral administration of SR 48692. Furthermore, intrastriatal microinjections of SR 48692 augmented L-DOPA-induced hyperlocomotion. These results demonstrate a dynamic regulation of striatal Nt expression by dopamine via D1 receptors in DD mice, and point to a physiological role for endogenous striatal NT in counteracting motor behaviors induced by an overactive dopamine system.

Chronic morphine leads to compensatory up-regulation of cAMP signaling pathways in numerous brain regions. One potential consequence of up-regulated cAMP signaling is increased phosphorylation of cAMP response element binding protein (CREB), a transcription factor that may regulate neuroadaptations related to morphine dependence. Altered gene expression within the nucleus accumbens (NAc), a ventral component of the striatum that receives substantial dopaminergic input, may play a role in some of the motivational aspects of opiate withdrawal. To determine if morphine withdrawal leads to increased CREB phosphorylation in striatal tissues, we examined the effects of naloxone-precipitated morphine withdrawal on CREB phosphorylation in primary cultures of rat striatal neurons. Precipitated morphine withdrawal was associated with enhanced dopamine-, SKF 82958 (D1 receptor agonist)-, and forskolin-induced CREB phosphorylation. During precipitated withdrawal, D1 receptor-mediated CREB phosphorylation was dependent on cAMP-dependent protein kinase (PKA). Precipitated withdrawal also led to up-regulation of c-fos mRNA in response to SKF 82958. CREB protein levels were not altered by acute or chronic morphine. These results suggest that D1 receptor-mediated signal transduction is enhanced during morphine withdrawal. Furthermore, they are consistent with in vivo evidence suggesting that increased CREB activation in portions of the striatum (e.g. the NAc) is related to dysphoric states associated with drug withdrawal.

Motor stereotypies are abnormally repetitive behaviors that can develop with excessive dopaminergic stimulation and are features of some neurologic disorders. To investigate the mechanisms required for the induction of stereotypy, we examined the responses of dopamine-deficient (DD) mice to increasing doses of the dopamine precursor L-DOPA. DD mice lack the ability to synthesize dopamine (DA) specifically in dopaminergic neurons yet exhibit robust hyperlocomotion relative to wild-type (WT) mice when treated with L-DOPA, which restores striatal DA tissue content to approximately 10% of WT levels. To further elevate brain DA content in DD mice, we administered the peripheral L-amino acid decarboxylase inhibitor carbidopa along with L-DOPA (C/l-DOPA). When striatal DA levels reached >50% of WT levels, a transition from hyperlocomotion to intense, focused stereotypy was observed that was correlated with an induction of c-fos mRNA in the ventrolateral and central striatum as well as the somatosensory cortex. WT mice were unaffected by C/L-DOPA treatments. A D1, but not a D2, receptor antagonist attenuated both the C/L-DOPA-induced stereotypy and the c-fos induction. Consistent with these results, stereotypy could be induced in DD mice by a D1, but not by a D2, receptor agonist, with neither agonist inducing stereotypy in WT mice. Intrastriatal injection of a D1 receptor antagonist ameliorated the stereotypy and c-fos induction by C/L-DOPA. These results indicate that activation of D1 receptors on a specific population of striatal neurons is required for the induction of stereotypy in DD mice.

Acute blockade of dopamine D(2) receptors by the typical antipsychotic drug haloperidol leads to alterations in neuronal gene expression and behavior. In the dorsolateral striatum, the levels of mRNA for the immediate-early gene c-fos and the neuropeptide gene neurotensin/neuromedin N (NT/N) are significantly increased by haloperidol. An acute behavioral response to haloperidol is catalepsy, considered to be a rodent correlate of some of the immediate extrapyramidal motor side effects seen in humans. Several lines of evidence suggest a link between neurotensin induction in the dorsolateral striatum and catalepsy. We hypothesize that both striatal gene induction and catalepsy elicited by haloperidol arise from the combined effect of excitatory adenosinergic and glutamatergic inputs acting at adenosine A(2A) and N-methyl-D-aspartate (NMDA) receptors, respectively. In agreement with our previous reports, adenosine antagonists reduced haloperidol-induced c-fos and neurotensin gene expression as well as catalepsy. In agreement with other reports, the noncompetitive NMDA receptor antagonist MK-801 also reduced gene expression and catalepsy in response to haloperidol. The competitive NMDA receptor antagonist LY235959 decreased haloperidol-induced catalepsy. We show here that blocking both A(2A) and NMDA receptors simultaneously in conjunction with haloperidol resulted in a combined effect on gene expression and behavior that was greater than that for block of either receptor alone. Both c-fos and NT/N mRNA levels were reduced, and catalepsy was completely abolished. These results indicate that the haloperidol-induced increases in c-fos and NT gene expression in the dorsolateral striatum and catalepsy are driven largely by adenosine and glutamatergic inputs acting at A(2A) and NMDA receptors.

The antipsychotic drug, haloperidol, elicits the expression of neurotensin and c-fos mRNA in the dorsal lateral region of the striatum and produces an acute cataleptic response in rodents that correlates with the motor side effects of haloperidol in humans. Mice harboring a targeted disruption of the RIIbeta subunit of protein kinase A have a profound deficit in cAMP-stimulated kinase activity in the striatum. When treated with haloperidol, RIIbeta mutant mice fail to induce either c-fos or neurotensin mRNA and the acute cataleptic response is blocked. However, both wild-type and mutant mice become cataleptic when neurotensin peptide is directly injected into the lateral ventricle, demonstrating that the kinase deficiency does not interfere with the action of neurotensin but rather its synthesis and release. These results establish a direct role for protein kinase A as a mediator of haloperidol induced gene induction and cataleptic behavior.

The proteins necessary for signal transduction in cells responding to ligands of the TGF-beta family are largely unknown. We have previously identified Mad (Mothers against dpp), a gene that interacts with the TGF-beta family member encoded by decapentaplegic (dpp) in Drosophila. Assay of Mad's role in the DPP-dependent events of embryonic midgut development demonstrates that Mad is required for any response of the visceral mesoderm or endoderm to DPP signals from the visceral mesoderm. Replacement of the normal DPP promoter with a heterologous (hsp70) promoter fails to restore DPP-dependent responses in Mad mutant midguts. Experiments utilizing Mad transgenes regulated by tissue-specific promoters show that MAD is required specifically in cells responding to DPP. Immunohistochemical studies localize MAD to the cytoplasm in all tissues examined. Experiments in Xenopus embryos demonstrate that Drosophila MAD can function in the signaling pathway of BMP-4, a vertebrate homolog of dpp. Based on these results, we propose that Mad is a highly conserved and essential element of the DPP signal transduction pathway.

The decapentaplegic (dpp) gene of Drosophila melanogaster encodes a growth factor that belongs to the transforming growth factor-beta (TGF-beta) superfamily and that plays a central role in multiple cell-cell signaling events throughout development. Through genetic screens we are seeking to identify other functions that act upstream, downstream or in concert with dpp to mediate its signaling role. We report here the genetic characterization and cloning of Mothers against dpp (Mad), a gene identified in two such screens. Mad loss-of-function mutations interact with dpp alleles to enhance embryonic dorsal-ventral patterning defects, as well as adult appendage defects, suggesting a role for Mad in mediating some aspect of dpp function. In support of this, homozygous Mad mutant animals exhibit defects in midgut morphogenesis, imaginal disk development and embryonic dorsal-ventral patterning that are very reminiscent of dpp mutant phenotypes. We cloned the Mad region and identified the Mad transcription unit through germline transformation rescue. We sequenced a Mad cDNA and identified three Mad point mutations that alter the coding information. The predicted MAD polypeptide lacks known protein motifs, but has strong sequence similarity to three polypeptides predicted from genomic sequence from the nematode Caenorhabditis elegans. Hence, MAD is a member of a novel, highly conserved protein family.