Blockage of glucocorticoid receptors during memory acquisition,retrieval and reconsolidation prevents the expression of morphine-induced conditioned place preferences in mice

Association between the reward caused by consuming drugs and the context in which they are consumed is essential in the formation of morphine-induced conditioned place preference(CPP).Glucocorticoid receptor(GRs)activation in different regions of the brain affects reward-based reinforcement and memory processing.A wide array of studies have demonstrated that blockage of GRs in some brain areas can have an effect on reward-related memory;however,to date there have been no systematic studies about the involvement of glucocorticoids(GCs)in morphine-related reward memory.Here,we used the GR antagonist RU38486 to investigate how GRs blockage affects the sensitization and CPP behavior during different phases of reward memory included acquisition,retrieval and reconsolidation.Interestingly,our results showed RU38486 has the ability to impair the acquisition,retrieval and reconsolidation of reward-based memory in CPP and sensitization behavior.But RU38486 by itself cannot induce CPP or conditioned place aversion(CPA)behavior.Our data provide a much more complete picture of the potential effects that glucocorticoids have on the reward memory of different phases and inhibit the sensitization behavior.

Effects of functional and transcriptional Hsp70 inhibitors on development and expres⁃sion of conditioned place preference induced by morphine in rats

OBJECTIVE To investigate the effects of heat shock protein 70(Hsp70)in the development as well as expression of morphine induced conditioned place preference(CPP)in rats using Hsp70 inhibitors.METHODS The unbi⁃ased procedures of CPP lasted for 7 d and included three phases:preconditioning(D1-D3),conditioning(D4-D6,6 sessions)and test(D7).Here,morphine 5 mg·kg-1 injected in a subcuta⁃neous(sc)manner can induce significant place preference.Inhibitors of Hsp70 were injected into the right lateral ventricle during either the condi⁃tioning phase or the test phase separately.Fur⁃thermore,the expression of Hsp70 in certain areas of the mesocorticolimbic system was also studied following the intervention of N-formyl-3,4-methylenedioxybenzylidine-γ-butyrolactam(KNK437),a transcriptional inhibitor of Hsp70.RESULTS Pifithrin-μ(PES),a selective functional inhibitor acting on the substrate binding domain(SBD)of Hsp70,dose-dependently suppressed both the acquisition and expression of morphine-induced CPP.Similar function was observed after the intracerebroventricular injection(icv)of KNK437.The other functional inhibitor methy⁃lene blue,targeting the nucleotide-binding area,showed a significant tendency of inhibitory phar⁃macological effect on the expressional and devel⁃opment phases of morphine-induced CPP.Following the interventions of KNK437,we found that the level of Hsp70 was significantly decreased in the NAcs both in the acquisition and expres⁃sion of morphine induced CPP.CONCLUSION Hsp70 in NAcs plays a critical role in mediating the psychological dependence induced by morphine.

Combined action of MK-801 and ceftriaxone impairs the acquisition and reinstatement of morphine-induced conditioned place preference,and delays morphine extinction in rats

Objective It is well established that glutamate and its receptors, particularly the N-methyl-D-aspartate receptor （NMDAR）, play a significant role in addiction and that the inhibition of glutamatergic hyperfunction reduces addictive behaviors in experimental animals. Specifically, NMDAR antagonists such as MK-801, and an inducer of the expression of glutamate transporter subtype-1 （GLT-1） （ceftriaxone） are known to inhibit addictive behavior. The purpose of this study was to determine whether the combined action of a low dose of MK-801 and a low dose of ceftriaxone provides better inhibition of the acquisition, extinction, and reinstatement of morphine-induced conditioned place preference （CPP） than either compound alone. Methods A morphine-paired CPP experiment was used to study the effects of low doses of MK-801, ceftriaxone and a combination of both on reward-related memory （acquisition, extinction, and reinstatement of morphine preference） in rats. Results A low dose of neither MK-801 （0.05 mg/kg, i.p.） nor ceftriaxone （25 mg/kg, i.p.） alone effectively impaired CPP behaviors. However, when applied in combination, they reduced the acquisition of morphine-induced CPP and completely prevented morphine reinstatement. Their combination also notably impaired the extinction of morphine-induced CPP. Conclusion The combined action of a low dose of an NMDAR antagonist （MK-801） and GLT-1 activation by ceftriaxone effectively changed different phases of CPP behavior.

Glu R2-3Y Inhibits the Acquisition and Reinstatement of Morphine-Induced Conditioned Place Preference in Rats

Accumulating evidence indicates that a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors（AMPARs） are involved in the relapse to abused drugs.However, the role of AMPARs containing the Glu R2 subunit in opiate addiction is still unclear. Glu R2-3Y,an interfering peptide, prevents the endocytosis of AMPARs containing the Glu R2 subunit. In this study, we explored the effect of intravenous injection of Glu R2-3Y on the acquisition, expression, and reinstatement of morphine-induced conditioned place preference（m CPP） in rats. We found that infusion of Glu R2-3Y（1.5 nmol/g） one hour before morphine during the conditioning phase inhibited the acquisition of m CPP, while an identical injection one hour before the post-conditioning test had no influence on the expression of m CPP. Injection of Glu R2-3Y（1.5 nmol/g） after m CPP extinction blocked the morphine-induced reinstatement of m CPP. Our results strongly support the hypothesis that inhibition of AMPAR endocytosis provides a new target for the treatment of opiate addiction.

Simvastatin Blocks Reinstatement of Cocaine-induced Conditioned Place Preference in Male Mice with Brain Lipidome Remodeling

Drug-associated reward memories are conducive to intense craving and often trigger relapse.Simvastatin has been shown to regulate lipids that are involved in memory formation but its influence on other cognitive processes is elusive.Here,we used a mass spectrometry-based lipidomic method to evaluate the impact of simvastatin on the mouse brain in a cocaine-induced reinstatement paradigm.We found that simvastatin blocked the reinstatement of cocaine-induced conditioned place preference(CPP)without affecting CPP acquisition.Specifically,only simvastatin administered during extinction prevented cocaine-primed reinstatement.Global lipidome analysis showed that the nucleus accumbens was the region with the greatest degree of change caused by simvastatin.The metabolism of fatty-acids,phospholipids,and triacylglycerol was profoundly affected.Simvastatin reversed most of the effects on phospholipids induced by cocaine.The correlation matrix showed that cocaine and simvastatin significantly reshaped the lipid metabolic pathways in specific brain regions.Furthermore,simvastatin almost reversed all changes in the fatty acyl profile and unsaturation caused by cocaine.In summary,pre-extinction treatment with simvastatin facilitates cocaine extinction and prevents cocaine relapse with brain lipidome remodeling.

Differences in cocaine-induced place preference persistence, locomotion and social behaviors between C57BL/6J and BALB/cJ mice

C57BL/6J and BALB/cJ mice display significant differences in sociability and response to drugs, but the phenotypic variability of their susceptibility to cocaine is still not well known. In this study, the differences between these two mice strains in the persistence of cocaine-induced conditioned place preference （CPP）, as well as the locomotion and social behaviors after the 24-hour withdrawal from a four-day cocaine （20 mg/kg/day） administration were investigated. The results showed that the cocaine-induced CPP persisted over two weeks in C57BL/6J mice, while it diminished within one week among BALB/cJ mice. After 24-hours of cocaine withdrawal, high levels of locomotion as well as low levels of social interaction and aggressive behavior were found in C57BL/6J mice, but no significant changes were found in BALB/cJ mice, indicating that cocaine-induced CPP persistence, locomotion and social behavior are not consistent between these two strains, and that overall C57BL/6J mice are more susceptible to cocaine than BALB/cJ mice at the tested doses.

Morphine pre-exposure facilitates reinstatement but attenuates retention of morphine-induced place preference

BACKGROUND： Drug-associated conditioned stimuli are a key factor to induce morphine relapse. To date, limited evidence is available regarding the impact of drug history on propensity or vulnerability to relapse after long-term abstinence. OBJECTIVE： To determine the effect of morphine pre-exposure on acquisition, maintenance and reinstatement of morphine-induced conditioned place preference （CPP） in rats. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Behavior Pharmacology, Institute of Psychology, the Chinese Academy of Sciences, from March to September, 2006. MATERIALS： Morphine hydrochloride was purchased from Qinghai Pharmaceutical, China; CPP software was designed and developed by Taiji Software Company, Beijing, China. METHODS： A total of 64 Sprague Dawley rats were randomly assigned to eight groups （n = 8）. Four morphine pretreatment regimens were used （subcutaneous injections, twice daily for 5 consecutive days and a total of 10 times）： （1） ＂intensive＂ （morphine injections with doses escalating from 10 to 60 mg/kg; （2） ＂moderate＂ （one morphine injection at 5 mg/kg dose and one saline injection at 1 mL/kg daily for 5 days）; and （3） ＂single＂ （nine saline injections at 1 mL/kg followed by one morphine injection at 5 mg/kg; （4） control （ten saline injections at 1 mL/kg）. At 5 days after morphine pretreatment, animals were divided into two subgroups that underwent morphine conditioned or saline conditioned training. The test for acquisition of CPP was performed 24 hours after CPP training. The retention of morphine CPP was measured by repeated tests performed weekly for 1 month after the initial test of place preference. After extinction by pairing each chamber with saline, the reinstatement of place preference by low doses of morphine （0.05, 0.15, 0.45 mg/kg） was tested. MAIN OUTCOME MEASURES： Acquisition, maintenance, and recovery response of CPP behavior. RESULTS： The acquisition magnitude of morphine-induced CPP was not affected by prior morphine exposure （F3, 56=0.17, P 〉 0.05）. However, rats treated with moderate or intensive morphine pretreatment showed a less persistent CPP （t = -1.36, P 〉 0.05; t = -1.18, P 〉 0.05）, but their place preference was reinstated by a low dose of morphine priming （t = -2.55, P 〈 0.05; t = -2.54, P 〈 0.05）. The retention and reinstatement of morphine-induced CPP did not differ between rats with single morphine pre-exposure and control rats. CONCLUSION： Morphine pretreatment enhanced reinstatement of morphine-induced CPP but with less persistence. Individuals with heavy drug exposure are more susceptible to drug relapse when re-exposed to addictive drugs.

PGE2 as a Morphine-Seeking Behavior Modulator in the Place Preference Model

The opioid epidemic has become one of the most concerning public health issues in the world, and currently does not have an adequate treatment available. It has been observed that, despite opioids being highly addictive, patients with chronic inflammation are less likely to develop an opioid dependence. This protective effect may be caused by Prostaglandin E2 (PGE2) as it has been found that non-painful carrageenan inflammation reduces morphine induced reward. Taking this into account, the aim of this study was to determine if the direct administration of PGE2 into the central nervous system could modulate the morphine-induced reward. We used the morphine-conditioned place preference (CPP) model with and without PGE2 or PGE2R antagonist in order to test the reward response. We found a significant reduction of morphine-induced reward after administering PGE2. Moreover, we found that this effect could be reversed by PGE2 receptor antagonism. Our data suggest that PGE2 may reduce morphine-induced reward making it an important drug-target research alternative to explore the possibility of modifying or even preventing opioid addiction.

Drug-Seeking Behavior Is Significantly Attenuated in Nutritionally Supplemented Cocaine Withdrawn Sprague-Dawley Rats

Aim: The effect of patented nutritional supplementation on drug-seeking behavior in cocaine addicted rats during acute drug withdrawal was investigated using a biased Conditioned Place Preference (CPP) paradigm. Method: Twenty-four (24) male Sprague-Dawley rats with pre-conditioned preference for the black chamber of the CPP box were randomly divided into Cocaine (COC) or Saline (SAL) treated groups. Rats (n = 12) treated with cocaine hydrochloride 20 mg/kg/ml, i.p. (COC group) were confined individually to the white chamber on days 1, 3, 5 and 7. On alternate days, they were given 1 ml saline vehicle, i.p. and confined to the black chamber. Control rats (SAL group, n = 12) received only vehicle on all 8 days and were confined on alternate days to the white or black chamber. Positive place preference was confirmed for COC rats, which subsequently received 6 increasing daily doses of cocaine. CPP performances of both COC and SAL rats were recorded following an acute 3-day withdrawal period. All animals were then randomly assigned to rats fed either chow reconstituted with the nutritional supplement (COC-S and SAL-S) or standard rat chow (COC-N and SAL-N) for 8 weeks, followed by final CPP performances. Results: Following supplementation, COC-S rats made significantly less entries and time spent in the white chamber (p < 0.05) compared with COC-N rats. COC-S rats exhibited significant place aversion to the white chamber similar to drug-naive animals;whereas COC-N continued to show positive place preference. Conclusion: Drug-seeking behavior that persisted during cocaine withdrawal was significantly diminished in the nutritionally supplemented.

Δ⁹-Tetrahydrocannabinol Produced Positive Place Preference in Mice without Significant <i>Ex-Vivo</i>Effect on Hepatic Arylamine N-Acetyltransferase Activity: Implications for Its Addictive Liability and Absence of Effect on Xenobiotic Metabolism

Aim: Δ9-Tetrahydrocannabinol (Δ9-THC) is a potentially addictive cannabinoid. Its impact on the activity of liver arylamine N-Acetyltransferase (NAT) has not been reported. This study investigated the rewarding effects of Δ9-THC in mice and whether Δ9-THC had any impact ex-vivo and in-vitro on NAT activity. Methods: Thirty-six Swiss albinomice randomly assigned to six groups (n = 6) completed a biased, 8-week Conditioned Place Preference (CPP) paradigm. Mice exhibiting ~80% preference for the black chamber at pre-conditioning were selected. Treatment groups were administered Δ9-THC (0.10, 0.50 or 2.0 mg/kg/mL, ip) or amphetamine (AMP, 5.0 mg/kg/mL, ip);while untreated groups (controls) received vehicle solutions (coconut oil or 0.9% saline). Entries and time spent in the white, drug-paired chamber during a 15-min post-conditioning exploration of the CPP apparatus were compared with the pre-conditioning exploratory scores. Livers from Δ9-THC treated and untreated mice were excised and NAT enzyme activity determined ex-vivo using a spectrophotometric assay with p-anisidine as substrate. The impact of varying concentrations of Δ9-THC (0.00 - 162 μM) on the activities of NAT from untreated mice livers were also investigated in-vitro. Results: Δ9-THC treated mice entered and spent significantly more time in the drug-paired CPP chamber (p ≤ 0.05) at post-conditioning vs pre-conditioning (F = 11.22). Mice treated with 2.0 mg/kg Δ9-THC made significantly more entries into the drug-paired chamber (p ≤ 0.05) as compared with their vehicle controls. AMP-treated mice displayed significant (p < 0.001) increases in both entries and time spent in the drug-paired chamber at post-conditioning (positive place preference). In-vitro NAT evaluations revealed a dose-dependent inhibitory impact of Δ9-THC on NAT activity with an IC50 value of 34.97 ±1.58 μM. Ex-vivo evaluations of livers from Δ9-THC treated mice showed no significant impact on liver NAT enzyme activity. Conclusion: Δ9-THC induced significant conditioned place preference (drug reward) and produced a moderate dose-dependent inhibition on NAT activity in-vitro, but not ex-vivo.

Blockade of the Dopamine D3 Receptor Attenuates Opioids-Induced Addictive Behaviours Associated with Inhibiting the Mesolimbic Dopamine System

Opioid use disorder(OUD)has become a considerable global public health challenge;however,potential medications for the management of OUD that are effective,safe,and nonaddictive are not available.Accumulating preclinical evidence indicates that antagonists of the dopamine D3 receptor(D3R)have effects on addiction in different animal models.We have previously reported that YQA14,a D3R antagonist,exhibits very high affinity and selectivity for D3Rs over D2Rs,and is able to inhibit cocaine-or methamphetamine-induced reinforcement and reinstatement in self-administration tests.In the present study,our results illustrated that YQA14 dose-dependently reduced infusions under the fixed-ratio 2 procedure and lowered the breakpoint under the progressive-ratio procedure in heroin self-administered rats,also attenuated heroin-induced reinstatement of drug-seeking behavior.On the other hand,YQA14 not only reduced morphine-induced expression of conditioned place preference but also facilitated the extinguishing process in mice.Moreover,we elucidated that YQA14 attenuated opioid-induced reward or reinforcement mainly by inhibiting morphine-induced up-regulation of dopaminergic neuron activity in the ventral tegmental area and decreasing dopamine release in the nucleus accumbens with a fiber photometry recording system.These findings suggest that D3R might play a very important role in opioid addiction,and YQA14 may have pharmacotherapeutic potential in attenuating opioid-induced addictive behaviors dependent on the dopamine system.

Disruption of dopamine D1 receptor phosphorylation at serine 421 attenuates cocaine-induced behaviors in mice

Dopamine D1 receptors（D1Rs） play a key role in cocaine addiction, and multiple protein kinases such as GRKs, PKA, and PKC are involved in their phosphorylation. Recently, we reported that protein kinase D1 phosphorylates the D1 R at S421 and promotes its membrane localization. Moreover, this phosphorylation of S421 is required for cocaineinduced behaviors in rats. In the present study, we generated transgenic mice over-expressing S421A-D1 R in the forebrain. These transgenic mice showed reduced phospho-D1R（S421） and its membrane localization, and reduced downstream ERK1/2 activation in the striatum. Importantly, acute and chronic cocaine-induced locomotor hyperactivity and conditioned place preference were significantly attenuated in these mice. These findings provide in vivo evidence for the critical role of S421 phosphorylation of the D1 R in its membrane localization and in cocaine-induced behaviors. Thus, S421 on the D1 R represents a potential pharmacotherapeutic target for cocaine addiction and other drug-abuse disorders.

Effects of Different Extinction for Morphine-CPP on Hippocampal EEG Power Spectrum in Mice

The relationship between hippocampal electroencephalogram(EEG)power spectrum and the extinction of addiction memory was investigated.Forty KM mice(Kunming mice)that had successfully established morphine(MOR)-conditioned place preference(CPP)were divided into four groups:saline-training extinction(SAL-TE),SAL-natural extinction(SAL-NE),MORTE,MOR-NE,for extinction treatment and EEG recording in the dorsal and ventral hippocampus(DH/VH).Results show that the CPP score of MOR-TE significantly decreased and the total,βandγbands power spectrum of MOR-TE was suppressed.Notably,the total power from the VH was more correlated with the extinction method,and the TE total power was always lower than NE in SAL or MOR group.Naloxone enhanced the EEG total power and all bands(DH)orβandγbands(VH)power in the SAL group.However,in the MOR-TE group,onlyβandγbands power in VH increased(P<0.05).The results suggest that TE can promote the extinction of opiate addictive memory more effectively than NE,and the extinction of MOR-CPP may have a stronger correlation with changes of VH-EEG power spectrum.