Functional antagonism between nociceptin/orphanin FQ (N/OFQ) and corticotropin-releasing factor (CRF) in the rat brain: evidence for involvement of the bed nucleus of the stria terminalis

Rationale Nociceptin/orphanin FQ (N/OFQ) has been proposed to be a functional antagonist of corticotropin-releasing factor (CRF) in relation to its anti-stress action and its ability to antagonize the anorectic effect of CRF in rats without exhibiting affinity for CRF receptors. The bed nucleus of the stria terminalis (BST) is highly sensitive to the inhibitory effect of N/OFQ on CRF-induced anorexia. Objective The present study was aimed at further evaluating the role of the BST in the functional antagonism between N/OFQ and CRF by examining it at molecular level and in the context of CRF-induced anxiety in the rat. Materials and methods First, in situ hybridization experiments investigated the expression of the pro-N/OFQ precursor and of NOP receptors in several brain areas 6 h after injection of CRF (0.2 and 1 μg/rat) into the lateral cerebroventricle (LV). Second, the elevated plus maze test was used to evaluate whether N/OFQ, injected into the BST (0.05 and 0.5 μg/rat) or into the LV (0.5, 1.8, and 2.4 μg/rat), inhibits the anxiogenic-like effect evoked by LV injection of CRF (1 μg/rat) in rats. Results The in situ hybridization study showed that LV injection of CRF 1 μg/rat increases NOP receptor expression in the BST, while no change of the N/OFQ precursor was observed. On the other hand, N/OFQ injection into the BST blocks the anxiogenic effect of CRF at doses lower than those required by LV injection (0.5 vs 1.8 μg/rat, respectively). Conclusion These data provide further support for the hypothesis that N/OFQ may behave as functional antagonist of CRF and suggest that this antagonism may occur within the BST.     

Nociceptin inhibits noradrenaline release in the mouse brain cortex via presynaptic ORL1 receptors

A fourth type of opioid receptor, termed ORL₁, has been cloned and nociceptin (also known as orphanin FQ) has been identified as an endogenous ligand at this receptor. We examined whether nociceptin affects the release of noradrenaline in the brain. For this purpose, cerebral cortex slices from the mouse, rat or guinea-pig were preincubated with [³H]noradrenaline and then superfused with medium containing desipramine and rauwolscine. Tritium overflow was evoked electrically (0.3 Hz) or by introduction of Ca²⁺ 1.3 mM into Ca²⁺-free K⁺-rich (15 mM) medium. Nociceptin 1 μM reduced the electrically evoked tritium overflow from mouse, rat and guinea-pig brain cortex slices by 80, 71 and 36%, respectively. Naloxone 10 μM did not change the effect of nociceptin. All subsequent experiments were performed on mouse brain cortex slices and in the presence of naloxone 10 μM. The concentration-response curve of nociceptin (maximum inhibition by 80%, pEC₅₀ 7.5) was shifted to the right by the non-selective ORL₁ receptor antagonist naloxone benzoylhydrazone and the selective ORL₁ receptor antagonist [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ (pA₂ 6.6 and 7.2, respectively). Naloxone benzoylhydrazone did not affect the evoked overflow by itself whereas [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ caused an inhibition by maximally 35% (pEC₅₀ 7.0; intrinsic activity α 0.45). The inhibitory effect of [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ was counteracted by naloxone benzoylhydrazone. Nociceptin also reduced the Ca ²⁺ -evoked tritium overflow in mouse brain cortex slices superfused in the presence of tetrodotoxin. This effect was also antagonized by naloxone benzoylhydrazone, which, by itself, did not affect the evoked tritium overflow. In conclusion, nociceptin inhibits noradrenaline release more markedly in the mouse than in the rat or guinea-pig brain cortex. The effect of nociceptin in the mouse brain cortex involves ORL₁ receptors, which are located presynaptically on noradrenergic neurones. Received: 19 June 1998 / Accepted: 17 July 1998     

Effect of nociceptin on alcohol intake in alcohol-preferring rats

The present study investigated the effect of nociceptin (NC), the endogenous ligand of the opioid-like orphan receptor ORL1, on ethanol intake in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats. Acute intracerebroventricular (ICV) injection of 250 or 500 ng/rat of NC, just before access to 10% ethanol (offered 2 h/day), significantly increased ethanol intake. Subchronic (7 days) ICV injection of 500 ng/rat of NC, given just before access to 10% ethanol (for 30 min/day), resulted in a progressive decrease in ethanol consumption. After the end of NC treatment, rats progressively recovered their usual ethanol intake. When NC, 500 or 1000 ng/rat, was tested versus the effect of ethanol in the place conditioning paradigm, NC significantly reduced the increase in time spent in the ethanol-paired compartment after conditioning. This finding suggests that NC reduces the rewarding properties of ethanol in msP rats; thus, they may respond to the acute NC administration by increasing their ethanol intake in an attempt to achieve the usual reinforcing effect of ethanol, whereas subchronic NC treatment may result in extinction of ethanol drinking. The results of the present study suggest that the brain NC mechanisms may represent an interesting target of pharmacological interventions for the treatment of alcoholism. Received: 11 August 1998/Final version: 15 October 1998     

ORL1 receptor-mediated inhibition by nociceptin of noradrenaline release from perivascular sympathetic nerve endings of the rat tail artery

In the present study the effect of the opioid heptadecapeptide nociceptin, also termed orphanin FQ, an endogenous ligand for the orphan receptor named ORL₁ (opioid receptor-like 1) receptor, was investigated on [³H]noradrenaline release induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery in the absence and presence of an α₂-adrenoceptor antagonist, rauwolscine 3 μM. Nociceptin inhibited the electrically-evoked tritiated noradrenaline release in a concentration-dependent manner from rat tail arteries. This inhibitory effect of nociceptin was enhanced in the presence of the α₂-adrenoceptor antagonist rauwolscine (maximum inhibition by 25% and 50% in the absence and presence of rauwolscine, respectively). At a supramaximal concentration (10 μM), the inhibitory action of DAGO, a selective μ-opioid receptor agonist, was less pronounced than that of nociceptin. The inhibitory effect of nociceptin was counteracted by naloxone benzoylhydrazone (3 μM) which by itself did not change the stimulation-evoked noradrenaline overflow. Naloxone (10 μM), a non-selective opioid receptor antagonist, did not affect the inhibitory effect of nociceptin whereas it abolished that of DAGO. In conclusion, these results suggest that nociceptin modulates noradrenergic neurotransmission by acting on prejunctional ORL₁ receptors located on nerve terminals innervating the rat tail artery. They also demonstrate that prejunctional ORL₁ receptors interact with prejunctional α₂-adrenoceptors. The physiological significance of this phenomenon remains to be determined. Received: 27 August 1998 / Accepted: 29 September 1998     

Distribution of ORL-1 receptor binding and receptor-activated G-proteins in rat forebrain and their experimental localization in anterior cingulate cortex

Opioid receptor-like (ORL-1) receptors and ORL-1-activated G-proteins are found in high levels in the forebrain, particularly cingulate cortex, an area involved in processing of nociceptive stimuli. [(3)H]nociceptin/orphanin FQ (N/OFQ) and N/OFQ-stimulated [(35)S]GTPgammaS autoradiography in rat brain were used to localize ORL-1 receptors and activated G-proteins, respectively. N/OFQ binding and activated G-proteins were highest in anterior cingulate, agranular insula, piriform, perirhinal and entorhinal cortices; midline and intralaminar thalamic nuclei; and subnuclei of the amygdala and hippocampus. In anterior cingulate area 24, [(3)H]N/OFQ and N/OFQ-stimulated [(35)S]GTPgammaS binding were highest in layers V and VI. The cellular localization of ORL-1 receptors and activated G-proteins in area 24 was examined using two strategies: ibotenic acid injection into the cortex or undercut lesions to remove afferent axons, followed by autoradiography. Ibotenic acid lesions that destroyed neurons in the anterior cingulate cortex decreased [(3)H]N/OFQ binding by 75-80% and reduced N/OFQ-stimulated [(35)S]GTPgammaS binding to basal levels seen in the absence of agonist. Deafferentation lesions increased [(3)H]N/OFQ binding by 40-50%, with no significant change in N/OFQ-stimulated [(35)S]GTPgammaS binding. These data demonstrate that ORL-1 receptors in layer V of anterior cingulate cortex are located on somatodendritic elements and that deafferentation increases ORL-1 receptor binding.     

Binding of the novel radioligand [3H]UFP-101 to recombinant human and native rat nociceptin/orphanin FQ receptors

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). Binding studies have relied on [leucyl-(3)H]N/OFQ, but as this is an agonist G-protein coupling will affect affinity. In this paper, we describe a new [(3)H]labeled NOP antagonist, [Nphe(1),4'-(3)H-Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2) ([(3)H]UFP-101). We have characterized [(3)H]UFP-101 at recombinant human NOP expressed in Chinese hamster ovary cells (CHO(hNOP)) and native rat NOP in cerebrocortex. Radioligand saturation and competition studies were performed on membranes, and [(3)H]UFP-101 (antagonist) was compared with [(3)H]N/OFQ (agonist). The effects of GTPgammaS (10 microM) and Na(+) were investigated alone and in combination in competition experiments with both radioligands. In CHO(hNOP), B (max), and pK (D), values were 561 and 580 fmol mg protein(-1) and 9.97 and 10.19 for [(3)H]UFP-101 and [leucyl-(3)H]N/OFQ, respectively. In rat cerebrocortex B (max) and pK (D), values were 65 and 88 fmol mg protein(-1) and 10.12 and 10.34 for [(3)H]UFP-101 and [leucyl-(3)H]N/OFQ. The binding of both radioligands was displaced by a range of peptide and non-peptide NOP ligands at both isoforms with good correlation (r (2) 0.92 in Rat and 0.97 in CHO(hNOP)). Naloxone was inactive. The binding of both radioligands was Na(+)-dependent with [(3)H]UFP-101 being more sensitive (IC(50) approximately20 mM). Unlike the agonist [leucyl-(3)H]N/OFQ, the antagonist [(3)H]UFP-101 was unaffected by GTPgammaS. [(3)H]UFP-101 binds to human and rat NOP with high affinity and good agreement with standard [leucyl-(3)H]N/OFQ in competition experiments. In addition, the binding of [(3)H]UFP-101 is unaffected by GTPgammaS. This radioligand will be useful to further characterize NOP in a range of binding paradigms.     

Reversal of stress- and CRF-induced anorexia in rats by the synthetic nociceptin/orphanin FQ receptor agonist, Ro 64-6198

RATIONALE: (1S,3aS)-8-(2,3,3,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (Ro 64-6198), a non-peptidic agonist for the opioid receptor-like1 (ORL1) receptor, exhibits anxiolytic properties in stressful conditions. OBJECTIVE: The present study was aimed at evaluating whether activation of ORL1 receptors by Ro 64-6198 may reverse the anorectic effect of restraint stress or intracerebroventricular (ICV) CRF injection. METHODS: In body restraint experiments, 20-h food deprived rats were treated with intraperitoneal (IP) injection of Ro 64-6198 or vehicle. Ten minutes later, they were confined in cylindrical Plexiglas tubes for 60 min and then returned to their cage with food. In CRF experiments, 20-h food deprived rats were IP injected with Ro 64-6198 or vehicle. Ten minutes later, they received ICV CRF, 200 ng/rat or vehicle; food was offered after 20 min. RESULTS: Intraperitoneal (IP) pretreatment with Ro 64-6198 reversed the hypophagic effect induced by both restraint or CRF; the effect was statistically significant at the three doses tested (0.3, 1.0 or 2.5 mg/kg). ICV administration of the selective ORL1 receptor antagonist [Nphe(1)]NC(1-13)NH(2)(two injections of 33 or 66 microg/rat) abolished the effect of Ro 64-6198 on CRF-induced anorexia. In freely feeding rats, Ro 64-6198 significantly increased feeding at 2.5, but not at 0.3 or 1.0 mg/kg; in food deprived rats, Ro 64-6198 (0.3 or 1.0 mg/kg) did not modify food intake. Thus, reversal of stress- and CRF-induced anorexia by Ro 64-6198 can be evoked at doses lower than those that are hyperphagic. Ro 64-6198 (1 or 2.5 mg/kg) did not modify the anorectic effect of E. coli lipopolysaccharide, suggesting that its effect is selective for stress- or CRF-induced anorexia. Lastly, the benzodiazepine diazepam was unable to reduce the anorectic effect of CRF at the anxiolytic dose of 0.3 mg/kg, and partially reduced it at the hyperphagic dose of 1 mg/kg. CONCLUSIONS: The results of this study show that the non-peptidic ORL1 receptor agonist Ro 64-6198 markedly and selectively inhibits the anorectic effect of stress and CRF, and provide evidence that this effect is mediated by ORL1 receptors. Thus, Ro 64-6198 may represent an interesting tool for treatment of stress-induced anorexia.     

Orphanin FQ/nociceptin attenuates motor stimulation and changes in nucleus accumbens extracellular dopamine induced by cocaine in rats

RATIONALE: Orphanin FQ (OFQ; also known as nociceptin), the endogenous ligand of the opioid receptor-like receptor, injected intracerebroventricularly (i.c.v.) decreases basal motor activity and basal extracellular levels of dopamine (DA) in the nucleus accumbens (Nuc Acc) in rats. OBJECTIVE: The present study was designed to determine if OFQ similarly attenuates cocaine-induced motor stimulation and to determine if this effect is dependent on attenuation of the increase in extracellular DA. METHODS: After a 1-h adaptation period, rats were injected with either artificial cerebrospinal fluid or OFQ (3-30 nmol, i.c.v.) 5 min prior to cocaine (10 mg/kg, i.p.) or apomorphine (3 mg/kg, i.p.) administration and the total distance traveled was measured for a further 1 h. In a separate experiment, changes in extracellular DA were monitored by microdialysis following cocaine and OFQ treatment in anesthetized rats. RESULTS: OFQ dose-dependently attenuated both basal and cocaine-induced motor stimulation. OFQ (30 nmol, i.c.v.) also attenuated both the basal and the cocaine-induced increase in extracellular DA in the Nuc Acc. OFQ, at the highest dose, also decreased the motor stimulation induced by apomorphine. CONCLUSIONS: Our results suggest that the modulatory effect of OFQ on locomotor activity is not solely due to its inhibitory action on extracellular DA in the Nuc Acc.     

Baclofen attenuates cue-induced reinstatement of alcohol-seeking behavior in Sardinian alcohol-preferring (sP) rats

The GABA_B receptor agonist, baclofen, suppressed alcohol deprivation effect (a proposed experimental model of alcohol relapse) in Sardinian alcohol-preferring rats. The present study was designed to extend the characterization of the “anti-relapse” properties of baclofen to the reinstatement of alcohol-seeking behavior (another proposed model of alcohol relapse). Rats of the sP line were first trained to lever press for alcohol under a fixed ratio 4 schedule of reinforcement. Subsequently, rats were exposed to two within-session 70-min extinction/reinstatement tests with saline or baclofen administered in a counterbalanced, within-subject design. After a 60-min extinction phase, an alcohol-associated stimulus complex was presented (reinstatement phase). Saline or baclofen (3 mg/kg) were administered via a permanent intraperitoneal catheter, 30 min before the reinstatement phase. During the reinstatement phase, baclofen administration: (a) reduced by approximately 60% responses on the previously active lever, (b) increased latency to the first response and (c) decreased the response rate. These results indicate that baclofen reduced cue-induced reinstatement of alcohol-seeking behavior in sP rats.     

Modulation of cocaine and food self-administration by low- and high-efficacy D1 agonists in squirrel monkeys

RATIONALE: Dopamine D1 ligands have been proposed as candidate medications for cocaine abuse. Previous studies have shown that the ability of D1 ligands to modulate the behavioral effects of cocaine may depend on agonist efficacy. OBJECTIVES: This study investigated the role of agonist efficacy in the ability of D1 ligands to modulate the reinforcing effects of cocaine in monkeys. METHODS: Squirrel monkeys trained to self-administer cocaine under a second-order schedule of reinforcement were treated daily with D1 agonists varying in efficacy from low to high (SKF 83959 < SKF 77434 < or = SKF 81297 < SKF 82958) and the D1 antagonist SCH 39166. RESULTS: D1 ligands, regardless of efficacy, produced dose-dependent reductions in responding maintained by a maximally effective dose of cocaine. Equivalent doses of each D1 ligand reduced responding for food under a similar second-order schedule, suggesting that the suppression was not specific to cocaine self-administration. When studied in combination with a range of cocaine doses, treatment with the agonists SKF 83959, SKF 77434, SKF 81297, and the antagonist SCH 39166 produced overall rightward and downward shifts in the dose-response function for cocaine self-administration. Treatment with the agonist SKF 82958, however, produced an overall suppression of responding, regardless of the dose of cocaine. CONCLUSIONS: In contrast to a high-efficacy agonist, low-efficacy D1 ligands modulated the reinforcing effects of cocaine in a manner consistent with at least a partial antagonism of cocaine self-administration. This delineation of the efficacy-dependent profile of effects for D1 ligands should guide research into their utility as cocaine pharmacotherapies.     

Free-choice responding for ethanol versus water in alcohol preferring (P) and unselected Wistar rats is differentially modified by naloxone,bromocriptine, and methysergide

The role of opioids, dopamine and serotonin in ethanol (EtOH) reward and preference was investigated in non-deprived, Alcohol-Preferring (P), and genetically heterogenous Wistar rats. Operant responding for ethanol was initiated using sweet-solution substitution procedures. The rats were then trained in 30-min daily sessiosn to respond for ethanol (10% v/v) versus water under a two-lever, free-choice contingency. All testing was conducted in the absence of water and food deprivation or addition of sweeteners to the ethanol drinking solution. Rats of both strains developed stable preferences in responding for ethanol over water and consumed ethanol at quantities sufficient to produce pharmacologically relevant mean blood alcohol concentrations (P-Rats: 98±19.6 mg%; unselected Wistars: 41.7±8.5 mg%). InP-rats, systemic naloxone (NAL; 0.125, 0.25 and 0.5 mg/kg) pretreatments resulted in a dose-dependent suppression in responding for both ethanol and water, but did not alter ethanol preference (expressed as percent ethanol of total intake). In contrast, bromocriptine (BRO; 1.0, 2.0 and 4.0 mg/kg) produced a significant, dose-dependent shift in preference from ethanol toward water by inhibiting responding for ethanol while enhancing water consumption. Inunselected Wistar rats, NAL and BRO treatments produced changes in ethanol preference patterns similar to those observed in P-rats. However, compared to P-rats, these changes were smaller and not consistently dose dependent. No changes in ethanol preference and water or ethanol intake were observed with methysergide (MET; 2.5, 5.0, 10.0 mg/kg) in either strain of rat. Together, the results suggest a possible involvement of dopaminergic mechanisms in the reinforcing properties of ethanol. In contrast, the response decrements observed with naloxone may reflect a more general depression in consummatory behavior.This is publication number 5768 BCR from the Research Institute of Scripps Clinic, La Jolla, California     

Effects of the Trace Amine Associated Receptor 1 Agonist RO5263397 on Abuse-Related Behavioral Indices of Methamphetamine in Rats

Background:

Methamphetamine is a major drug of abuse with no effective pharmacotherapy available. Trace amine associated receptor 1 is implicated in cocaine addiction and represents a potential therapeutic target. However, the effects of trace amine associated receptor 1 agonists on addiction-related behavioral effects of methamphetamine are unknown.

Methods:

This study examined the effects of a trace amine associated receptor 1 agonist RO5263397 on methamphetamine-induced behavioral sensitization, methamphetamine self-administration, cue- and methamphetamine-induced reinstatement of drug seeking, and cue-induced reinstatement of sucrose-seeking behaviors in rats. Male Sprague-Dawley rats were used to examine the effects of methamphetamine alone and in combination with the trace amine associated receptor 1 agonist RO5263397 (3.2–10mg/kg).

Results:

RO5263397 dose-dependently attenuated the expression of behavioral sensitization to methamphetamine, reduced methamphetamine self-administration, and decreased both cue- and a priming dose of methamphetamine-induced reinstatement of drug-seeking behaviors. However, RO5263397 did not alter cue-induced reinstatement of sucrose-seeking behavior.

Conclusions:

Taken together, trace amine associated receptor 1 agonists attenuate some abuse-related behavioral effects of methamphetamine, strongly suggesting that drugs activating trace amine associated receptor 1 may be potentially useful for the treatment of methamphetamine addiction and warrant further studies.     

Attenuation of cocaine self-administration in squirrel monkeys following repeated administration of the mGluR5 antagonist MPEP: comparison with dizocilpine

RATIONALE: The mGluR5 antagonist MPEP has effects that suggest potential as a pharmacotherapy for cocaine addiction. MPEP can attenuate self-administration of cocaine in animals; however, studies usually involved only acute treatment with MPEP and a single dose of self-administered cocaine. Cocaine addicts use varied amounts of cocaine over long periods of time, and an effective pharmacotherapy would almost certainly require more chronic treatment. OBJECTIVES: The present study (1) compared the effects of repeated treatment with MPEP or the NMDA receptor antagonist dizocilpine on the reinforcing effects of a range of doses of cocaine and (2) determined the pharmacological specificity of the effects of the drugs in attenuating cocaine self-administration compared to food-reinforced behavior. An effective pharmacotherapy should selectively reduce cocaine self-administration. MATERIALS AND METHODS: Groups of monkeys responded under a fixed-ratio schedule of i.v. cocaine self-administration or food-pellet delivery. The effects of daily treatment with MPEP and dizocilpine were determined under both the schedule of i.v. cocaine injection and food delivery. RESULTS: Treatment with MPEP and dizocilpine significantly reduced cocaine self-administration, producing rightward and downward shifts in the ascending limb of the cocaine dose-response function. MPEP and dizocilpine selectively and significantly attenuated self-administration of a low reinforcing dose of cocaine compared to food without evidence of tolerance. CONCLUSIONS: Both MPEP and dizocilpine functioned as partially surmountable antagonists of the reinforcing effects of cocaine. The similar effects of the two drugs raises the possibility that MPEP attenuated the reinforcing effects of cocaine, at least in part, via mGluR5-mediated inhibition of NMDA receptor activity.     

Naltrexone reduces ethanol- and sucrose-reinforced responding in rhesus monkeys

 These experiments evaluated the ability of naltrexone (NTX) to reduce selectively oral and IV ethanol-reinforced responding, and examined the ethanol-NTX interaction in terms of the competitive opioid antagonist property of NTX. Five rhesus monkeys self-administered ethanol or sucrose and concurrently available water. Ethanol concentration was varied from 0.25% to 8% (w/v). Naltrexone (0.032–0.32 mg/kg) or saline was given IM 30 min prior to some drinking sessions. NTX (0.32 mg/kg) reduced ethanol-reinforced responding at the concentration that maintained the most responding (1% or 2%). NTX (0.1 mg/kg) reduced ethanol-reinforced responding, both at a low ethanol concentration (0.25%) that produced little ethanol intake (g/kg), and at a higher concentration (4%) with an appreciable intake. Thus, NTX (0.1 mg/kg) shifted the ethanol concentration-consumption curve down, in an insurmountable manner. NTX (0.1 and 0.32 mg/kg) also reduced reinforced responding for sucrose 100 g/l. In another experiment, three rhesus monkeys were given opportunities to self-administer ethanol IV. NTX (0.1 mg/kg) reduced the number of ethanol injections obtained by the monkeys at all ethanol doses tested (0.01, 0.032, and 0.1 g/kg per injection).The dose-effect curve was also shifted down. These results showed that NTX reduced behavior maintained by either ethanol or sucrose non-selectively. Furthermore, the ability of NTX to suppress ethanol-reinforced responding did not depend on the route of ethanol administration and was not overcome by increasing the concentration or dose per injection of ethanol. Received: 11 November 1997 / Final version: 19 March 1998     

Cannabinoid modulation of the reinforcing and motivational properties of heroin and heroin-associated cues in rats

Rationale Recently, we provided evidence for a cannabinoid mechanism in relapse to cocaine seeking in rats. There is also increasing evidence for functional cross-talk between cannabinoid and opioid systems in several physiological processes. Objectives This study was designed to evaluate whether the cannabinoid system plays a role in mediating the reinforcing and motivational effects of heroin and heroin-paired stimuli. Methods Male Wistar rats were trained to self-administer heroin (50 μg/kg per infusion) on fixed (FR5) or progressive ratio schedules of reinforcement in the presence of a discriminative and discrete heroin-associated cue. The selective cannabinoid CB1 antagonist SR141716A was given 30 min before the session to determine its effect on responding for heroin. Separate groups of rats were subjected to extinction training during which heroin-associated cues were absent and no heroin was delivered. During subsequent reinstatement tests, the effects of the cannabinoid agonist HU210 and the antagonist SR141716A on reinstatement of heroin seeking were evaluated. Results The cannabinoid antagonist dose-dependently reduced responding for heroin on the FR5 schedule and to a greater extent on the progressive ratio schedule. HU210 (20 μg/kg) reinstated heroin seeking behaviour following a 2-week extinction period, whereas SR141716A dose-dependently attenuated heroin seeking that was provoked by a priming injection of heroin (0.25 mg/kg) and heroin seeking that was triggered by re-exposure to heroin paired stimuli. Conclusions The results show that the reinforcing and motivational effects of heroin and heroin-paired stimuli are mediated, at least in part, by activation of cannabinoid CB1 receptors. Therefore, the present study provides a rationale for the use of cannabinoid antagonists in the treatment of opiate addiction.     

Increase of brain endocannabinoid anandamide levels by FAAH inhibition and alcohol abuse behaviours in the rat

RATIONALE: A major clinical concern with the use of cannabinoid receptor 1 (CB1) direct agonists is that these compounds increase alcohol drinking and drug abuse-related behaviours. As an alternative approach, CB1-receptor-mediated activity can be facilitated by increasing anandamide levels with the use of hydrolase fatty acid amide hydrolase (FAAH) inhibitors. OBJECTIVE: Using the selective FAAH inhibitor URB597, we investigated whether activation of the endogenous cannabinoid tone increases alcohol abuse liability, as what happens with the CB1 receptor direct agonists. MATERIALS AND METHODS: URB597 was tested on alcohol self-administration in Wistar rats and on homecage alcohol drinking in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats. In Wistar rats, URB597 effects on alcohol-induced anxiety and on stress-, yohimbine- and cue-induced reinstatement of alcohol seeking were also evaluated. For comparison, the effect of the CB1 receptor antagonist rimonabant on ethanol self-administration was also tested. RESULTS: Under our experimental condition, intraperitoneal (IP) administration of URB597 (0.0, 0.3 and 1.0 mg/kg) neither increased voluntary homecage alcohol drinking in msP rats nor facilitated fixed ratio 1 and progressive ratio alcohol self-administration in nonselected Wistars. In the reinstatement tests, the compound did not have effects on cue-, footshock stress- and yohimbine-induced relapse. Conversely, URB597 completely abolished the anxiogenic response measured during withdrawal after an acute IP administration of alcohol (3.0 g/kg). Rimonabant (0.0, 0.3, 1.0 and 3.0 mg/kg) significantly reduced ethanol self-administration. CONCLUSIONS: Results demonstrate that activation of the endocannabinoid anandamide system by selective inhibition of FAAH does not increase alcohol abuse risks but does reduce anxiety associated to alcohol withdrawal. We thus can speculate that medication based on the use of endocannabinoid system modulators such as URB597 may offer important advantages compared to treatment with direct CB1 receptor activators.     

Reinstatement of ethanol and sucrose seeking by the neurosteroid allopregnanolone in C57BL/6 mice

Rationale  Recent work in our laboratory documented that the “sipper” method of operant ethanol self-administration produced high ethanol intake and blood ethanol concentrations as well as the typical extinction “burst” in responding under nonreinforced conditions in male C57BL/6 mice. However, the neurochemical basis for reinstatement of responding following extinction has not been examined in mice with this model. Objectives  Based on findings that the GABAergic neurosteroid allopregnanolone (ALLO) significantly increased the consummatory phase of ethanol self-administration, the present study determined the effect of ALLO on the reinstatement of extinguished ethanol-seeking behavior and compared this effect to the reinstatement of responding for sucrose reward. Materials and methods  Separate groups of male C57BL/6 mice were trained to lever press for access to a 10% ethanol (10E) or a 5% sucrose (5S) solution. A single response requirement of 16 presses (RR16) on an active lever resulted in 30 min of continuous access to the 10E or 5S solution. After the animals responded on the RR16 schedule for 14 weeks, mice were exposed to 30 min extinction sessions where responding had no scheduled consequence. Once responding stabilized below the preextinction baseline, mice received an intraperitoneal injection of ALLO (0, 3.2, 5.6, 10, or 17 mg/kg) 15 min prior to the extinction session in a within-subjects design. Results  ALLO produced a dose-dependent increase in responding under nonreinforced conditions in both the 10E and 5S groups. Additional work documented the ability of a conditioned cue light or a compound cue (light+lever retraction) to reinstate nonreinforced responding on the previously active lever. Conclusions  These findings definitively show that conditioned cues and priming with ALLO are potent stimuli for reinstating both ethanol- and sucrose-seeking behavior in C57BL/6 mice.     

Differential effects of ethanol in the nucleus accumbens shell of alcohol-preferring (P), alcohol-non-preferring (NP) and Wistar rats: A proteomics study

The objective of this study was to determine the effects of ethanol injections on protein expression in the nucleus accumbens shell (ACB-sh) of alcohol-preferring (P), alcohol-non-preferring (NP) and Wistar (W) rats. Rats were injected for 5 consecutive days with either saline or 1 g/kg ethanol; 24 h after the last injection, rats were killed and brains obtained. Micro-punch samples of the ACB-sh were homogenized; extracted proteins were subjected to trypsin digestion and analyzed with a liquid chromatography-mass spectrometer procedure. Ethanol changed expression levels (1.15-fold or higher) of 128 proteins in NP rats, 22 proteins in P, and 28 proteins in W rats. Few of the changes observed with ethanol treatment for NP rats were observed for P and W rats. Many of the changes occurred in calcium-calmodulin signaling systems, G-protein signaling systems, synaptic structure and histones. Approximately half the changes observed in the ACB-sh of P rats were also observed for W rats. Overall, the results indicate a unique response to ethanol of the ACB-sh of NP rats compared to P and W rats; this unique response may reflect changes in neuronal function in the ACB-sh that could contribute to the low alcohol drinking behavior of the NP line.     

Neuropeptide Y (NPY)-induced reductions in alcohol intake during continuous access and following alcohol deprivation are not altered by restraint stress in alcohol-preferring (P) rats

Administration of neuropeptide Y (NPY) reduces anxiety-like behavior and alcohol intake in alcohol-preferring rats. The present experiment examined whether the effects of NPY on alcohol drinking are modulated by stress exposure during continuous access or following ethanol deprivation. Female P rats underwent 6 weeks of continuous access to 15% v/v ethanol and water prior to intracerebroventricular (ICV) cannula implantation. Deprived rats underwent two cycles of 5 days of ethanol exposure followed by 2 days of ethanol deprivation, while non-deprived rats had uninterrupted access to ethanol. Stressed rats in both ethanol access groups were exposed to restraint stress for 1 h 4-6 h after ethanol was removed from the deprived group in both cycles. ICV infusions of 5.0 μg NPY or aCSF were administered 48 h following the deprivation/stress procedure, after which ethanol was returned. Rats showed increased ethanol intake following ethanol deprivation compared to non-deprived controls. Food and water intake were increased, while ethanol intake was decreased, in rats infused with NPY. Stress did not increase ethanol intake or alter the response to NPY. Although no stress effects were found, the present experiment replicates previous findings regarding the effectiveness of NPY in reducing ethanol consumption. Future studies aimed at determining the extent to which stress may affect relapse to ethanol drinking and response to NPY would benefit from implementing different stress paradigms and varying the pattern of ethanol access.     

Effects of unconditioned and conditioned social defeat on alcohol self-administration and reinstatement of alcohol seeking in rats

Rationale and objectives We and others have shown that a stressor commonly used in laboratory studies, intermittent footshock, reinstates alcohol seeking in a rat relapse model. The effects of more ethologically relevant stressors on reinstatement have not been examined. Here, we characterized the effects of social defeat (a naturalistic stressor) or a cue associated with the defeat experience on reinstatement of alcohol seeking. We also examined the effect of unconditioned and conditioned social defeat on alcohol self-administration.Methods Rats were trained to self-administer alcohol (12% w/v, 1 h day⁻¹), and after stable responding, one group of animals received five exposures to social defeat paired with peppermint odor prior to daily self-administration sessions. After three more self-administration sessions, these rats were tested for the effects of the peppermint odor cue on self-administration. In another group of rats, the effects of three daily exposures to social defeat paired with peppermint odor on extinction of responding were examined. After further extinction sessions, the effect of the odor cue on reinstatement was tested in these animals. The acute effect of social defeat on reinstatement was examined in another group of animals.Results Acute exposure to social defeat decreased alcohol self-administration, reduced rates of responding during extinction, and did not reinstate alcohol seeking. Exposure to a discrete odor cue previously paired with social defeat decreased alcohol self-administration but induced modest reinstatement of alcohol seeking.Conclusions Results provide the first demonstration of reinstatement of alcohol seeking by a cue paired with social defeat and are also in agreement with previous findings on the suppressive effect of social defeat stress on alcohol self-administration.