Cue-induced alcohol-seeking behaviour is reduced by disrupting the reconsolidation of alcohol-related memories

Rationale  In humans, the retrieval of memories associated with an alcohol-related experience frequently evokes alcohol-seeking behaviour. The reconsolidation hypothesis states that a consolidated memory could again become labile and susceptible to disruption after memory retrieval. Objectives  The aim of our study was to examine whether retrieval of alcohol-related memories undergoes a reconsolidation process. Methods  For this purpose, male Wistar rats were trained to self-administer ethanol in the presence of specific conditioned stimuli. Thereafter, animals were left undisturbed in their home cages for the following 21 days. Memory retrieval was performed in a single 5-min exposure to all alcohol-associated stimuli. The protein synthesis inhibitor anisomycin, the non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 and acamprosate, a clinically used drug known to reduce a hyper-glutamatergic state, were given immediately after retrieval of alcohol-related memories. The impact of drug treatment on cue-induced alcohol-seeking behaviour was measured on the following day and 7 days later. Results  Administration of both anisomycin and MK-801 reduced cue-induced alcohol-seeking behaviour, showing that memory reconsolidation was disrupted by these compounds. However, acamprosate had no effect on the reconsolidation process, suggesting that this process is not dependent on a hyper-glutamatergic state but is more related to protein synthesis and NMDA receptor activity. Conclusions  Pharmacological disruption of reconsolidation of alcohol-associated memories can be achieved by the use of NMDA antagonists and protein synthesis inhibitors and may thus provide a potential new therapeutic strategy for the prevention of relapse in alcohol addiction. C. von der Goltz and V. Vengeliene contributed equally to this work.     

Pharmacological manipulation of memory reconsolidation: towards a novel treatment of pathogenic memories

Well-consolidated memories, when retrieved, may return to a transiently fragile state, and need to be consolidated again in order to be maintained. This process has been referred to as memory reconsolidation and presumably serves to modify or strengthen memory traces. In recent years, our understanding of the neurobiological mechanisms underlying this phenomenon has increased rapidly. Here, we will briefly review some of the pharmacological evidence, stressing a crucial role for the brain's major neurotransmitter systems, such as glutamate and noradrenaline, in memory reconsolidation. Pharmacological intervention of reconsolidation processes may have clinical relevance, especially for the treatment of psychiatric disorders that are characterized by pathological memories, including post-traumatic stress disorder and addictive behaviour.     

Involvement of amygdalar protein kinase A, but not calcium/calmodulin-dependent protein kinase II, in the reconsolidation of cocaine-related contextual memories in rats

Rationale

Contextual control over drug relapse depends on the successful reconsolidation and retention of context-response–cocaine associations in long-term memory stores. The basolateral amygdala (BLA) plays a critical role in cocaine memory reconsolidation and subsequent drug context-induced cocaine-seeking behavior; however, less is known about the cellular mechanisms of this phenomenon.

Objectives

The present study evaluated the hypothesis that protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) activation in the BLA is necessary for the reconsolidation of context-response–cocaine memories that promote subsequent drug context-induced cocaine-seeking behavior.

Methods

Rats were trained to lever-press for cocaine infusions in a distinct context, followed by extinction training in a different context. Rats were then briefly re-exposed to the previously cocaine-paired context or an unpaired context in order to reactivate cocaine-related contextual memories and initiate their reconsolidation or to provide a similar behavioral experience without explicit cocaine-related memory reactivation, respectively. Immediately after this session, rats received bilateral microinfusions of vehicle, the PKA inhibitor, Rp-adenosine 3’,5’-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS), or the CaMKII inhibitor, KN-93, into the BLA or the posterior caudate putamen (anatomical control region). Rats were then tested for cocaine-seeking behavior (responses on the previously cocaine-paired lever) in the cocaine-paired context and the extinction context.

Results

Intra-BLA infusion of Rp-cAMPS, but not KN-93, following cocaine memory reconsolidation impaired subsequent cocaine-seeking behavior in a dose-dependent, site-specific, and memory reactivation-dependent fashion.

Conclusions

PKA, but not CaMKII, activation in the BLA is critical for cocaine memory re-stabilization processes that facilitate subsequent drug context-induced instrumental cocaine-seeking behavior.     

Nitrous oxide may interfere with the reconsolidation of drinking memories in hazardous drinkers in a prediction-error-dependent manner

Weakening drinking-related reward memories by blocking their reconsolidation is a potential novel strategy for treating alcohol use disorders. However, few viable pharmacological options exist for reconsolidation interference in humans. We therefore examined whether the NMDA receptor antagonising gas, Nitrous Oxide (N₂O) could reduce drinking by preventing the post-retrieval restabilisation of alcohol memories in a group of hazardous drinkers. Critically, we focussed on whether prediction error (PE; a key determinant of reconsolidation) was experienced at retrieval. Sixty hazardous drinkers were randomised to one of three groups that retrieved alcohol memories either with negative PE (Retrieval + PE), no PE (Retrieval no PE) or non-alcohol memory retrieval with PE (No-retrieval +PE). All participants then inhaled 50% N₂O for 30?min. The primary outcome was change in beer consumption and alcohol cue-driven urge to drink from the week preceding manipulation (baseline) to the week following manipulation (test). The manipulation did not affect drinking following the intended retrieval+/- PE conditions However, a manipulation check, using a measure of subjective surprise, revealed that the group-level manipulation did not achieve the intended differences in PE at retrieval. Assessment of outcomes according to whether alcohol-relevant PE was actually experienced at retrieval, showed N₂O produced reductions in drinking in a retrieval and PE-dependent fashion. These preliminary findings highlight the importance of directly testing assumptions about memory reactivation procedures in reconsolidation research and suggest that N₂O should be further investigated as a potential reconsolidation-blocking agent.     

Acetaminophen nephrotoxicity in male Wistar rats

Acute acetaminophen (APAP) nephrotoxicity was studied in male Wistar rats 1 h after different APAP single doses (200, 500 and 1000 mg/kg body wt, i.p.). Significant impairments in glomerular filtration rate (GFR) and clearance ofp-aminohippuric acid (Cl_PAH) were observed in a dose-dependent way, although tubular parameters measured, water and electrolyte fractional excretion, remained at control values, while the urine to plasma osmolality ratios (U_osm/P_osm) were diminished in APAP-1000 rats (control=2.93 ±0.20, APAP-1000=1.40±0.04). The time course of renal function was also studied in APAP-1000 mg/kg-treated animals; parallel impairments were observed in GFR, Cl_PAH and tubular functions. Maximal alteration was observed at 16 h and restorement began at 24 h post-injection. Glucose renal handling, either at low or at high tubular glucose loads, remained at control values. Thus, our data suggest that the early stage of acetaminophen nephrotoxicity might be due to renal hemodynamic changes which might induce an alteration in tubular function principally in distal structures of medullary tissue, as shown by the U_osm/P_osm results. These effects occurred coupled with a diminution in hepatic glutathione (GSH) levels at every APAP dose and in renal GSH levels in APAP-1000 mg/kg-treated rats. Moreover, renal damage was observed both in the presence or absence of hepatic damage.     

Toxicokinetics of p-tert-octylphenol in male Wistar rats

Only weak oestrogenic activity has been reported for p-alkylphenols compared with the physiological hormone 17β-estradiol. Despite the low potency, there is concern that due to bioaccumulation oestrogenically efficient blood levels could be reached in humans exposed to trace levels of p-alkylphenols. To address these concerns, toxicokinetic studies with p-tert-octylphenol [OP; p-(1,1,3,3-tetramethylbutyl)-phenol] as a model compound have been conducted in male Wistar rats. OP blood concentrations were determined by GC-MS in rats receiving either single oral (gavage) applications of 50 or 200mg OP/kg body wt or a single intravenous injection of 5mg/kg body wt. The OP blood concentration was ∼1970ng/ml immediately after a single intravenous application, decreased rapidly within 30 min, and was no longer detectable 6–8h after application. The curve of blood concentration vs time was used to calculate an elimination half-life of 310min. OP was detected in blood as early as 10min after gavage administration, indicating rapid initial uptake from the gastrointestinal tract; maximal blood levels reached 40 and 130ng/ml after applications of 50 and 200mg/kg, respectively. Using the area under the curve (AUC) of blood concentration vs time, low oral bioavailabilities of 2 and 10% were calculated for the 50 and 200mg/kg groups, respectively. OP toxicokinetics after repeated administration was investigated in male Wistar rats receiving daily gavage administrations of 50 or 200mg OP/kg body wt for 14 consecutive days. Profiles of OP blood concentration vs time determined on day 1 and day 14 were similar, indicating that repeated oral gavage administration did not lead to increased blood concentrations. Another group of rats received OP via drinking water saturated with OP (∼8mg/l, corresponding to a mean daily dose of ∼800μg/kg) over a period of up to 28 days. OP was not detected in any blood sample from animals treated via drinking water (detection limit was 1–5ng/ml blood). OP concentrations were also analysed in tissues obtained from the repeated gavage (14 days) and drinking water groups (14 and 28 days). In the 50mg/kg group, low OP concentrations were detected in fat and liver from some animals at average concentrations of 10 and 7ng/g tissue, respectively. OP was not detected in the other tissues analysed from this group. In the 200mg/kg group, OP was found in all tissues analysed except testes (fat, liver, kidney, muscle, brain and lung had average concentrations of 1285, 87, 71, 43, 9 and 7ng/g tissue, respectively). OP was not detected in tissues of animals receiving OP via drinking water for 14 or 28 days, except in muscle and kidney tissue of one single animal receiving OP for 14 days. Using rat liver fractions it was demonstrated that OP was conjugated via glucuronidation and sulphation in vitro. A V _max of 11.24 nmol/(min * mg microsomal protein) and a K _m of 8.77μmol/l were calculated for enzyme-catalysed OP glucuronidation. For enzyme-catalysed sulphation, a V _max of 2.85nmol/(minT15*mg protein) and a K _m of 11.35μmol/l were calculated. The results indicate that OP does not bioaccumulate in rats receiving low oral doses, in agreement with the hypothesis of a rapid first-pass elimination of OP by the liver after oral ingestion, via glucuronidation and sulphation. Only if these detoxification pathways are saturated may excessive doses lead to bioaccumulation. Received: 22 March 1996/Accepted: 12 June 1996     

Developmental toxicity of homobrassinolide in Wistar rats

Brassinosteroids (BRs) are close analogues of animal cholesterol. Brassinosteroids have shown their great value as yield promoters of a variety of plants. In view of its steroidal moiety and recent use in agriculture in many countries, the teratogenic potential of homobrassinolide (HBR) was evaluated in Wistar rats. Homobrassinolide was administered by oral gavage at doses 0, 100, and 1000 mg/kg body weight in water during gestation days (GD) 6 to 15 in groups of 20 mated females. Maternal and embryo-fetal toxicity was analyzed by studying the effects such as clinical signs, mortality/morbidity, abortions, body weight, feed consumption, and pregnancy data, gravid uterine weights, implantation losses, litter size, external, visceral, and skeletal malformations. No treatment-related effect was observed on any of the maternal/fetal end points in any dose group. From the results, it can be concluded that HBR is nonteratogenic at doses as high as up to 1000 mg/kg body weight in Wistar rats.     

Metabolism of myosmine in Wistar rats.

The alkaloid myosmine is present not only in tobacco products but also in various foods. Myosmine is easily nitrosated, yielding 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and the esophageal tobacco carcinogen N'-nitrosonornicotine. Due to its widespread occurrence, investigations on the metabolism and activation of myosmine are needed for risk assessment. Therefore, the metabolism of myosmine has been studied in Wistar rats treated with single oral doses of [pyridine-5-3H]myosmine at 0.001, 0.005, 0.5, and 50 micromol/kg body weight. Oral administration was achieved by feeding a labeled apple bite. Radioactivity was completely recovered in urine and feces within 48 h. At the two lower doses, 0.001 and 0.005 micromol/kg, a higher percentage of the radioactivity was excreted in urine (86.2 +/- 4.9% and 88.9 +/- 1.7%) as compared with the higher doses, 0.5 and 50 micromol/kg, where only 77.8 +/- 7.3% and 75.4 +/- 6.6% of the dose was found in urine. Within 24 h, urinary excretion of radioactivity was nearly complete with less than 4% of the total urinary output appearing between 24 and 48 h. The two major metabolites accounting for >70% of total radioactivity in urine were identified as 3-pyridylacetic acid (20-26%) and 4-oxo-4-(3-pyridyl)butyric acid (keto acid, 50-63%) using UV-diode array detection and gas chromatography-mass spectrometry measurements. 3-Pyridylmethanol (3-5%), 3'-hydroxymyosmine (2%) and HPB (1-3%) were detected as minor metabolites. 3'-Hydroxymyosmine is exclusively formed from myosmine and therefore might be used as a urinary biomarker for myosmine exposure in the future.     

Polymorphism in diazepam metabolism in Wistar rats

We observed variations in the metabolism of diazepam in Wistar rats. We studied these variations carefully, and found that the variations are dimorphic and about 17% of male rats of Wistar strain we examined showed two times higher diazepam metabolic activities in their liver microsomes than the rest of animals at the substrate concentrations less than 5 microM. We classified them as extensive metabolizer (EM) and poor metabolizer (PM) of diazepam. No sex difference was observed in the frequency of appearance of EM. Activities of the primary metabolic pathways of diazepam were examined to elucidate the cause of this polymorphism in male Wistar rats. No significant differences were observed in activities of neither diazepam 3-hydroxylation or N-desmethylation between EM and PM rats, while activity of diazepam p-hydroxylation was markedly (more than 200 times) higher in EM rats, indicating that this reaction is responsible for the polymorphism of diazepam metabolism in Wistar rats. We examined the expression levels of CYP2D1, which was reported to catalyze diazepam p-hydroxylation in Wistar rats to find no differences in the expression levels of CYP2D1 between EM and PM rats. The kinetic study on diazepam metabolism in male Wistar rats revealed that EM rats had markedly higher V(max) and smaller K(m) in diazepam p-hydroxylation than those of PM rats, indicating the presence of high affinity high capacity p-hydroxylase enzyme in EM rats. As a consequence, at low concentrations of diazepam, major pathways of diazepam metabolism were p-hydroxylation and 3-hydroxylation in male EM rats, while in male PM rats, 3-hydroxylation followed by N-desmethylation. Due to this kinetic nature of p-hydroxylase activity, EM rats had markedly higher total CL(int) of diazepam than that of PM rats. Polymorphism in diazepam metabolism in humans is well documented, but this is the first report revealing the presence of the polymorphism in diazepam metabolism in rats. The current results infer polymorphic expression of new diazepam p-hydroxylating enzyme with lower K(m) than CYP2D1 in EM Wistar rats.     

Reproductive adverse effects of fipronil in Wistar rats

The purpose of the present study was to investigate possible reproductive adverse effects of fipronil (Frontline TopSpot) in female Wistar rats. The pesticide was topically applied to rats (single dose) at different concentrations (70, 140 and 280 mg/kg) and hormonal analysis, estrous cycle, and pregnancy and outcome data were determined. Treatment with fipronil altered cyclicity of female rats lengthening the estrous cycle (days) after a single topic administration of 70 mg/kg (9.7+/-1.18) or 280 mg/kg (14.5+/-1.45) when compared to control (4.8+/-0.17). In the mating study fipronil reduced the pregnancy index (67%) in the highest dose group (280 mg/kg). Plasma progesterone and estradiol levels, obtained in different periods after treatment with fipronil (70 mg/kg), were significantly different 96 h after treatment, when compared to controls. In summary, the results of the present study indicate that fipronil may alter the normal functioning of the endocrine system and cause adverse reproductive effects in female rats.     

Niridazole-mediated modulation of suppressor cells in Wistar rats

The antischistosomal drug niridazole has been shown to inhibit inductive (Vadas and Bernard, 1981) as well as effector phases of delayed hypersensitivity (Sainis et al., 1983). Furthermore, it also abrogates help for delayed hypersensitivity in antigen-primed animals (Sainis et al., 1983). The effect of this drug on antigen-induced suppression was examined in the present studies. Profound suppression of delayed hypersensitivity to sheep erythrocytes was obtained in Wistar rats given 10(8) erythrocytes (i.v.) 6 days before the immunizing dose (2 x 10(9) erythrocytes, i.p.). When these rats were orally administered niridazole (50 mg/kg) 7 days before the tolerising dose of antigen, suppression of delayed hypersensitivity was not obtained. Splenic lymphocytes of rats given the tolerising dose 6 days earlier adoptively transferred the suppression to inbred recipients. Treatment of these afferent suppressor cells with sera from niridazole-treated unimmunized rats abrogated their function. Likewise, the efferent suppressor cells obtained from fully tolerised rats did not suppress the delayed hypersensitivity when co-transferred with immune lymphocytes, if they were pretreated with niridazole-active serum. The metabolite of niridazole present in this serum seems to impair the suppressor cells functionally. Niridazole may thus prove to be a versatile immunomodulator for effector, helper and suppressor T-cells.     

Teratogenic effects of diphenyl diselenide in Wistar rats

Diphenyl diselenide is an organoselenium compound with potential therapeutic use. The present study evaluates the effects of single maternal subcutaneous injection of 50 and 100 mg/kg diphenyl diselenide [(PhSe)₂] at gestational days (GD) 6, 10 or 17 in Wistar rats. The highest dose of (PhSe)₂ was also administered at GD 7–12. External and internal fetal soft-tissue examination was performed at GD 20. No mortality was observed in fetuses or dams at any (PhSe)₂ treatment group. Neither did exposure to (PhSe)₂ cause significant changes to fetal body weight, organ weight, or fetal size when administered at GD 6–8, 10–12 or 17. Exposure to 100 mg/kg (PhSe)₂ at GD 9 produced significant changes in fetal biometry (crown-rump (CR) length) and body weight. No significant increase in the proportion of fetuses with external visible abnormalities was observed in groups exposed to (PhSe)₂. Skeletal anomalies were observed in fetuses in the GD 9–11 treatment groups and included incomplete ossification of cranial bones, misshapen and incomplete ossification of sternebrae, reduced sternebrae number, wavy and extra ribs, incomplete ossification of fore and hindpaw bones and incomplete ossification of sacral and caudal bones. We conclude that maternal administration of (PhSe)₂ during GD 7–12 led to increased incidences of these skeletal variations or anomalies, but did not cause externally visible malformations in rat fetuses.     

Methylphenidate reduces impulsive behaviour in juvenile Wistar rats, but not in adult Wistar, SHR and WKY rats

Rationale Impulsivity is a core symptom of attention deficit/hyperactivity disorder (ADHD). The spontaneously hypertensive rats (SHR) is a strain commonly used as an animal model of ADHD. However, there is no clear evidence that psychostimulants, which are used for treatment of ADHD, reduce impulsivity in SHR. Because ADHD mainly affects children, it may be relevant to study psychostimulants on juvenile animals. Objectives Using tolerance to delay of reward as index of impulsivity, the effects of methylphenidate were assessed in adult SHR, Wistar Kyoto (WKY) and Wistar rats and in juvenile Wistar rats. Materials and methods Animals were trained in a T-maze to choose between a small-but-immediate and a large-but-delayed reward. Adult SHR, WKY and Wistar rats were compared for their ability to tolerate a 15-s delay. The effect of methylphenidate on the tolerance to a 30-s delay was studied in adult rats of the three strains and in juvenile (4.5 to 6.5-week-old) Wistar rats. Results In adult rats, the waiting ability was lower in SHR than in control strains. Waiting ability was improved by methylphenidate (3 and 5 mg/kg) in juveniles, but not by methylphenidate (3 mg/kg) in adults. Conclusions These data support the idea that SHR are more impulsive than control strains. However, at the dose studied, methylphenidate fails to improve tolerance to delay in adult rats whatever the strain used. The reduction of impulsivity induced by methylphenidate in juvenile Wistar rats indicates that juvenile animals may be suitable for testing the therapeutic potential of drugs intended to the treatment of ADHD in children.     

Assessment of pimozide's motor and hedonic effects on operant behavior in rats

The present study examined the effects of the neuroleptic pimozide on several measures of motor capacity and reinforcement efficacy in rats trained to respond according to a multiple random interval (RI) food reinforcement schedule (mean interreinforcement intervals of 10, 20, 40, 80, and 160 sec). Pimozide (0.125, 0.25, 0.5, and 1.0 mg/kg) produced a dose-dependent suppression of response rates for all five RI schedules and a dose-dependent increase in response duration. An independent measure of motor activity in photocell activity chambers also was decreased by pimozide in a dose-dependent manner. Photocell activity was significantly correlated with response duration and with the Matching Equation parameter k. Thus, all three measures of motor performance revealed similar decreases in motor capacity at the high dose of pimozide. Reinforcement efficacy also was reduced by the 1.0 mg/kg dose of pimozide as indicated by an increase in the Matching Equation parameter Re. The parameters k and Re were not significantly correlated, suggesting that these two Matching Equation parameters do provide independent measures of motor capacity and reinforcement efficacy, respectively. The present results demonstrate the importance of obtaining measures other than simple response rates in order to assess drug effects on operant behavior.     

Stress modulation of reconsolidation

Memories are consolidated and are inscribed as stable traces in the brain; however, once they are retrieved, they are rendered labile and can be modified in a process termed reconsolidation. Studies illustrate the power of behavioral stress and stress hormones to modulate memory processes while focusing on consolidation. However, sparse evidence indicates a critical role of stress in modulating reconsolidation. In this review, we discuss the effects of stress and stress-related neurotransmitter systems on reconsolidation of emotional and non-emotional types of memories. We show that although some general features underlie consolidation and reconsolidation, there is a possible dissimilarity between the two processes that may be dependent on factors such as the cognitive task employed, specific type of stressor, and the arousal state of the animal. The ability to disrupt or facilitate the reconsolidation of emotional and drug-related memories by stress exposure has important implications for the treatment of anxiety disorders linked to traumatic memories, such as post-traumatic stress disorder and of drug-of-abuse memories.     

Effects of sleep deprivation on retrieval and reconsolidation of morphine reward memory in rats

Relapse induced by exposure to cues associated with drugs of abuse is a major challenge to the treatment of drug addiction. Drug seeking can be inhibited by manipulation of the reconsolidation of drug-related memory. Sleep has been proposed to be involved in various memory processes. However, the role of sleep in drug reward memory is not clear. The present study used conditioned place preference to examine the effects of total sleep deprivation on retrieval and reconsolidation of morphine reward memory in rats. Six-hour total sleep deprivation had no effect on the retrieval of morphine reward memory. However, sleep deprivation from 0-6 h, but not 6-12 h, after re-exposure disrupted the reconsolidation of morphine reward memory. This impairment was not attributable to the formation of an aversive associative memory between the drug-paired context and sleep deprivation. Our findings suggest that sleep plays a critical role in morphine reward memory reconsolidation, and sleep deprivation may be a potential non-pharmacotherapy for the management of relapse associated with drug-related memory.     

Effects of scopolamine and ketamine on reconsolidation of morphine conditioned place preference in rats

Persistent memory associated with addictive drugs contributes to the relapse of drug abuse. The current study was conducted to examine the effects of scopolamine and ketamine on reconsolidation of morphine-induced conditioned place preference (CPP). In experiment 1, after morphine CPP was acquired, rats were injected with ketamine (60 mg/kg, intraperitoneally) and scopolamine (2 mg/kg, intraperitoneally), respectively, after reexposure to an earlier morphine-paired context or in their home cages. The CPP was reassessed 24 and 48 h after reexposure. An additional group of rats received saline following reexposure to the earlier morphine-paired context. In experiment 2, two groups of rats were only given saline during the CPP training and subsequent administration of ketamine or scopolamine during the reexposure. In experiment 1, rats failed to exhibit morphine CPP when ketamine and scopolamine were administered only after reexposure to a morphine-paired context. CPP was not abolished by ketamine or scopolamine administration in the animals' home cages. Also, the animals receiving only saline injections showed strong morphine CPP 24 h after a short exposure to the morphine-paired context. In experiment 2, ketamine or scopolamine treatment alone did not induce CPP or aversion. Administration of scopolamine and ketamine, after reexposure to a drug-paired context, resulted in the disruption of morphine CPP, suggesting the potential effects of scopolamine and ketamine in disrupting memory associated with environmental cues and addictive drugs.