Paradoxical anxiolytic effect of the 'bath salt' synthetic cathinone MDPV during early abstinence is inhibited by a chemokine CXCR4 or CCR5 receptor antagonist.

Chemokine CXCR4 and CCR5 receptors are best known as HIV co-entry receptors, but evidence that CXCR4 or CCR5 blockade reduces rewarding and locomotor-stimulant effects of psychostimulants in rats suggests a role in psychostimulant use disorders. We investigated the impact of CXCR4 or CCR5 receptor antagonism on anxiety-related effects of the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) in the elevated zero-maze (EZM) assay. Rats exposed to a 4-day MDPV binge dosing paradigm and tested 24 or 72 h post-treatment spent more time in the open compartment at the 24-h time point but less time at the 72-h post-binge time point. Daily administration of AMD 3100, a CXCR4 antagonist (10 mg/kg), or maraviroc, a CCR5 antagonist (2.5 mg/kg), during MDPV treatment inhibited the MDPV-induced increase in time spent in the open compartment. Neither antagonist affected the MDPV-induced reduction in time spent in the open compartment at the 72-h post-binge time point. Cocaine, administered in the same paradigm as MDPV, did not increase time spent in the open compartment 24-h post-binge, suggesting specificity to MDPV. The present results identify a surprising anxiolytic-like effect of MDPV 24 h after cessation of repeated exposure that is sensitive to chemokine CXCR4 and CCR5 receptor activity.

CRF-5-HT interactions in the dorsal raphe nucleus and motivation for stress-induced opioid reinstatement.

RATIONALE: The serotonin (5-hydroxytryptamine, 5-HT) system plays an important role in stress-related psychiatric disorders and substance abuse. Our previous data show that stressors can inhibit 5-HT neuronal activity and release by stimulating the release of the stress neurohormone corticotropin-releasing factor (CRF) within the serotonergic dorsal raphe nucleus (DRN). The inhibitory effects of CRF on 5-HT DRN neurons are indirect, mediated by CRF-R1 receptors located on GABAergic afferents. OBJECTIVES: We tested the hypothesis that DRN CRF-R1 receptors contribute to stress-induced reinstatement of morphine-conditioned place preference (CPP). We also examined the role of this circuitry in stress-induced negative affective state with 22-kHz distress ultrasonic vocalizations (USVs), which are naturally emitted by rats in response to environmental challenges such as pain, stress, and drug withdrawal. METHODS: First, we tested if activation of CRF-R1 receptors in the DRN with the CRF-R1-preferring agonist ovine CRF (oCRF) would reinstate morphine CPP and then if blockade of CRF-R1 receptors in the DRN with the CRF-R1 antagonist NBI 35965 would attenuate swim stress-induced reinstatement of morphine CPP. Second, we tested if intra-DRN pretreatment with NBI 35965 would attenuate foot shock stress-induced 22-kHz USVs. RESULTS: Intra-DRN injection of oCRF reinstated morphine CPP, while intra-DRN injection of NBI 35965 attenuated swim stress-induced reinstatement. Moreover, intra-DRN pretreatment with NBI 35965 significantly reduced 22-kHz distress calls induced by foot shock. CONCLUSIONS: These data provide evidence that stress-induced negative affective state is mediated by DRN CRF-R1 receptors and may contribute to reinstatement of morphine CPP.

Cocaine abuse and midbrain circuits: Functional anatomy of hypocretin/orexin transmission and therapeutic prospect.

Cocaine abuse remains a pervasive public health problem, and treatments thus far have proven ineffective for long-term abstinence maintenance. Intensive research on the neurobiology underlying drug abuse has led to the consideration of many candidate transmitter systems to target for intervention. Among these, the hypocretin/orexin (hcrt/ox) neuropeptide system holds largely untapped yet clinically viable therapeutic potential. Hcrt/ox originates from the hypothalamus and projects widely across the mammalian central nervous system to produce neuroexcitatory actions via two excitatory G-protein coupled receptor subtypes. Functionally, hcrt/ox promotes arousal/wakefulness and facilitates energy homeostasis. In the early 2000s, hcrt/ox transmission was shown to underlie mating behavior in male rats suggesting a novel role in reward-seeking. Soon thereafter, hcrt/ox neurons were shown to respond to drug-associated stimuli, and hcrt/ox transmission was found to facilitate motivated responding for intravenous cocaine. Notably, blocking hcrt/ox transmission using systemic or site-directed pharmacological antagonists markedly reduced motivated drug-taking as well as drug-seeking in tests of relapse. This review will unfold the current state of knowledge implicating hcrt/ox receptor transmission in the context of cocaine abuse and provide detailed background on animal models and underlying midbrain circuits. Specifically, attention will be paid to the mesoaccumbens, tegmental, habenular, pallidal and preoptic circuits. The review will conclude with discussion of recent preclinical studies assessing utility of suvorexant - the first and only FDA-approved hcrt/ox receptor antagonist - against cocaine-associated behaviors.

Indices of dentate gyrus neurogenesis are unaffected immediately after or following withdrawal from morphine self-administration compared to saline self-administering control male rats.

Opiates - including morphine - are powerful analgesics with high abuse potential. In rodents, chronic opiate exposure or self-administration negatively impacts hippocampal-dependent function, an effect perhaps due in part to the well-documented opiate-induced inhibition of dentate gyrus (DG) precursor proliferation and neurogenesis. Recently, however, intravenous (i.v.) morphine self-administration (MSA) was reported to enhance the survival of new rat DG neurons. To reconcile these disparate results, we used rat i.v. MSA to assess 1) whether a slightly-higher dose MSA paradigm also increases new DG neuron survival; 2) how MSA influences cells in different stages of DG neurogenesis, particularly maturation and survival; and 3) if MSA-induced changes in DG neurogenesis persist through a period of abstinence. To label basal levels of proliferation, rats received the S-phase marker bromodeoxyuridine (BrdU, i.p.) 24 -h prior to 21 days (D) of i.v. MSA or saline self-administration (SSA). Either immediately after SA (0-D) or after 4 weeks in the home cage (28-D withdrawal), stereology was used to quantify DG proliferating precursors (or cells in cell cycle; Ki67+ cells), neuroblast/immature neurons (DCX+ cells), and surviving DG granule cells (BrdU+ cells). Analysis revealed the number of DG cells immunopositive for these neurogenesis-relevant markers was similar between MSA and SSA rats at the 0-D or 28-D timepoints. These negative data highlight the impact experimental parameters, timepoint selection, and quantification approach have on neurogenesis results, and are discussed in the context of the large literature showing the negative impact of opiates on DG neurogenesis.

Image-guided cranial irradiation-induced ablation of dentate gyrus neurogenesis impairs extinction of recent morphine reward memories.

Dentate gyrus adult neurogenesis is implicated in the formation of hippocampal-dependent contextual associations. However, the role of adult neurogenesis during reward-based context-dependent paradigms-such as conditioned place preference (CPP)-is understudied. Therefore, we used image-guided, hippocampal-targeted X-ray irradiation (IG-IR) and morphine CPP to explore whether dentate gyrus adult neurogenesis plays a role in reward memories created in adult C57BL/6J male mice. In addition, as adult neurogenesis appears to participate to a greater extent in retrieval and extinction of recent (<48 hr posttraining) versus remote (>1 week posttraining) memories, we specifically examined the role of adult neurogenesis in reward-associated contextual memories probed at recent and remote timepoints. Six weeks post-IG-IR or Sham treatment, mice underwent morphine CPP. Using separate groups, retrieval of recent and remote reward memories was found to be similar between IG-IR and Sham treatments. Interestingly, IG-IR mice showed impaired extinction-or increased persistence-of the morphine-associated reward memory when it was probed 24-hr (recent) but not 3-weeks (remote) postconditioning relative to Sham mice. Taken together, these data show that hippocampal-directed irradiation and the associated decrease in dentate gyrus adult neurogenesis affect the persistence of recently-but not remotely-probed reward memory. These data indicate a novel role for adult neurogenesis in reward-based memories and particularly the extinction rate of these memories. Consideration of this work may lead to better understanding of extinction-based behavioral interventions for psychiatric conditions characterized by dysregulated reward processing.

Synthetic cathinone MDPV enhances reward function through purinergic P2X7 receptor-dependent pathway and increases P2X7 gene expression in nucleus accumbens.

BACKGROUND AND PURPOSE: Purinergic P2X7 receptors are present on neurons, astrocytes and microglia and activated by extracellular ATP. Since P2X7 receptor activation releases endogenous substrates (e.g., pro-inflammatory cytokines, dopamine, and glutamate) that facilitate psychostimulant reward and reinforcement, we investigated the hypothesis that the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) produces rewarding effects that are dependent on active P2X7 receptors. METHODS: Reward function was measured in male mice using intracranial self-stimulation (ICSS). MDPV (0.1, 0.3, 0.5 mg/kg, SC) and a selective P2X7 antagonist (A438079) (5, 10, 50 mg/kg, IP) were tested alone and in combination. In separate mice, gene and protein expression of P2X7 and mitochondrial adenosine triphosphate (ATP) synthase (an enzyme that catalyzes synthesis of ATP, an endogenous ligand for P2X7 receptors) in the nucleus accumbens (NAcc) were quantified following MDPV exposure (0.1, 0.5, 5 mg/kg, SC). KEY RESULTS: MDPV (0.5 mg/kg, SC) facilitated ICSS as quantified by a significant reduction in brain reward threshold. A438079 (5, 10, 50 mg/kg, IP) did not affect ICSS by itself; however, for combined administration, A438079 (10 mg/kg, IP) inhibited facilitation of ICSS by MDPV (0.5 mg/kg, SC). At the cellular level, MDPV exposure increased gene and protein expression of P2X7 and ATP synthase in the NAcc. CONCLUSION AND IMPLICATION: We provide evidence that a psychostimulant drug produces reward enhancement that is influenced by P2X7 receptor activity and enhances P2X7 receptor expression in the brain reward circuit.

Synthetic cathinone adulteration of illegal drugs.

RATIONALE: Current prevalence estimates of synthetic cathinone ("bath salt") use may be underestimates given that traditional metrics (e.g., surveys, urinalysis) often fail to capture the emergent issue of synthetic cathinone adulteration of more common illegal drugs, such as ecstasy (3,4-methylenedioxymethamphetamine). OBJECTIVES: This review examines the evolution of synthetic cathinones and prevalence of use over the past decade in the United States. We also review methods of self-report and biological testing of these compounds as well as adverse outcomes associated with adulterated drug use. RESULTS: Synthetic cathinone use emerged in the United States by 2009 with use associated with tens of thousands of poisonings. Reported poisonings and self-reported use have substantially decreased over the past five years. However, our review suggests that current estimates of use are underestimates due to underreporting stemming primarily from unknown or unintentional use of adulterated formulations of relatively popular illegal drugs, such as ecstasy. CONCLUSIONS: While intentional synthetic cathinone use has decreased in recent years, evidence suggests that prevalence of use is underestimated. Testing of drugs and/or biological specimens can improve the accuracy of synthetic cathinone use estimates. Furthermore, we advocate that researchers and clinicians should become better aware that exposure to these potent compounds (e.g., as adulterants) often occurs unknowingly or unintentionally. To improve our understanding of synthetic cathinone adulteration, research utilizing a combinatorial approach (survey and biological testing) will help more accurately estimate the prevalence and impact of this public health issue.

DARK Classics in Chemical Neuroscience: Cathinone-Derived Psychostimulants.

Cathinone is a plant alkaloid found in khat leaves of perennial shrubs grown in East Africa. Similar to cocaine, cathinone elicits psychostimulant effects which are in part attributed to its amphetamine-like structure. Around 2010, home laboratories began altering the parent structure of cathinone to synthesize derivatives with mechanisms of action, potencies, and pharmacokinetics permitting high abuse potential and toxicity. These "synthetic cathinones" include 4-methylmethcathinone (mephedrone), 3,4-methylenedioxypyrovalerone (MDPV), and the empathogenic agent 3,4-methylenedioxymethcathinone (methylone) which collectively gained international popularity following aggressive online marketing as well as availability in various retail outlets. Case reports made clear the health risks associated with these agents and, in 2012, the Drug Enforcement Agency of the United States placed a series of synthetic cathinones on Schedule I under emergency order. Mechanistically, cathinone and synthetic derivatives work by augmenting monoamine transmission through release facilitation and/or presynaptic transport inhibition. Animal studies confirm the rewarding and reinforcing properties of synthetic cathinones by utilizing self-administration, place conditioning, and intracranial self-stimulation assays and additionally show persistent neuropathological features which demonstrate a clear need to better understand this class of drugs. This Review will thus detail (i) historical context of cathinone use and the rise of "dark" synthetic derivatives, (ii) structural features and mechanisms of synthetic cathinones, (iii) behavioral effects observed clinically and in animals under controlled laboratory conditions, and (iv) neurotransmitters and circuits that may be targeted to manage synthetic cathinone abuse in humans.

Chemokines and 'bath salts': CXCR4 receptor antagonist reduces rewarding and locomotor-stimulant effects of the designer cathinone MDPV in rats.

BACKGROUND AND PURPOSE: Little is known about how chemokine systems influence the behavioral effects of designer cathinones and psychostimulants. The chemokine CXCL12 and its principal receptor target, CXCR4, are of particular interest because CXCR4 activation enhances mesolimbic dopamine output that facilitates psychostimulant reward, reinforcement, and locomotor activation. Repeated cocaine enhances CXCL12 gene expression in the midbrain and produces conditioned place preference (CPP) that is inhibited by a CXCR4 antagonist. Yet, interactions between chemokines and synthetic cathinones remain elusive. METHODS: We tested the hypothesis that an FDA-approved CXCR4 antagonist (AMD3100) inhibits MDPV-induced reward, locomotor activation and positive affective state in rats using a triad of behavioral assays (CPP, open field, and 50-kHz ultrasonic vocalizations [USVs]). KEY RESULTS: AMD3100 (1, 2.5, 5, 10 mg/kg, ip) significantly reduced MDPV (2 mg/kg, ip)-evoked hyper-locomotion in a dose-related manner. AMD3100 (1, 5, 10 mg/kg) administered during CPP conditioning caused a significant, dose-dependent reduction of MDPV (2 mg/kg x 4 days) place preference. MDPV injection elicited significantly greater 50 kHz USVs in vehicle-pretreated rats but not in AMD3100-pretreated rats. CONCLUSION AND IMPLICATION: A CXCR4 antagonist reduced the rewarding and locomotor-activating effects of MDPV. Our results identify the existence of chemokine/cathinone interactions and suggest the rewarding and stimulant effects of MDPV, similar to cocaine, require an active CXCL12/CXCR4 system.

Effects of Suvorexant, a Dual Orexin/Hypocretin Receptor Antagonist, on Impulsive Behavior Associated with Cocaine.

Hypothalamic hypocretin (orexin) peptides mediate arousal, attention, and reward processing. Fibers containing orexins project to brain structures that govern motivated behavior, including the ventral tegmental area (VTA). A number of psychiatric conditions, including attention deficit hyperactivity disorder (ADHD) and substance use disorders, are characterized by deficits in impulse control, however the relationship between orexin and impulsive behavior is incompletely characterized. The effects of systemic or centrally administered orexin receptor (OXR) antagonists on measures of impulsive-like behavior in rats were evaluated using the five-choice serial reaction time task (5-CSRTT) and delay discounting procedures. These paradigms were also used to test the capacity of OXR antagonists to attenuate acute cocaine-evoked impulsivity. Finally, immunohistochemistry and calcium imaging were used to assess potential cellular mechanisms by which OXR blockade may influence motor impulsivity. Suvorexant, a dual (OX1/2R) orexin receptor antagonist, reduced cocaine-evoked premature responses in 5-CSRTT when administered systemically or directly into VTA. Neither suvorexant nor OX1R- or OX2R-selective compounds (SB334867 or TCS-OX2-29, respectively) altered delay discounting. Finally, suvorexant did not alter Fos-immunoreactivity within tyrosine hydroxylase-immunolabeled neurons of VTA, but did attenuate cocaine- and orexin-induced increases in calcium transient amplitude within neurons of VTA. Results from the present studies suggest potential therapeutic utility of OXR antagonists in reducing psychostimulant-induced motor impulsivity. These findings also support the view that orexin transmission is closely involved in executive function in normal and pathological conditions.

Suvorexant, an orexin/hypocretin receptor antagonist, attenuates motivational and hedonic properties of cocaine.

Orexins ('hypocretins') are peptides produced by neurons of the hypothalamus that project to structures implicated in reward and emotion processing. Converging evidence demonstrates functional roles of orexin signaling in arousal, sleep/wakefulness and motivated behaviors for natural and drug rewards. Suvorexant, a dual orexin receptor antagonist, recently received approval from the US Food and Drug Administration to treat insomnia. In Experiment 1, rats self-administered cocaine under a progressive-ratio schedule of reinforcement and the effects of suvorexant on motivation to self-administer cocaine were measured. In Experiment 2, the effects of suvorexant on cocaine reward were assessed by using a place conditioning paradigm, and 50-kHz ultrasonic vocalizations were also recorded to track changes in hedonic reactivity to cocaine. To rule out potentially confounding effects of suvorexant-induced somnolence, locomotor activity was also measured. In Experiment 3, the effects of suvorexant on cocaine-evoked elevations in ventral striatal dopamine were examined. Data reveal that suvorexant (i) reduced the number of cocaine infusions earned during progressive-ratio self-administration; (ii) attenuated initial positive hedonic reactivity to cocaine and prevented cocaine place preference; (iii) did not affect cocaine-induced hyperlocomotion and (iv) reduced cocaine-induced elevations in extracellular ventral striatal dopamine. The present study examined the therapeutic potential of suvorexant in rodent models of cocaine use disorder. These results contribute toward a growing literature supporting therapeutic roles of orexin receptor antagonists in treating substance use disorders.

Comparing rewarding and reinforcing properties between 'bath salt' 3,4-methylenedioxypyrovalerone (MDPV) and cocaine using ultrasonic vocalizations in rats.

Abuse of synthetic psychostimulants like synthetic cathinones has risen in recent years. 3,4-Methylenedioxypyrovalerone (MDPV) is one such synthetic cathinone that demonstrates a mechanism of action similar to cocaine. Compared to cocaine, MDPV is more potent at blocking dopamine and norepinephrine reuptake and is readily self-administered by rodents. The present study compared the rewarding and reinforcing properties of MDPV and cocaine using systemic injection dose-response and self-administration models. Fifty kilohertz ultrasonic vocalizations (USVs) were recorded as an index of positive affect throughout experiments. In Experiment 1, MDPV and cocaine dose-dependently elicited 50-kHz USVs upon systemic injection, but MDPV increased USVs at greater rates and with greater persistence relative to cocaine. In Experiment 2, latency to begin MDPV self-administration was shorter than latency to begin cocaine self-administration, and self-administered MDPV elicited greater and more persistent rates of 50-kHz USVs versus cocaine. MDPV-elicited 50-kHz USVs were sustained over the course of drug load-up whereas cocaine-elicited USVs waned following initial infusions. Notably, we observed a robust presence of context-elicited 50-kHz USVs from both MDPV and cocaine self-administering rats. Collectively, these data suggest that MDPV has powerfully rewarding and reinforcing effects relative to cocaine at one-tenth doses. Consistent with prior work, we additionally interpret these data in supporting that MDPV has significant abuse risk based on its potency and subjectively positive effects. Future studies will be needed to better refine therapeutic strategies targeted at reducing the rewarding effects of cathinone analogs in efforts to ultimately reduce abuse liability.

Stereoselective Differences between the Reinforcing and Motivational Effects of Cathinone-Derived 4-Methylmethcathinone (Mephedrone) In Self-Administering Rats.

Mephedrone (4-methylmethcathinone (4-MMC)) (MEPH) is a new psychoactive substance (NPS) of the synthetic cathinone class. MEPH has a chiral center and exists as two enantiomers (R-,S-MEPH), yet stereospecific effects of MEPH have not been extensively investigated in preclinical assays. Because significant behavioral and neurochemical differences can exist between enantiomers, probing effects of stereochemistry on biological activity enables separation of adverse and therapeutic effects. Our prior work showed that R-MEPH, relative to S-MEPH, produced greater locomotor activation, place preference, and facilitation of brain reward thresholds in rodents. The present study sought to determine if MEPH enantiomers display stereospecific reward and reinforcement in rat self-administration assays. In Experiment 1, rats were trained to self-administer racemic MEPH (0.50 mg/kg/inf), and dose substitution effects of R-MEPH (0.50 mg/kg/inf) and S-MEPH (0.25, 0.50, 2.00 mg/kg/inf) were examined. In Experiment 2, separate rats were trained to self-administer R-MEPH (0.25, 0.50, 2.00 mg/kg/inf) or S-MEPH (0.25, 0.50, 2.00 mg/kg/inf) and were thereafter evaluated under progressive-ratio access conditions. Within this cohort, 50 kHz ultrasonic vocalizations (USVs) were recorded to measure potential differences in subjective positive affect associated with MEPH enantiomer self-administration. We identified enantiomer- and dose-dependent effects on infusions earned during self-administration following acquisition of racemic MEPH, with greatest infusions under low-effort, fixed-ratio 1 access conditions from low-dose S-MEPH self-administration. When taxed with progressive-ratio access conditions, rats trained to self-administer R-MEPH showed higher break points than those of rats trained to self-administer S-MEPH. Additionally, R-MEPH elicited greatest rates of 50 kHz USVs compared to S-MEPH. Taken together, these data suggest that the R-enantiomer of MEPH is primarily responsible for the rewarding, reinforcing, and motivational properties of racemic MEPH, which increases our understanding of stereospecific preferences pertaining to MEPH abuse.

Role of hypocretin/orexin receptor blockade on drug-taking and ultrasonic vocalizations (USVs) associated with low-effort self-administration of cathinone-derived 3,4-methylenedioxypyrovalerone (MDPV) in rats.

RATIONALE: Synthetic psychostimulant abuse, including cathinone-derived 3,4-methylenedioxypyrovalerone (MDPV), continues to increase in many countries. Similar to cocaine but with greater potency, MDPV elicits a transient sympathomimetic response by blocking cellular uptake of dopamine (DA) and norepinephrine (NE)-administration in some users is reported as euphoria-inducing much like cocaine and amphetamine. Pharmacological agents that disrupt excitatory transmission onto midbrain DA-producing neurons, including hypothalamic hypocretin/orexin (hcrt/ox) receptor antagonists, present attractive targets to aide abstinence maintenance by reducing psychostimulant-associated reward and reinforcement. OBJECTIVE: The present study sought to assess the degree to which suvorexant, a dual hcrt/ox receptor antagonist, influences drug-taking as well as ultrasonic vocalizations (USVs) associated with MDPV self-administration. METHODS: Rats were trained to self-administer MDPV (~0.03 mg/kg/inf, 3-s) for 14 days under a fixed-ratio 1 schedule of reinforcement, and effects of suvorexant (0, 3, 10, 30 mg/kg, i.p.) on drug-taking was assessed. USVs were recorded during a 30-min pre-lever period as well as during 2-h of MDPV self-administration. RESULTS: We observed that suvorexant modestly suppressed the number of MDPV infusions earned. Notably, we observed that suvorexant reduced 50-kHz USVs associated with pre- and post-lever time-points but did not noticeably alter call type profiles. Upon comparison of the two measures, we observed trending positive associations between suvorexant-induced changes in drug-taking and 50-kHz USVs. CONCLUSIONS: Results from this exploratory study provide support for the following: (1) studying how suvorexant may provide benefit to humans with stimulant use disorders, (2) identifying a potential role for orexin transmission in cathinone abuse, and (3) further interrogating the potential utility of rat USVs to predict drug consumption in preclinical models of substance use disorders.

Effects of ceftriaxone on conditioned nicotine reward in rats.

Nicotine is the addictive compound in tobacco products which exerts psychosomatic effects that contribute to abuse and to low rates of abstinence in treatment-seeking smokers. At present, the most successful smoking cessation aide helps one in four individuals quit smoking at 1 year postcessation. New adjunctive therapies are needed to improve status of smoking-related public health crises, and ß-lactam antibiotics are one class of potential therapies as they favorably augment extrasynaptic glutamate clearance. Our study used two-chamber place conditioning to assess effects of ceftriaxone (CTX) on persistence of conditioned nicotine reward. Rats were conditioned to associate nicotine (0.4 mg/kg, subcutaneous) with one context and vehicle with an alternative context. After initial post-test, rats received either daily ceftriaxone (200 mg/kg, intraperitoneal) or saline. All rats showed nicotine place preference during post-test 1. CTX-treated rats meeting extinction criterion by post-test 7 showed significantly reduced preference for the nicotine-paired context during post-test 2 compared with vehicle-treated rats. We interpret these data to support the further study of CTX as a smoking cessation aide. Our results suggest that CTX reduces persistence of conditioned nicotine reward and may be helpful for improving abstinence rates in a subset of treatment-seeking smokers.

Nicotinic receptor blockade decreases fos immunoreactivity within orexin/hypocretin-expressing neurons of nicotine-exposed rats.

Tobacco smoking is the leading cause of preventable death in the United States. Nicotine is the principal psychoactive ingredient in tobacco that causes addiction. The structures governing nicotine addiction, including those underlying withdrawal, are still being explored. Nicotine withdrawal is characterized by negative affective and cognitive symptoms that enhance relapse susceptibility, and suppressed dopaminergic transmission from ventral tegmental area (VTA) to target structures underlies behavioral symptoms of nicotine withdrawal. Agonist and partial agonist therapies help 1 in 4 treatment-seeking smokers at one-year post-cessation, and new targets are needed to more effectively aid smokers attempting to quit. Hypothalamic orexin/hypocretin neurons send excitatory projections to dopamine (DA)-producing neurons of VTA and modulate mesoaccumbal DA release. The effects of nicotinic receptor blockade, which is commonly used to precipitate withdrawal, on orexin neurons remain poorly investigated and present an attractive target for intervention. The present study sought to investigate the effects of nicotinic receptor blockade on hypothalamic orexin neurons using mecamylamine to precipitate withdrawal in rats. Separate groups of rats were treated with either chronic nicotine or saline for 7-days at which point effects of mecamylamine or saline on somatic signs and anxiety-like behavior were assessed. Finally, tissue from rats was harvested for immunofluorescent analysis of Fos within orexin neurons. Results demonstrate that nicotinic receptor blockade leads to reduced orexin cell activity, as indicated by lowered Fos-immunoreactivity, and suggest that this underlying cellular activity may be associated with symptoms of nicotine withdrawal as effects were most prominently observed in rats given chronic nicotine. We conclude from this study that orexin transmission becomes suppressed in rats upon nicotinic receptor blockade, and that behavioral symptoms associated with nicotine withdrawal may be aided by intervention upon orexinergic transmission.

Ultrasonic Vocalizations as a Measure of Affect in Preclinical Models of Drug Abuse: A Review of Current Findings.

The present review describes ways in which ultrasonic vocalizations (USVs) have been used in studies of substance abuse. Accordingly, studies are reviewed which demonstrate roles for affective processing in response to the presentation of drug-related cues, experimenter- and self-administered drug, drug withdrawal, and during tests of relapse/reinstatement. The review focuses on data collected from studies using cocaine and amphetamine, where a large body of evidence has been collected. Data suggest that USVs capture animals' initial positive reactions to psychostimulant administration and are capable of identifying individual differences in affective responding. Moreover, USVs have been used to demonstrate that positive affect becomes sensitized to psychostimulants over acute exposure before eventually exhibiting signs of tolerance. In the drug-dependent animal, a mixture of USVs suggesting positive and negative affect is observed, illustrating mixed responses to psychostimulants. This mixture is predominantly characterized by an initial bout of positive affect followed by an opponent negative emotional state, mirroring affective responses observed in human addicts. During drug withdrawal, USVs demonstrate the presence of negative affective withdrawal symptoms. Finally, it has been shown that drug-paired cues produce a learned, positive anticipatory response during training, and that presentation of drug-paired cues following abstinence produces both positive affect and reinstatement behavior. Thus, USVs are a useful tool for obtaining an objective measurement of affective states in animal models of substance abuse and can increase the information extracted from drug administration studies. USVs enable detection of subtle differences in a behavioral response that might otherwise be missed using traditional measures.

Involvement of neuronal ß2 subunit-containing nicotinic acetylcholine receptors in nicotine reward and withdrawal: implications for pharmacotherapies.

WHAT IS KNOWN AND OBJECTIVE: Tobacco smoking remains a major health problem. Nicotine binds to nicotinic acetylcholine receptors (nAChRs), which can cause addiction and withdrawal symptoms upon cessation of nicotine administration. Pharmacotherapies for nicotine addiction target brain alterations that underlie withdrawal symptoms. This review will delineate the involvement of the ß2 subunit of neuronal nAChRs in nicotine reward and in generating withdrawal symptoms to better understand the efficacy of smoking cessation pharmacotherapies. COMMENT: Chronic nicotine desensitizes and upregulates ß2 subunit-containing nAChRs, and the prolonged upregulation of receptors may underlie symptoms of withdrawal. Experimental research has demonstrated that the ß2 subunit of neuronal nAChRs is necessary for generating nicotine reward and withdrawal symptoms. WHAT IS NEW AND CONCLUSION: Smoking cessation pharmacotherapies act on ß2 subunit-containing nAChRs to reduce nicotine reward and withdrawal symptom severity.

Nicotine shifts the temporal activation of hippocampal protein kinase A and extracellular signal-regulated kinase 1/2 to enhance long-term, but not short-term, hippocampus-dependent memory.

Acute nicotine enhances hippocampus-dependent learning through nicotine binding to ß2-containing nicotinic acetylcholine receptors (nAChRs), but it is unclear if nicotine is targeting processes involved in short-term memory (STM) leading to a strong long-term memory (LTM) or directly targeting LTM. In addition, the molecular mechanisms involved in the effects of nicotine on learning are unknown. Previous research indicates that protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein synthesis are crucial for LTM. Therefore, the present study examined the effects of nicotine on STM and LTM and the involvement of PKA, ERK1/2, and protein synthesis in the nicotine-induced enhancement of hippocampus-dependent contextual learning in C57BL/6J mice. The protein synthesis inhibitor anisomycin impaired contextual conditioning assessed at 4 h but not 2 h post-training, delineating time points for STM (2 h) and LTM (4 h and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24 h but not 2 h post-training, indicating nicotine specifically enhances LTM but not STM. Furthermore, nicotine did not rescue deficits in contextual conditioning produced by anisomycin, suggesting that the nicotine enhancement of contextual conditioning occurs through a protein synthesis-dependent mechanism. In addition, inhibition of dorsal hippocampal PKA activity blocked the effect of acute nicotine on learning, and nicotine shifted the timing of learning-related PKA and ERK1/2 activity in the dorsal and ventral hippocampus. Thus, the present results suggest that nicotine specifically enhances LTM through altering the timing of PKA and ERK1/2 signaling in the hippocampus, and suggests that the timing of PKA and ERK1/2 activity could contribute to the strength of memories.

Ultrasonic vocalizations: evidence for an affective opponent process during cocaine self-administration.

RATIONALE: Preclinical models of cocaine addiction in the rodent have shown that cocaine induces both positive and negative affective states. These observations have led to the notion that the initial positive/euphoric state induced by cocaine administration may be followed by an opposing, negative process. In the rodent, one method for inferring positive and negative affective states involves measuring their ultrasonic vocalizations (USVs). Previous USV recordings from our laboratory suggested that the transition between positive and negative affect might involve decaying or sub-satiety levels of self-administered cocaine. OBJECTIVES: In order to explicitly test the role of cocaine levels on these affective states, the present study examined USVs when calculated body levels of cocaine were clamped (i.e., held at a constant level via experimenter-controlled infusions) at, below, or above subjects' self-determined drug satiety thresholds. RESULTS: USVs indicated that (1) positive affect was predominantly observed during the drug loading period, but declined quickly to near zero during maintenance and exhibited little relation to calculated drug level, and (2) in contrast, negative affect was observed at sub-satiety cocaine levels, but was relatively absent when body levels of cocaine were clamped at or above subjects' satiety thresholds. CONCLUSIONS: The results reinforce the opponent-process hypothesis of addiction and suggest that an understanding of the mechanisms underlying negative affect might serve to inform behavioral and pharmacological therapies.