Changes of CREB in rat hippocampus, prefrontal cortex and nucleus accumbens during three phases of morphine induced conditioned place preference in rats

Objective: To investigate the changes in CREB (cAMP response element binding protein) in hippocampus, PFC (prefrontal cortex) and NAc (nucleus accumbens) during three phases of morphine induced CPP (conditioned place preference) in rats, and to elucidate the role of CREB during the progress of conditioned place preference. Methods: Morphine induced CPP acquisition, extinction and drug primed reinstatement model was established, and CREB expression in each brain area was measured by Western Blot methods. Results: Eight alternating injections of morphine (10 mg/kg) induced CPP, and 8 d saline extinction training that extinguished CPP. CPP was reinstated following a priming injection of morphine (2.5 mg/kg). During the phases of CPP acquisition and reinstatement, the level of CREB expression was significantly changed in different brain areas. Conclusion: It was proved that CPP model can be used as an effective tool to investigate the mechanisms underlying drug-induced reinstatement of drug seeking after extinction, and that morphine induced CPP and drug primed reinstatement may involve acti-vation of the transcription factor CREB in several brain areas, suggesting that the CREB and its target gene regulation pathway may mediate the basic mechanism underlying opioid dependence and its drug seeking behavior.

Ohmefentanyl stereoisomers induce changes of CREB phosphorylation in hippocampus of mice in conditioned place preference paradigm

The present study was designed to determine the changes of phosphorylation of cAMP- response ele-ment binding protein (CREB) in hippocampus induced by ohmefentanyl stereoisomers (F9202 and F9204)in conditioned place preference (CPP) paradigm. The results showed that mice receiving F9202 and F9204displayed obvious CPP. They could all significantly stimulate CREB phosphorylation and maintained for along time without affecting total CREB protein levels. The effect of F9204 was similar to morphine whicheffect was more potent and longer than F9202. We also examined the effects of ketamine, a noncompetitiveN-mthyl-D-aspartate receptor (NR) antagonist, on morphine-, F9202- and F9204- induced CPP and phos-phorylation of CREB in hippocampus. Ketamine could suppress not only the place preference but also thephosphorylation of CREB produced by morphine, F9202 and F9204. These findings suggest that alterationsin the phosphorylation of CREB be relevant to opiates signaling and the development of opiates dependence.NR antagonists may interfere with opiates dependence and may have potential therapeutic implications.

Microinjection of Ghrelin into the Ventral Tegmental Area Potentiates Cocaine-Induced Conditioned Place Preference

Prior work has shown that systemic cocaine pretreatment augments cocaine conditioned place preference (CPP) in rats. In contrast, ghrelin receptor antagonism attenuates cocaine and amphetamine-induced CPP. In order to further investigate ghrelin’s role in dopamine-mediated reward, the present report examined whether pretreament with ghrelin, administered directly into the ventral tegmental area (VTA) of the midbrain, would potentiate the rewarding properties of cocaine as measured by CPP. Adult male Sprague-Dawley rats were given access to either side of the CPP chamber in order to determine initial side preferences. The rats were then restricted to either their non-preferred or preferred side over the course of conditioning which lasted for a total of 16 consecutive days. This was followed by a final test day to then reassess preference. On days where rats were confined to their non-preferred side, ghrelin (30-300 pmol) and cocaine (0.625-10 mg/kg IP) were administered immediately prior to the conditioning trial. On alternate days rats were treated with vehicle and placed into what was initially determined to be their preferred side. CPP was calculated as the difference in percentage of total time spent in the treatment-paired compartment during the post-conditioning session and the pre-conditioning session. Our results indicated that both cocaine and ghrelin elicited CPP and that ghrelin pretreatment potentiated the effect of cocaine on place preference. Overall, these findings provide additional support for the argument that ghrelin signaling within the VTA enhances the rewarding effects of psychostimulant compounds.

The FDA Mandate to Reassess Benzodiazepines: Alprazolam Induces a Positive Conditioned Place-Preference in Male Rats

On September 23, 2020, in order “To address the serious risks of abuse, addiction, physical dependence, and withdrawal reactions, the U.S. Food and Drug Administration (FDA) is requiring the Boxed Warning be updated for all benzodiazepine medicines”. With this announcement, the FDA proclaimed that much more needs to be known about the initiation, continuation, and discontinuation of these widely-used drugs. Unfortunately, relevant information is lacking, since for many years, there has been a notable sparsity in the funding and conduct of basic and clinical research on these drugs. In order to begin to fill the void, it is valuable to (re)examine animal models. We here describe a model of conditioned place-preference (CPP) for rats and for the first time, to our knowledge, show that the representative benzodiazepine alprazolam induces positive place-preference in male rats.

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.

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.

Amphetamine Conditioned Place Preference in Planarians

Meth- and other amphetamines currently present major drug-abuse concerns. However, the demonstration and study of abuse-related behaviors expressed in animal models is expensive and time-consuming. We previously reported a novel model of conditioned place preference (CPP), which is a standard tool in abuse research, in invertebrates (planarians). In the present study, planarians were tested for light/dark preference, then exposed for 5 min to either d-amphetamine or vehicle (water) in light and then re-tested for place preference (light vs dark). The planarians’ natural strong preference for dark (15 of 16) was significantly altered by amphetamine experience, such that 12 of 16 preferred the unnatural, but amphetamine-associated, light side. These results extend the demonstration of CPP to this invertebrate species and provide further evidence in support of this model to testing/screening amphetamine-like and possibly other drugs of abuse.

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.

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.

不同剂量天麻素对甲基苯丙胺依赖CPP大鼠海马中TLR4/MyD88/NF-κB炎症信号通路的影响

目的探讨不同剂量的天麻素(Gastrodin,Gas)对甲基苯丙胺(Methamphetamine,Meth)依赖条件性位置偏爱(Conditioned place preference,CPP)大鼠海马中TLR4/MyD88/NF-κB通路的作用。方法建立Meth(10 mg/kg,ip,14 d)依赖大鼠CPP模型,采用低中高剂量的天麻素(10、30、100 mg/kg)干预14 d后,采用Western blotting和RT-qPCR方法检测TLR4/MyD88/NF-κB信号通路中关键因子的蛋白和mRNA在海马中的表达水平。结果天麻素干预后TLR4、MyD88、NF-κB p65、TRAF6的蛋白和mRNA表达呈剂量依赖性降低(P<0.001、P<0.01或P<0.05)、p-NF-κB p65的蛋白表达呈剂量依赖性降低(P<0.01或P<0.05)、IκB-α的蛋白和mRNA表达呈剂量依赖性升高(P<0.01或P<0.05),天麻素干预后p-IκB-α的蛋白表达降低(P<0.01)。结论天麻素干预对甲基苯丙胺依赖CPP大鼠海马的神经炎症具有保护作用,且与TLR4/MyD88/NF-κB信号通路密切相关。

TLR4/MyD88/NF-κB信号通路对甲基苯丙胺依赖CPP大鼠海马的影响

目的 研究TLR4/MyD88/NF-κB信号通路对甲基苯丙胺(methamphetamine,MA)依赖的条件性位置偏好(conditioned place preference,CPP)大鼠海马的影响,同时采用特异性抑制剂TAK-242抑制Toll样4受体(Toll-like receptor 4,TLR4),减轻MA依赖诱导的海马神经炎症。方法 建立MA(10 mg/kg,ip,14 d)依赖大鼠CPP模型,分别为生理盐水组、MA组、TAK-242组、MA+TAK-242组。TAK-242组和MA+TAK-242组先分别腹腔注射抑制剂TAK-242(3 mg/kg),1h后MA+TAK-242组再腹腔注射MA(10 mg/kg)。采用Western Blot实验和采用荧光定量PCR实验检测MA依赖CPP大鼠海马中TLR4、MyD88、TRAF6、IκB-α、p-IκB-α、NF-κBp65、p-NF-κBp65蛋白的表达和mRNA表达。结果 与生理盐水组相比,MA组TLR4、MyD88、TRAF6、NF-κBp65的蛋白和mRNA表达均升高(P <0.001或P <0.01),IκB-α的蛋白和mRNA表达下降(P <0.01),p-IκB-α、p-NF-κBp65的表达升高(P <0.01或P <0.05);与MA组相比,MA+TAK-242组TLR4、MyD88、TRAF6、NF-κBp65的蛋白和mRNA表达均下降(P <0.001、P<0.01或P <0.05),IκB-α的蛋白和mRNA表达升高(P <0.01),p-IκB-α、p-NF-κBp65表达下降(P <0.01或P <0.05)。结论 MA依赖可通过激活TLR4/MyD88/NF-κB信号通路,诱导CPP大鼠海马神经炎症的发生,采用特异性TLR4抑制剂可以减轻MA诱导的神经炎症。

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.

Effect of Pingan Fang, a Traditional Chinese Medicine compound, on behavioral sensitization and conditioned place preference induced by ethanol in mice

OBJECTIVE:To observe the effect of Pingan Fang(PG)on behavioral sensitization and conditioned place preference(CPP)induced by ethanol in mice,and to determine the intervention mechanism of PG on alcohol addiction.METHODS:A behavioral sensitization mouse model induced by ethanol was established to observe the effect of PG on the development and expression of behavioral sensitization induced by ethanol by recording the spontaneous activity of mice.The resident time of mice in a white box was measured to evaluate the effect of PG on developing CPP induced by ethanol.Concentrations of dopamine(DA),Glutamate(Glu),and?-aminobutyric acid(GABA)in the corresponding mesolimbic region of mice were determined by enzyme-linked immunosorbent assay.RESULTS:Although PG did not alter spontaneous activity in mice,it reduced the growth of spontaneous activity stimulated by ethanol.The residence time in the white box after-ethanol-training of mice in CPP experiments was decreased.CONCLUSION:Our data suggested that PG blocked the development and expression of behavioral sensitization induced by ethanol and the development of CPP in mice.The mechanism might be related to the decreased content of DA and Glu and increased content of GABA in the mesolimbic dopamine system.This suggests that PG might be useful for the prevention and treatment of alcohol addiction.

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.

海洛因诱导CPP大鼠伏隔核脑电活动的无线遥测研究

目的:分析海洛因诱导条件性位置偏爱(CPP)大鼠的伏隔核(NAc)脑电活动在海洛因依赖不同时期的变化特征,探讨其在药物依赖形成过程中的作用。方法:通过立体定位向大鼠NAc埋藏电极,建立海洛因依赖大鼠模型,用无线遥测方法分别记录海洛因给药前、给药后即刻及戒断状态3个时期内大鼠NAc神经元放电活动,分析NAc神经元脑电活动的频率(0.5 Hz^30 Hz)分布及波幅。结果:CPP系统建立海洛因依赖模型有效,大鼠戒断症状明显。海洛因给药5~10 d,大鼠陆续出现白箱CPP,戒断2 d时戒断症状达高峰。各时期内大鼠NAc脑电活动δ波比例皆最高,α2波比例最低,不同频段脑电的分布梯度相似。与给药前和戒断期相比,给药后即刻状态NAc低频放电(δ波)比例明显升高(P<0.01),高频放电(β波)比例明显降低(P<0.01);戒断期α1波比例明显小于其它各期(P<0.01)。与给药前相比,给药后即刻和戒断期NAc脑电活动平均波幅均显著增大(P<0.01),但戒断期的平均波幅比给药后即刻期的明显降低(P<0.01)。结论:海洛因依赖不同时期内大鼠NAc的脑电活动出现明显变化,提示NAc神经元活动可能参与药物依赖的调节。