Cingulate circuits are associated with escalation of heroin use and naloxone-induced increases in heroin self-administration.

Opioid use disorder (OUD) is defined as a compulsion to seek and take opioids, loss of control over intake and the development of a negative emotional state when access to opioids is denied. Using functional magnetic resonance imaging (fMRI) data in a rat model of OUD, we demonstrate that the escalation of heroin self-administration (SA) and the increased heroin SA following an injection of an opioid receptor antagonist (naloxone) are associated with changes in distinct brain circuits, centered on the cingulate cortex (Cg). Here, SA escalation score was negatively associated with changes in resting state functional connectivity (rsFC) between the Cg and the dorsal striatum. Conversely, increased heroin SA following naloxone injection, was associated with increased connectivity between the Cg and the extended amygdala and hypothalamus. Naloxone-induced increased SA was also positively associated with changes in the amplitude of low frequency fluctuations within the Cg, a measure of spontaneous neuronal activity. Characterizing the distinct brain circuit and behavior changes associated with different facets of addiction increases our understanding of OUD and may provide insight into addiction prevention and treatment.

Fentanyl vapor self-administration model in mice to study opioid addiction.

Intravenous drug self-administration is considered the "gold standard" model to investigate the neurobiology of drug addiction in rodents. However, its use in mice is limited by frequent complications of intravenous catheterization. Given the many advantages of using mice in biomedical research, we developed a noninvasive mouse model of opioid self-administration using vaporized fentanyl. Mice readily self-administered fentanyl vapor, titrated their drug intake, and exhibited addiction-like behaviors, including escalation of drug intake, somatic signs of withdrawal, drug intake despite punishment, and reinstatement of drug seeking. Electrophysiological recordings from ventral tegmental area dopamine neurons showed a lower amplitude of GABAB receptor-dependent currents during protracted abstinence from fentanyl vapor self-administration. This mouse model of fentanyl self-administration recapitulates key features of opioid addiction, overcomes limitations of the intravenous model, and allows investigation of the neurobiology of opioid addiction in unprecedented ways.

Development and initial characterization of a novel ghrelin receptor CRISPR/Cas9 knockout wistar rat model.

BACKGROUND/OBJECTIVES: Ghrelin, a stomach-derived hormone implicated in numerous behaviors including feeding, reward, stress, and addictive behaviors, acts by binding to the growth hormone secretagogue receptor (GHSR). Here, we present the development, verification, and initial characterization of a novel GHSR knockout (KO) Wistar rat model created with CRISPR genome editing. METHODS: Using CRISPR/Cas9, we developed a GHSR KO in a Wistar background. Loss of GHSR mRNA expression was histologically verified using RNAscope in wild-type (WT; n = 2) and KO (n = 2) rats. We tested the effects of intraperitoneal acyl-ghrelin administration on food consumption and plasma growth hormone (GH) concentrations in WT (n = 8) and KO (n = 8) rats. We also analyzed locomotion, food consumption, and body fat composition in these animals. Body weight was monitored from early development to adulthood. RESULTS: The RNAscope analysis revealed an abundance of GHSR mRNA expression in the hypothalamus, midbrain, and hippocampus in WTs, and no observed probe binding in KOs. Ghrelin administration increased plasma GH levels (p = 0.0067) and food consumption (p = 0.0448) in WT rats but not KOs. KO rats consumed less food overall at basal conditions and weighed significantly less compared with WTs throughout development (p = 0.0001). Compared with WTs, KOs presented higher concentrations of brown adipose tissue (BAT; p = 0.0322). CONCLUSIONS: We have verified GHSR deletion in our KO model using histological, physiological, neuroendocrinological, and behavioral measures. Our findings indicate that GHSR deletion in rats is not only associated with a lack of response to ghrelin, but also associated with decreases in daily food consumption and body growth, and increases in BAT. This GHSR KO Wistar rat model provides a novel tool for studying the role of the ghrelin system in obesity and in a wide range of medical and neuropsychiatric disorders.

A relationship between the aldosterone-mineralocorticoid receptor pathway and alcohol drinking: preliminary translational findings across rats, monkeys and humans.

Aldosterone regulates electrolyte and fluid homeostasis through binding to the mineralocorticoid receptors (MRs). Previous work provides evidence for a role of aldosterone in alcohol use disorders (AUDs). We tested the hypothesis that high functional activity of the mineralocorticoid endocrine pathway contributes to vulnerability for AUDs. In Study 1, we investigated the relationship between plasma aldosterone levels, ethanol self-administration and the expression of CYP11B2 and MR (NR3C2) genes in the prefrontal cortex area (PFC) and central nucleus of the amygdala (CeA) in monkeys. Aldosterone significantly increased after 6- and 12-month ethanol self-administration. NR3C2 expression in the CeA was negatively correlated to average ethanol intake during the 12 months. In Study 2, we measured Nr3c2 mRNA levels in the PFC and CeA of dependent and nondependent rats and the correlates with ethanol drinking during acute withdrawal. Low Nr3c2 expression levels in the CeA were significantly associated with increased anxiety-like behavior and compulsive-like drinking in dependent rats. In Study 3, the relationship between plasma aldosterone levels, alcohol drinking and craving was investigated in alcohol-dependent patients. Non-abstinent patients had significantly higher aldosterone levels than abstinent patients. Aldosterone levels positively correlated with the number of drinks consumed, craving and anxiety scores. These findings support a relationship between ethanol drinking and the aldosterone/MR pathway in three different species.

A synthetic small-molecule Isoxazole-9 protects against methamphetamine relapse.

Adult neurogenesis in the dentate gyrus (DG) is strongly influenced by drug-taking behavior and may have a role in the etiology of drug-seeking behavior. However, mechanistic studies on the relationship of neurogenesis on drug seeking are limited. Outbred Wistar rats experienced extended access methamphetamine self-administration and individual differences in drug taking defined animals with higher preferred and lower preferred levels of drug intake. Forced abstinence from higher preferred levels of drug taking enhanced neurogenesis and neuronal activation of granule cell neurons (GCNs) in the DG and produced compulsive-like drug reinstatement. Systemic treatment with the drug Isoxazole-9 (a synthetic small molecule known to modulate neurogenesis in the adult rodent brain) during abstinence blocked compulsive-like context-driven methamphetamine reinstatement. Isoxazole-9 modulated neurogenesis, neuronal activation and structural plasticity of GCNs, and expression of synaptic proteins associated with learning and memory in the DG. These findings identify a subset of newly born GCNs within the DG that could directly contribute to drug-seeking behavior. Taken together, these results support a direct role for the importance of adult neurogenesis during abstinence in compulsive-like drug reinstatement.

Methylomic profiling and replication implicates deregulation of PCSK9 in alcohol use disorder.

Alcohol use disorder (AUD) is a common and chronic disorder with substantial effects on personal and public health. The underlying pathophysiology is poorly understood but strong evidence suggests significant roles of both genetic and epigenetic components. Given that alcohol affects many organ systems, we performed a cross-tissue and cross-phenotypic analysis of genome-wide methylomic variation in AUD using samples from 3 discovery, 4 replication, and 2 translational cohorts. We identified a differentially methylated region in the promoter of the proprotein convertase subtilisin/kexin 9 (PCSK9) gene that was associated with disease phenotypes. Biological validation showed that PCSK9 promoter methylation is conserved across tissues and positively correlated with expression. Replication in AUD datasets confirmed PCSK9 hypomethylation and a translational mouse model of AUD showed that alcohol exposure leads to PCSK9 downregulation. PCSK9 is primarily expressed in the liver and regulates low-density lipoprotein cholesterol (LDL-C). Our finding of alcohol-induced epigenetic regulation of PCSK9 represents one of the underlying mechanisms between the well-known effects of alcohol on lipid metabolism and cardiovascular risk, with light alcohol use generally being protective while chronic heavy use has detrimental health outcomes.

11ß-hydroxysteroid dehydrogenase inhibition as a new potential therapeutic target for alcohol abuse.

The identification of new and more effective treatments for alcohol abuse remains a priority. Alcohol intake activates glucocorticoids, which have a key role in alcohol's reinforcing properties. Glucocorticoid effects are modulated in part by the activity of 11ß-hydroxysteroid dehydrogenases (11ß-HSD) acting as pre-receptors. Here, we tested the effects on alcohol intake of the 11ß-HSD inhibitor carbenoxolone (CBX, 18ß-glycyrrhetinic acid 3ß-O-hemisuccinate), which has been extensively used in the clinic for the treatment of gastritis and peptic ulcer and is active on both 11ß-HSD1 and 11ß-HSD2 isoforms. We observed that CBX reduces both baseline and excessive drinking in rats and mice. The CBX diastereomer 18α-glycyrrhetinic acid 3ß-O-hemisuccinate (αCBX), which we found to be selective for 11ß-HSD2, was also effective in reducing alcohol drinking in mice. Thus, 11ß-HSD inhibitors may be a promising new class of candidate alcohol abuse medications, and existing 11ß-HSD inhibitor drugs may be potentially re-purposed for alcohol abuse treatment.

Traumatic stress reactivity promotes excessive alcohol drinking and alters the balance of prefrontal cortex-amygdala activity.

Post-traumatic stress disorder (PTSD) and alcoholism are highly comorbid in humans and have partially overlapping symptomatic profiles. The aim of these studies was to examine the effects of traumatic stress (and stress reactivity) on alcohol-related behaviors and neuronal activation patterns. Male Wistar rats were trained to respond for alcohol, were exposed to predator odor (bobcat urine) paired with context and were tested for short- and long-term avoidance of the predator odor-paired context, alcohol self-administration and compulsivity of alcohol responding. Rats were re-exposed to the odor-paired context for western blot analysis of ERK phosphorylation in subregions of the medial prefrontal cortex (mPFC) and the amygdala. Rats that avoided the predator-paired chamber (Avoiders) exhibited persistent avoidance up to 6 weeks post conditioning. Avoiders exhibited increases in operant alcohol responding over weeks, as well as more compulsive-like responding for alcohol adulterated with quinine. Following re-exposure to the predator odor-paired context, Avoiders and Non-Avoiders exhibited unique patterns of neuronal activation in subregions of the mPFC and the amygdala, which were correlated with changes in avoidance and alcohol drinking. Furthermore, activity of upstream regions was differentially predictive of downstream regional activity in the Avoiders versus Non-Avoiders. An animal model for assessing the effect of traumatic stress on alcohol drinking reveals individual differences in neuronal activation patterns associated with re-exposure to traumatic stress-related stimuli, and may provide insight into the neural mechanisms underlying excessive alcohol consumption in humans with PTSD.

Effects of the specific α4ß2 nAChR antagonist, 2-fluoro-3-(4-nitrophenyl) deschloroepibatidine, on nicotine reward-related behaviors in rats and mice.

RATIONALE: Alleviating addiction to tobacco products could prevent millions of deaths. Investigating novel compounds selectively targeting α4ß2 nAChRs hypothesized to have a key role in the rewarding effects of nicotine may be a useful approach for future treatment. OBJECTIVES: The present study was designed to evaluate 2-fluoro-3-(4-nitrophenyl) deschloroepibatidine (4-nitro-PFEB), a potent competitive antagonist of neuronal α4ß2 nAChRs, in several animal models related to nicotine reward: drug discrimination, intracranial self-stimulation (ICSS), conditioned place preference, and limited access to self-administration. METHODS: Long Evans rats were trained in a two-lever discrimination procedure to discriminate 0.4 mg/kg nicotine (s.c.) from saline. Male Sprague-Dawley rats were stereotaxically implanted with electrodes and trained to respond for direct electrical stimulation of the medial forebrain bundle. ICR mice were evaluated using an unbiased place preference paradigm, and finally, male Wistar rats were implanted with intrajugular catheters and tested for nicotine self-administration under limited access (1 h/day). RESULTS: 4-Nitro-PFEB attenuated the discriminative stimulus effects of nicotine, but alone did not produce nicotine-like discriminative stimulus effects. Nicotine-induced facilitation of ICSS reward thresholds was reversed by 4-nitro-PFEB, which alone had no effect on thresholds. 4-Nitro-PFEB also blocked the conditioned place preference produced by nicotine, but alone had no effect on conditioned place preference. Finally, 4-nitro-PFEB dose-dependently decreased nicotine self-administration. CONCLUSIONS: These results support the hypothesis that neuronal α4ß2 nAChRs play a key role in mediating the rewarding effects of nicotine and further suggest that targeting α4ß2 nAChRs may yield a potential candidate for the treatment of nicotine dependence.

Understanding the Global Problem of Drug Addiction is a Challenge for IDARS Scientists.

IDARS is an acronym for the International Drug Abuse Research Society. Apart from our scientific and educational purposes, we communicate information to the general and scientific community about substance abuse and addiction science and treatment potential. Members of IDARS are research scientists and clinicians from around the world, with scheduled meetings across the globe. IDARS is developing a vibrant and exciting international mechanism not only for scientific interactions in the domain of addiction between countries but also ultimately as a resource for informing public policy across nations. Nonetheless, a lot more research needs to be done to better understand the neurobiological basis of drug addiction - A challenge for IDARS scientists.

Systemic urocortin 2, but not urocortin 1 or stressin 1-A, suppresses feeding via CRF2 receptors without malaise and stress.

BACKGROUND AND PURPOSE: Infusion of corticotropin-releasing factor (CRF)/urocortin (Ucn) family peptides suppresses feeding in mice. We examined whether rats show peripheral CRF/Ucn-induced anorexia and determined its behavioural and pharmacological bases. EXPERIMENTAL APPROACH: Male Wistar rats (n= 5-12 per group) were administered (i.p.) CRF receptor agonists with different subtype affinities. Food intake, formation of conditioned taste aversion and corticosterone levels were assessed. In addition, Ucn 1- and Ucn 2-induced anorexia was studied in fasted CRF(2) knockout (n= 11) and wild-type (n= 13) mice. KEY RESULTS: Ucn 1, non-selective CRF receptor agonist, reduced food intake most potently (~0.32 nmol·kg(-1) ) and efficaciously (up to 70% reduction) in fasted and fed rats. The peptides' rank-order of anorexic potency was Ucn 1 ≥ Ucn 2 > >stressin(1) -A > Ucn 3, and efficacy, Ucn 1 > stressin(1) -A > Ucn 2 = Ucn 3. Ucn 1 reduced meal frequency and size, facilitated feeding bout termination and slowed eating rate. Stressin(1) -A (CRF(1) agonist) reduced meal size; Ucn 2 (CRF(2) agonist) reduced meal frequency. Stressin(1) -A and Ucn 1, but not Ucn 2, produced a conditioned taste aversion, reduced feeding efficiency and weight regain and elicited diarrhoea. Ucn 1, but not Ucn 2, also increased corticosterone levels. Ucn 1 and Ucn 2 reduced feeding in wild-type, but not CRF(2) knockout, mice. CONCLUSIONS AND IMPLICATIONS: CRF(1) agonists, Ucn 1 and stressin(1) -A, reduced feeding and induced interoceptive stress, whereas Ucn 2 potently suppressed feeding via a CRF(2) -dependent mechanism without eliciting malaise. Consistent with their pharmacological differences, peripheral urocortins have diverse effects on appetite.

Dynamics of neuronal circuits in addiction: reward, antireward, and emotional memory.

Drug addiction is conceptualized as chronic, relapsing compulsive use of drugs with significant dysregulation of brain hedonic systems. Compulsive drug use is accompanied by decreased function of brain substrates for drug positive reinforcement and recruitment of brain substrates mediating the negative reinforcement of motivational withdrawal. The neural substrates for motivational withdrawal ("dark side" of addiction) involve recruitment of elements of the extended amygdala and the brain stress systems, including corticotropin-releasing factor and norepinephrine. These changes, combined with decreased reward function, are hypothesized to persist in the form of an allostatic state that forms a powerful motivational background for relapse. Relapse also involves a key role for the basolateral amygdala in mediating the motivational effects of stimuli previously paired with drug seeking and drug motivational withdrawal. The basolateral amygdala has a key role in mediating emotional memories in general. The hypothesis argued here is that brain stress systems activated by the motivational consequences of drug withdrawal can not only form the basis for negative reinforcement that drives drug seeking, but also potentiate associative mechanisms that perpetuate the emotional state and help drive the allostatic state of addiction.

Dependence-induced alcohol drinking by alcohol-preferring (P) rats and outbred Wistar rats.

BACKGROUND: Chronic intermittent alcohol vapor exposure and selective breeding procedures have been used separately for many years to model specific aspects of alcohol dependence. The purpose of the present investigation was to combine these 2 approaches by exposing alcohol-preferring (P) rats to chronic intermittent alcohol vapor for extended periods of time and then testing them for operant alcohol responding in parallel with a group of outbred Wistar rats at multiple time points following the termination of vapor exposure. METHODS: P rats (n = 20) and Wistar rats (n = 18) were trained to respond for 10% (w/v) ethanol in an operant situation, then divided into groups matched for intake levels. Animals were then exposed to chronic intermittent alcohol vapor (14 hours ON/10 hours OFF) or air for 8 weeks. Rats were then tested for operant alcohol responding under various conditions and at multiple time points during alcohol withdrawal (6 hours) and protracted abstinence (1 to 15 days). RESULTS: Chronic alcohol vapor exposure produced similar increases in operant alcohol responding in P rats and Wistar rats during acute withdrawal and protracted abstinence. CONCLUSIONS: These results illustrate the separate and combined effects of genetic selection for high alcohol preference and dependence on alcohol drinking behavior. Furthermore, these results confirm past findings that dependent rats consume more alcohol than nondependent controls well into abstinence following extended periods of alcohol vapor exposure.

Role of the corticotropin-releasing factor receptor type 2 in the control of food intake in mice: a meal pattern analysis.

The actions of corticotropin-releasing factor (CRF) and related peptides are mediated by two receptors (CRF(1) and CRF(2)). The respective role of each subtype in the control of food intake remains poorly known. In the present study, we examined the quantity and microstructure of ingestive behavior of knockout (KO) mice lacking CRF(2) receptors and their wild-type (WT) littermates. Under basal conditions, CRF(2) KO mice showed increased nocturnal food intake, evident as an increased zenith in circadian cosinor analysis of food intake. Microstructure analysis revealed that this greater food intake reflected increased meal size, rather than meal frequency, suggesting a decreased satiating value of food. Following acute restraint stress, CRF(2) KO mice showed an intact immediate anorectic response with increased latency to eat and decreased meal size. However, CRF(2) deletion abolished the prolonged phase of restraint-induced anorexia. CRF(2) KO mice did not differ from WT controls in feeding responses to food deprivation or injection of ghrelin receptor agonists. Independent of genotype, food deprivation increased food intake, with dramatic changes in meal size, meal frequency, water : food ratio and eating rate. Acyl-ghrelin or BIM-28131, a potent ghrelin analog, dose-dependently stimulated food intake by increasing meal size (ghrelin, BIM-28131) and meal number (BIM-28131), while slowing the average eating rate (BIM-28131) similarly in WT and KO mice. These results suggest that the CRF(2) receptor is involved in the control of meal size during the active phase of eating and following acute exposure to stress.

Epipregnanolone and a novel synthetic neuroactive steroid reduce alcohol self-administration in rats.

This study was designed to compare the effects of several neuroactive steroids with varying patterns of modulation of gamma-aminobutyric acid (GABA)(A) and NMDA receptors on operant self-administration of ethanol or water. Once stable responding for 10% (w/v) ethanol was achieved, separate test sessions were conducted in which male Wistar rats were allowed to self-administer ethanol or water following pre-treatment with vehicle or one of the following neuroactive steroids: (3beta,5beta)-3-hydroxypregnan-20-one (epipregnanolone; 5, 10, 20 mg/kg; n=12), (3alpha,5beta)-20-oxo-pregnane-3-carboxylic acid (PCA; 10, 20, 30 mg/kg n=10), (3alpha,5beta)-3-hydroxypregnan-20-one hemisuccinate (pregnanolone hemisuccinate; 5, 10, 20 mg/kg; n=12) and (3alpha,5alpha)-3-hydroxyandrostan-17-one hemisuccinate (androsterone hemisuccinate; 5, 10, 20 mg/kg; n=11). The effect of the 3beta-epimer of PCA, (3beta,5beta)-20-oxo-pregnane-3-carboxylic acid (10, 20, 30 mg/kg; n=9), on ethanol self-administration was also examined. The compounds were administered using a Latin-square design 45 min prior to the weekly test sessions. The effects of the 30 mg/kg dose of the steroidal hemisuccinates on ethanol intake were also examined 5 min after administration of these drugs. Both epipregnanolone and PCA attenuated ethanol self-administration. However, neither of the hemisuccinate compounds significantly altered this behavior. The steroidal hemisuccinates (30 mg/kg; n=7) were also tested 5 min before behavior testing and had no effect on ethanol intake 5 min after administration. The 3beta-epimer of PCA also failed to alter ethanol intake. None of the test compounds altered water intake. In electrophysiological studies, the effects of PCA and androsterone hemisuccinate on evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (GABA(A)-IPSCs) was examined in brain slices of the amygdala. PCA had a stimulatory effect at concentrations of 5 and 25 muM. Androsterone hemisuccinate had no agonist activity. The ability of epipregnanolone and PCA to alter ethanol intake appears to be related to different inhibitory actions of these compounds on either GABA(A) or NMDA receptors, respectively. Thus, dual modulation of these systems by selected neuroactive steroids may offer potential for modifying the reinforcing effects of alcohol.

A non-invasive gating device for continuous drug delivery that allows control over the timing and duration of spontaneous opiate withdrawal.

Opiate dependence in laboratory animals is commonly induced by two methods: (1) subcutaneous (s.c.) insertion of morphine pellets, and (2) daily injections of increasing doses of opiates. While both of these methods reliably induce opiate dependence, they do not allow one to discontinue, and subsequently reestablish steady state opiate plasma levels with minimal invasive procedures. We developed an "ON-OFF" gating device for repeatedly and non-invasively turning ON or OFF opiate delivery by standard osmotic minipumps. The reliability of this "device" was tested utilizing naloxone (NAL)-precipitated somatic signs of withdrawal, and body mass index (BMI) as measures of withdrawal. Rats were implanted with osmotic minipumps equipped with the gating device, containing heroin (2.66 mg per day). Three days after surgery, somatic signs of withdrawal were precipitated every 48 h by NAL (0.3mg/kg), with minipumps gated ON or OFF. For BMI, spontaneous withdrawal was repeatedly (three times) induced by turning OFF and ON the gating devices every 48 h. Body weights were measured every 4h from 06:00 to 22:00 h daily. Results show that NAL precipitated intense somatic signs of withdrawal when gating devices were ON. This effect was almost abolished when gating devices were OFF. BMI rapidly decreased after the gating devices were turned OFF with maximum weight loss occuring 12 h post-OFF position, and gradually returning to baseline values after gating devices were turned back ON. These results demonstrate the validity of the "ON-OFF" gating device for non-invasively and repeatedly inducing physical dependence to opiates over a prolonged time.

Effect of bilateral 6-hydroxydopamine lesions of the medial forebrain bundle on reaction time.

Overt symptoms of Parkinson's disease do not manifest themselves until there is a substantial loss of the dopaminergic nigrostriatal projection. However, as neuroprotective strategies are developed, it will be essential to detect the disease in its preclinical phase. Performance on conditioned reaction time tasks is known to be impaired by extensive 6-hydroxydopamine-induced lesions of the nigrostriatal dopamine pathway. However, the effect of smaller lesions on a reaction time task has not been systematically assessed. We, therefore, used this test to examine behavioral deficits as a function of striatal dopamine loss. When injected at doses that produced striatal DA depletion <50%, 6-hydroxydopamine infused in the medial forebrain bundle produced no reliable impairment in the reaction time task. Higher doses producing > or = 60% DA depletion in the striatum produced a decrease in the percent correct responding throughout the 5 week testing period and akinetic deficits expressed by an increase in delayed responding. In addition, larger DA depletions (> or = 95%) profoundly altered motor control with decreases in percent correct responses, increases in delayed responses and increases in reaction time. These results suggest that reaction time may be a relatively sensitive measure of preclinical or subtle deficits, although it might be even more useful in quantitating the severity of depletion once overt deficits or symptoms appear and has the advantage of measuring such deficits over time to follow recovery of function. Furthermore since reaction time deficits required extensive loss of dopamine, these results are consistent with a predominant role of extrasynaptic dopamine in the mediation of relatively skilled motor tasks.

Time-dependent induction of anxiogenic-like effects after central infusion of urocortin or corticotropin-releasing factor in the rat.

RATIONALE: Corticotropin-releasing factor (CRF) and urocortin (Ucn) belong to the CRF-related family, share a high degree of structural homology and bind to CRF receptors. However, compared with CRF, Ucn was shown to display either weaker or similar anxiogenic-like effects in vivo. OBJECTIVE: To compare the anxiogenic-like responses of rats injected intracerebroventricularly (ICV) with different doses of either rat/human CRF (r/hCRF) or rat Ucn (rUcn) at different intervals after injection. METHODS: Rats were tested on three validated paradigms of emotional behavior [i.e. elevated plus-maze (EPM), defensive withdrawal (DW) and conflict test (CT)] 5 and 30 min after treatment. RESULTS: In the EPM test only r/hCRF, but not rUcn, produced anxiogenic-like effects at the dose of 1.0 microg, when the peptides were injected 5 min before testing. At 30 min after injection, both peptides caused a significant reduction of open arms exploration, rUcn being effective at 0.01 microg. In the DW test both peptides were equally potent in decreasing the exploratory behavior and increasing the time spent in the chamber at the dose of 1.0 microg when tested 30 min after injection. In the CT both rUcn (0.25-1.0 microg) and r/hCRF (0.75-1.0 microg) decreased significantly the responding in the punished component. However, rUcn reduced food responding also in the unpunished component possibly due to its powerful anorectic activity. CONCLUSIONS: Comparison of anxiogenic-like activities of r/hCRF and rUcn at doses up to 1.0 microg revealed striking differential effects that depended on the time of testing after ICV peptide injection, and on the paradigm of anxiety used. These results suggest that the onset of r/hCRF and rUcn actions related to behavioral responses to anxiety is likely to depend on brain peptide-specific mechanisms including binding properties to CRF-receptors, differential distribution to specific functional brain sites and the distribution and effectiveness of binding-protein interactions.

Increased ethanol self-administration in delta-opioid receptor knockout mice.

BACKGROUND: The role of the delta-opioid receptor in ethanol drinking has remained unclear despite the use of traditional pharmacological and correlational approaches. The results of several studies suggest that pharmacological blockade of these receptors results in decreases in ethanol drinking behavior, but an approximately equal number of reports have failed to observe an effect of delta-receptor antagonism on ethanol drinking. It is clear that alternative approaches to understanding opioid-receptor involvement in ethanol drinking are needed. METHODS: In this study, ethanol drinking was examined in delta-opioid receptor knockout (KO) mice by using first a two-bottle-choice test, then an operant self-administration paradigm and a second two-bottle-choice test, in that order. In addition, because KO mice were previously shown to display enhanced anxiety-like behavior relative to wild-type (WT) mice, the effect of ethanol self-administration on anxiety-like responses was determined. RESULTS: delta KO mice initially showed no evidence of a preference for ethanol in the first two-bottle-choice drinking test; however, after an experience of operant self-administration of ethanol, a preference for ethanol developed in the second two-bottle-choice test. KO mice also showed a preference for ethanol over water and self-administered more ethanol than WT mice in the operant self-administration paradigm. The ethanol self-administered in this procedure was sufficient to reverse the innate anxiety-like response observed in this strain. CONCLUSIONS: delta KO mice showed a greater preference for ethanol and consumed more ethanol than their WT counterparts, suggesting that a decrease in delta-receptor activity is associated with increased ethanol-drinking behavior. It is hypothesized that delta receptors may influence ethanol self-administration at least partly through an effect of these receptors on anxiety-like behavior.