Conditioned Place Preference Paradigm Research Articles

The Nociceptin Receptor (NOP) Agonist AT-312 Blocks Acquisition of Morphine- and Cocaine-Induced Conditioned Place Preference in Mice.

Treatment of drug addiction remains an unmet medical need due to the dearth of approved pharmacotherapies. There are no approved treatments for cocaine addiction, whereas the current opioid crisis has revealed the stark reality of the limited options to treat prescription and illicit opioid abuse. Preclinical studies in rodents and nonhuman primates have shown that orphanin FQ/nociceptin (N/OFQ), the endogenous ligand for the nociceptin opioid receptor (NOP) reduces the rewarding effects of several abused substances, including opioids, psychostimulants and alcohol. A few nonpeptide small-molecule NOP agonists have also shown efficacy in attenuating the rewarding effects of various abused drugs. We previously demonstrated that a high affinity small-molecule NOP agonist AT-312 selectively reduced the rewarding effects of ethanol in the conditioned place preference paradigm in mice. In the present study, we examined if AT-312 (3 mg/kg, i.p. or s.c. respectively), would alter the rewarding action of morphine (7.5 mg/kg, s.c.) or cocaine (15 mg/kg, i.p.). The effect of AT-312 on morphine- and cocaine-induced motor stimulation was also assessed on the conditioning days. The role of the NOP receptor in the effects of AT-312 was further confirmed by conducting the place conditioning experiments in NOP knockout mice and compared to their wild-type controls. Our results showed that AT-312 significantly reduced the acquisition of morphine and cocaine CPP in wild-type mice but not in mice lacking NOP receptors. AT-312 also suppressed morphine-induced and completely abolished cocaine-induced motor stimulation in NOP wild-type mice, but not in NOP knockout mice. These results show that small-molecule NOP receptor agonists have promising efficacy for attenuating the rewarding effects of morphine and cocaine, and may have potential as pharmacotherapy for opioid and psychostimulant addiction or for treating polydrug addiction.

PI3K activation within ventromedial prefrontal cortex regulates the expression of drug-seeking in two rodent species.

Phosphatidylinositide 3-kinases (PI3Ks) are intracellular signal transducer enzymes that recruit protein kinase B (aka Akt) to the cell membrane, the subsequent activation of which regulates many cellular functions. PI3K/Akt activity is up-regulated within mesocorticolimbic structures in animal models of alcoholism, but less is known regarding PI3K/Akt activity in animal models of cocaine addiction. Given that prefrontal cortex (PFC) is grossly dysregulated in addiction, we studied how cocaine affects protein indices of PFC PI3K/Akt activity in rat and mouse models and examined the relevance of PI3K activity for cocaine-related learning. Immunoblotting of mouse medial PFC at 3weeks withdrawal from a cocaine-sensitization regimen (seven injections of 30mg/kg, intraperitoneal [IP]) revealed increased kinase activity, as did immunoblotting of tissue from the ventral PFC of rats with a history of long-access intravenous cocaine self-administration (0.25mg/0.1mL infusion; 10days of 6h/d cocaine access). Interestingly, increased Akt phosphorylation was observed in rat ventromedial PFC at both 3- and 30-day withdrawal only in animals re-exposed to cocaine-associated cues. A conditioned place-preference paradigm in mice and a cue-elicited drug-seeking test in rats were conducted to determine the functional relevance for elevated PI3K activity for addiction-related behavior. In both cases, an intra-PFC infusion of the PI3K inhibitor wortmannin (50μM) reduced drug-seeking behavior. Taken together, this cross-species, interdisciplinary, study provides convincing evidence that cocaine history produces an enduring increase in PI3K/Akt-dependent signaling within the more ventral aspect of the PFC that is relevant to behavioral reactivity to drug-associated cues/contexts. As such, PI3K inhibitors may well serve as an effective strategy for reducing drug cue reactivity and craving in cocaine addiction.

Removal of microglial-specific MyD88 signaling alters dentate gyrus doublecortin and enhances opioid addiction-like behaviors

Drugs of abuse promote a potent immune response in central nervous system (CNS) via the activation of microglia and astrocytes. However, the molecular mechanisms underlying microglial activation during addiction are not well known. We developed and functionally characterized a novel transgenic mouse (Cx3cr1-CreBTtg/0:MyD88f/f [Cretg/0]) wherein the immune signaling adaptor gene, MyD88, was specifically deleted in microglia. To test the downstream effects of loss of microglia-specific MyD88 signaling in morphine addiction, Cretg/0 and Cre0/0 mice were tested for reward learning, extinction, and reinstatement using a conditioned place preference (CPP) paradigm. There were no differences in drug acquisition, but Cretg/0 mice had prolonged extinction and enhanced reinstatement compared to Cre0/0 controls. Furthermore, morphine-treated Cretg/0 mice showed increased doublecortin (DCX) signal relative to Cre0/0 control mice in the hippocampus, indicative of increased number of immature neurons. Additionally, there was an increase in colocalization of microglial lysosomal marker CD68 with DCX+cells in morphine-treated Cretg/0 mice but not in Cre0/0 or drug-naїve mice, suggesting a specific role for microglial MyD88 signaling in neuronal phagocytosis in the hippocampus. Our results show that MyD88 deletion in microglia may negatively impact maturing neurons within the adult hippocampus and thus reward memories, suggesting a novel protective role for microglia in opioid addiction.

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats.

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Intra-hippocampal administration of orexin receptor antagonists dose-dependently attenuates reinstatement of morphine seeking behavior in extinguished rats

It has been shown that the hippocampus plays an essential role in the regulation of reward and memory as indicated by the conditioned place preference (CPP) paradigm. Morphine-induced CPP is a common method to consider motivational properties of morphine in animals. Recently, this model has been used in many laboratories to investigate neuronal mechanisms underlying reinstatement of morphine seeking induced by drug re-exposure. Our previous studies indicate that the hippocampus especially CA1 region is involved in reinstatement of drug-seeking behaviors. Also, several studies have shown that orexin attenuates key functional and behavioral effects of its co-transmitter dynorphin. The present study evaluates the role of orexinergic receptors within the CA1 region of the hippocampus in the reinstatement of morphine-induced CPP. Therefore, after the extinction period, the different doses (SB 334867; 0.3, 3, and 30 nM/0.5 μl DMSO) of either orexin-1 or -2 receptor antagonists were bilaterally microinjected into the CA1, 15 min before receiving an effective priming dose of morphine (1 mg/kg). The results revealed that administration of both SB 334867 and TCS OX2 29 prior to injection of the priming dose of morphine significantly reduced the reinstatement of morphine-induced CPP without altering the animal's locomotor activity. Also, the 50% effective dose value of SB 334867 on the reinstatement of morphine seeking behavior was close three times more than that in TCS OX2 29 treatment group. Therefore, the consequences suggested that both orexin receptors in the CA1 play a considerable role in the reinstatement of morphine-induced CPP.

Fluoxetine exposure in adolescent and adult female mice decreases cocaine and sucrose preference later in life.

Preclinical evidence from male subjects indicates that exposure to psychotropic medications, during early development, results in long-lasting altered responses to reward-related stimuli. However, it is not known if exposure to the antidepressant fluoxetine, in female subjects specifically, changes sensitivity to natural and drug rewards, later in life. The aim of this work was to investigate if exposure to fluoxetine mediates enduring changes in sensitivity to the rewarding properties of cocaine and sucrose, using female mice as a model system. We exposed C57BL/6 female mice to fluoxetine (250 mg/L in their drinking water) for 15 consecutive days, either during adolescence (postnatal day 35-49) or adulthood (postnatal day 70-84). Twenty-one days later, mice were examined on their behavioral reactivity to cocaine (0, 2.5, 5, 7.5 mg/kg) using the conditioned place preference paradigm, or assessed on the two-bottle sucrose (1%) test. We found that regardless of age of antidepressant exposure, female mice pre-exposed to fluoxetine displayed reliable conditioning to the cocaine-paired compartment. However, when compared to respective age-matched controls, antidepressant pre-exposure decreased the magnitude of conditioning at the 5 and 7.5 mg/kg cocaine doses. Furthermore, fluoxetine pre-exposure reduced sucrose preference without altering total liquid intake. The data suggest that pre-exposure to fluoxetine, during adolescence or adulthood, results in a prolonged decrease in sensitivity to the rewarding properties of both natural and drug rewards in female C57BL/6 mice.

Substitution therapy with amphetamine-isotherapic attenuates amphetamine toxicological aspects of addiction

Introductionamphetamine (AMPH) is related to development of addiction, anxiety-like behaviors and impairments of memory after chronic use. In the current experiment, an ultra-high dilution (10−24mg/mL) of AMPH was used, consisting of the AMPH isotherapic (AMPH-ISO), which can be used as a replacement therapy to treat AMPH addiction. AimTo verify the influence of AMPH-ISO on toxicological aspects of AMPH addiction. MethodsRats received d,l-AMPH (4.0 mg/kg, i.p.) in the conditioned place preference (CPP) paradigm (8 days). Then, half of each experimental group (AMPH or saline) received AMPH-ISO/vehicle (0.2 mL per rat, once a day), for fourteen days. On the fifteenth day, animals were re-assessed in the CPP paradigm (to verify relapse behaviors) after a single dose of AMPH (2.0 mg/kg). Subsequently, anxiety-like behaviors were quantified, followed by ex vivo assays in the pre-frontal cortex. ResultsAMPH-ISO prevented relapse-like behavior of AMPH and reduced anxiety-like behavior per se in animals co-treated with AMPH. Molecular analysis evidenced that AMPH-ISO modulated dopaminergic targets (dopamine transporter, tyrosine hydroxylase and D1-R), whose immunoreactivity was increased by AMPH. Also, AMPH-ISO increased catalase activity and NPSH levels and reduced lipid peroxidation and protein carbonyl levels in the prefrontal cortex. ConclusionThis study shows that an ultra-high dilution of AMPH may be a useful alternative which can contribute with AMPH addiction treatment.

β-lactams modulate astroglial glutamate transporters and attenuate dependence to CP 55,940, a CB1 receptor agonist, in rat model

Studies on cannabinoids have reported contradictory findings, showing both aversion and rewarding outcomes in conditioned place preference (CPP). Various possibilities have been suggested to explain the aversive properties of cannabinoids, including the pharmacokinetics profile and dose selection. In this study, we have established a CPP method to investigate the effects of modulating astroglial glutamate transporters in cannabinoid dependence using a cannabinoid receptor 1 (CB1R) agonist, CP 55,940 (CP). Previous reports using CPP paradigm demonstrated the involvement of glutamatergic system in seeking behavior of several drugs of abuse such as cocaine, heroin and nicotine. Glutamate homeostasis is maintained by several astroglial glutamate transporters, such as glutamate transporter 1 (GLT-1), cystine/glutamate transporter (xCT) and glutamate aspartate transporter (GLAST). In this study, we investigated the effects of Ampicillin/Sulbactam, β-lactam compounds known to upregulate GLT-1 and xCT, on cannabinoid seeking behavior using CP. We found first that one prime dose of CP induced CP reinstatement; this effect was associated, in part, with significant downregulation of xCT expression in the nucleus accumbens, dorsomedial prefrontal cortex and amygdala. Moreover, GLT-1 expression was downregulated in the amygdala. Importantly, Ampicillin/Sulbactam treatment during the extinction phase attenuated CP-induced reinstatement and restored the expression of GLT-1 and xCT in mesocorticolimbic brain regions. These findings suggest that β-lactams may play a potential therapeutic role in attenuating dependence to cannabinoids, in part, through upregulation of GLT-1 and xCT.

Involvement of orexinergic receptors in the nucleus accumbens, in the effect of forced swim stress on the reinstatement of morphine seeking behaviors

Orexinergic system is involved in primary rewards; the neural circuit of the ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex and amygdala represents overlapping elements mediating the rewarding effects of drugs and stressful experiences. The NAc integrates reward-related information from the VTA. Also, it has been indicated that orexinergic system activates the mesolimbic dopamine projecting neurons to the NAc and promotes the development of reward in rodents. Therefore, in the present study, the conditioned place preference (CPP) paradigm was used to determine the role of the two types of orexin receptors (OXR) in the NAc in forced swim stress (FSS), as physical stress, and/or priming-induced reinstatement of morphine. The CPP was induced by injecting morphine (5 mg/kg, SC for 3 days) and lasted for eight free-morphine days; the reinstatement was induced by administration of effective priming dose of morphine (1 mg/kg; sc). The extinguished rats received intra-NAc injection of SB334867 as OX1R antagonist or TCSOX229 as OX2R antagonist before effective priming dose injection of morphine (1 mg/kg; sc). In others, the extinguished rats were given intra-NAc injection of SB334867 or TCSOX229 and then, they underwent FSS before injection of ineffective priming dose of morphine (0.5 mg/kg; sc). Our results showed that intra-accumbal administration of SB334867 or TCSOX229 could inhibit morphine priming- and FSS-induced reinstatement of extinguished morphine-seeking in the rats. It seems that OXR in the NAc may be involved in reward and could play an important role in the effect of stress on reinstatement of morphine-seeking behaviors in this area.

Modulatory role of the intra-accumbal CB1 receptor in protein level of the c-fos and pCREB/CREB ratio in the nucleus accumbens and ventral tegmental area in extinction and morphine seeking in the rats

Brain reward and motivation circuit begin from the ventral tegmental area (VTA) that its dopaminergic terminals project to various regions of the brain including the nucleus accumbens (NAc). This reward circuit is influenced by drugs of abuse such as morphine and cannabinoid. The present study tried to investigate the role of the intra-accumbal CB1 receptor in the c-fos level and pCREB/CREB ratio in the NAc and the VTA during reinstatement phase of morphine-induced conditioned place preference (CPP) by western blotting. The present data reveals that intra-accumbal administration of CB1 agonist, WIN55,212-2 (0.5, 1 and 2 mM/0.5 μl DMSO) before/during extinction period of morphine-induced CPP, significantly decreased the NAc and the VTA c-fos protein level in the reinstatement phase; whereas the pre-reinstatement administration of the CB1 agonist, increased the c-fos protein level. Intra-accumbal administration of the CB1 agonist during the extinction period of morphine-induced CPP reduced the pCREB/CREB ratio in the NAc. Also, the present data show that intra-accumbal administration of CB1 antagonist, AM251 (15, 45 and 90 μM/0.5 μl DMSO) during/after extinction period of morphine-induced CPP affects the NAc and the VTA c-fos protein level in the reinstatement phase. Also, intra-NAc microinjection of AM251 during the extinction period reduced pCREB/CREB ratio in these regions. In conclusion, the results presented here provide compelling evidence of the modulation and involvement of the c-fos and the CREB molecules in the cannabinoid-opioid interaction of the brain reward system in the CPP paradigm.

The histone demethylase KDM6B in the medial prefrontal cortex epigenetically regulates cocaine reward memory

Epigenetic remodeling contributes to synaptic plasticity via modification of gene expression, which underlies cocaine-induced long-term memory. A prevailing hypothesis in drug addiction is that drugs of abuse rejuvenate developmental machinery to render reward circuitry highly plastic and thus engender drug memories to be highly stable. Identification and reversal of these pathological pathways are therefore critical for cocaine abuse treatment. Previous studies revealed an interesting finding in which the mRNA of histone lysine demethylase, KDM6B, is upregulated in the medial prefrontal cortex (mPFC) during early cocaine withdrawal. However, whether and how it contributes to drug-seeking behavior remain unknown. Here we used a conditioned place preference paradigm to investigate the potential role of KDM6B in drug-associated memory. We found that KDM6B protein levels selectively increased in the mPFC during cocaine withdrawal. Notably, systemic injection of KDM6B inhibitor, GSK-J4, disrupted both reconsolidation of cocaine-conditioned memory and cocaine-primed reinstatement, suggesting dual effects of KDM6B in cocaine reward memory. In addition, we found that NMDAR expression and function were both enhanced during early cocaine withdrawal in mPFC. Injection of GSK-J4 selectively reversed this cocaine-induced increase of NR2A expression and synaptic function, suggesting that mal-adaptation of cocaine-induced synaptic plasticity in mPFC largely underlies KDM6B-mediated cocaine-associated memory. Altogether, these data suggest that KDM6B plays an essential role in cocaine-associated memory, which mainly acts through enhancing cocaine-induced synaptic plasticity in the mPFC. Our findings revealed a novel role of KDM6B in cocaine-associated memory and inhibition of KDM6B is a potential strategy to alleviate drug-seeking behavior.

Intracerebroventricular microinjection of the 5-HT1F receptor agonist LY 344864 inhibits methamphetamine conditioned place preference reinstatement in rats

Relapse following a prolonged period of drug cessation is a key barrier in the treatment of methamphetamine (METH) addiction, for which pharmacological treatment exhibits little efficacy. Previous studies have suggested that this process involves alterations in levels of serotonin (5-HT) in the brain. Although the 5-HT1F receptor has been implicated in the reward pathway, its physiological functions remain unknown. In the present study, we examined the effect of the 5-HT1F agonist LY 344864 on the reinstatement of METH-seeking behavior in rats using a conditioned place preference (CPP) paradigm. The CPP paradigm was first used to determine the effective doses of LY and METH. Four groups were then conditioned with METH (5 mg/kg; i.p.), while the sham group received saline. METH-induced CPP was subsequently extinguished. On the 13th day of extinction, the rats received either METH (0, 1, or 2.5 mg/kg; i.p.) plus vehicle or priming METH plus LY (2 μg/5 μL; i.c.v.). On reinstatement day, preference scores were calculated as the difference in time spent in the drug-paired and vehicle-paired compartments. Rats conditioned with the lowest effective dose of METH (5 mg/kg) exhibited significant differences in pre- and post-testing preference scores. Preference scores were significantly higher in the saline + METH group than in the control group. Furthermore, preference scores were significantly higher in rats that had received priming METH treatment, and pre-treatment with LY significantly attenuated the reinstatement of METH-seeking behavior. These findings suggest that future studies should evaluate the therapeutic potential of 5-HT1F agonists for preventing relapse in individuals with METH addiction.

Cannabidiol does not display drug abuse potential in mice behavior.

Recent evidence suggests that cannabidiol (CBD) may be useful for the treatment of different neuropsychiatric disorders. However, some controversy regarding its profile as a drug of abuse hampers the further development of basic and clinical studies. In this study, the behavioral profile of CBD as a potential drug of abuse was evaluated in C57BL/6J mice. Reinforcing properties of CBD (15, 30, and 60 mg/kg; i.p.) were assessed using the conditioned place preference (CPP) paradigm. Spontaneous withdrawal symptoms and motor activity in the open field were examined 12 h after the last CBD administration (30 mg/kg/12 h, i.p., 6 days). CBD plasma concentrations were measured at 2, 4, 8, 12, and 24 h after the administration of CBD (30 mg/kg, i.p.). Furthermore, an oral CBD self-administration paradigm (50 mg/kg; CBD water-soluble 1.2 mg/mL) was performed to evaluate whether this drug produced any effects on motivation compared with a non-reinforcing substance (water). We found that CBD failed to induce CPP, withdrawal symptoms, or altered motor behavior 12 h after its administration. At that time, only traces of CBD were detected, ensuring that the lack of alterations in somatic signs and locomotor activity was not due to residual drug in plasma. Interestingly, mice displayed similar motivation and consumption of CBD and water. Taken together, these results show that CBD lacks activity as a drug of abuse and should stimulate the development of the basic and clinical studies needed to elucidate its potential therapeutic use for the treatment of neuropsychiatric and drug use disorders.

Examining affective structure in chickens: valence, intensity, persistence and generalization measured using a Conditioned Place Preference Test

When measuring animals’ valenced behavioural responses to stimuli, the Conditioned Place Preference (CPP) test goes a step further than many approach-based and avoidance-based tests by establishing whether a learned preference for, or aversion to, the location in which the stimulus was encountered can be generated. We designed a novel, four-chambered CPP test to extend the capability of the usual CPP paradigm to provide information on four key features of animals’ affective responses: valence, scale, persistence and generalization. Using this test, we investigated the affective responses of domestic chickens (Gallus gallus domesticus) to four potentially aversive stimuli: 1. Puffs of air; 2. Sight of (robotic) snake; 3. Sprays of water; 4. Sound of conspecific alarm calls. We found conditioned avoidance of locations associated with the air puffs and water sprays (Friedman’s χ2(3) = 13.323 p > .005; χ2(3) = 14.235 p > .005), but not with the snake and alarm calls. The scale of the learned avoidance was similar for the air puff and water spray stimuli, but persistence and generalization differed. We conclude that the four chambered CPP test can have a valuable role to play in making multi-feature measurements of stimulus-generated affective responses, and we highlight the value of such measurements for improving our understanding of the structure of affect in chickens and other animals.

BFGF expression is differentially regulated by cocaine seeking versus extinction in learning-related brain regions.

In cocaine use disorder, relapse can be elicited by drug-associated cues despite long periods of abstinence. The persistence of drug-associated cues in eliciting drug seeking suggests enduring changes in structural and functional plasticity, which may be mediated by basic fibroblast growth factor (bFGF, FGF2). Stimulant drug use increases bFGF expression in reward- and learning-related brain regions, such as the infralimbic medial-prefrontal cortex (IL-mPFC), and we previously found that this increase was reversed by extinction. However, whether bFGF expression is similarly modified in other brain regions is unknown. Therefore, we used the conditioned place preference (CPP) paradigm to assess bFGF expression following cocaine-associated CPP or extinction of that CPP within the mPFC, nucleus accumbens (NAc), hippocampus (Hipp), and basolateral amygdala (BLA). bFGF expression was increased in IL-mPFC and NAc-Core and -Shell following a cocaine-associated CPP, an effect reversed by extinction. Conversely, bFGF expression was increased in BLA following extinction, but no significant changes were observed in PL-mPFC or either dorsal or ventral Hipp. These results demonstrate differential regulation of bFGF following cocaine-associated CPP or extinction of that CPP in discrete brain regions. Changes in bFGF expression may regulate long-lasting drug-induced plasticity that underlies persistent drug-associated memories, and therefore present potential prophylactic targets.

Effects of concurrent blockade of OX2 and CB1 receptors in the ventral tegmental area on nicotine-induced place preference in rats

In this study, the role of orexin-2 (OX2) and cannabinoid-1 (CB1) receptors and their potential interaction within the ventral tegmental area (VTA) on nicotine-induced place preference, was examined in male rats. A 5-day conditioned place preference (CPP) paradigm was used. Nicotine (0.5 mg/kg; s.c.) induced a significant CPP, without any effect on the locomotor activity during the testing phase. TCS-OX2-29 (0.4, 0.8 and 4 μg/rat), as a selective OX2 receptor antagonist and AM251 (0.2, 1 and 2 μg/rat), as a selective cannabinoid CB1 receptor antagonist, individually or simultaneously were microinjected bilaterally into the VTA. The results showed that administration of AM251 (1 and 2 μg/rat) or TCS-OX2-29 (0.4, 0.8 and 4 μg/rat) into the VTA, during the 3-day conditioning phase or testing day, could dose-dependently inhibit the development of nicotine-induced CPP, in the acquisition or expression, respectively. Concurrent administration of ineffective doses of TCS-OX2-29 and AM251 into the VTA could not affect conditioning scores. The findings of this study support the possible role of OX2 and CB1 receptors in the VTA, in the acquisition and the expression of nicotine-induced place preference. Furthermore, our data suggest that there is a possible interaction between the VTA orexinergic and cannabinoid systems in nicotine-induced place preference.

Activation of GABAergic Neurons in the Nucleus Accumbens Mediates the Expression of Cocaine-Associated Memory.

Cocaine-associated environmental cues elicit craving and relapse to cocaine use by recalling the rewarding memory of cocaine. However, the neuronal mechanisms underlying the expression of cocaine-associated memory are not fully understood. Here, we investigated the possible contribution of γ-aminobutyrate (GABA)ergic neurons in the nucleus accumbens (NAc), a key brain region associated with the rewarding and reinforcing effects of cocaine, to the expression of cocaine-associated memory using the conditioned place preference (CPP) paradigm combined with designer receptors exclusively activated by designer drugs (DREADD) technology. The inhibitory DREADD hM4Di was selectively expressed in NAc GABAergic neurons of vesicular GABA transporter-Cre (vGAT-Cre) mice by infusing adeno-associated virus (AAV) vectors. Ex vivo electrophysiological recordings revealed that bath application of clozapine-N-oxide (CNO) significantly hyperpolarized membrane potentials and reduced the number of spikes induced by depolarizing current injections in hM4Di-positive NAc neurons. Additionally, systemic CNO injections into cocaine-conditioned mice 30 min before posttest session significantly reduced CPP scores compared to saline-injected mice. These results indicate that chemogenetic inhibition of NAc GABAergic neurons attenuated the expression of cocaine CPP, suggesting that NAc GABAergic neuronal activation is required for the environmental context-induced expression of cocaine-associated memory.

Baseline prepulse inhibition of the startle reflex predicts the sensitivity to the conditioned rewarding effects of cocaine in male and female mice.

Prepulse inhibition (PPI) of the startle reflex is a model of pre-attentional inhibitory function. The dopamine baseline in the nucleus accumbens plays a key role in PPI regulation as well as in the rewarding effects of cocaine. The aim of this study was to evaluate the predictive ability of PPI to identify the more vulnerable mice of both sexes to the conditioned rewarding effects of cocaine. Male and female OF1 mice were first tested in the PPI paradigm to classify them as high or low PPI. Afterwards, they were evaluated in the conditioned place preference (CPP) paradigm induced by cocaine (1, 6 and 12mg/kg). Moreover, the D1R and D2R protein expressions in the striatum of high and low PPI animals were analysed by Western blot. Only high-PPI mice acquired CPP induced by low doses of cocaine (1 and 6mg/kg), while the low-PPI mice needed a higher dose of cocaine (12mg/kg) to acquire the CPP, but once mice were conditioned, males did not extinguish the conditioned preference and females reinstated the preference with lower doses of cocaine than their control counterparts. Low-PPI animals, especially females, showed higher basal levels of D2R than those with a higher PPI. Low-PPI mice presented a lower sensitivity to the conditioned rewarding effects of cocaine, but once they were conditioned with a higher dose, they displayed a stronger, perseverant conditioned preference. The predictive capacity of PPI to detect the more vulnerable mice to the conditioned effects of cocaine is discussed.

Role of orexin-1 and orexin-2 receptors in the CA1 region of hippocampus in the forced swim stress- and food deprivation-induced reinstatement of morphine seeking behaviors in rats

Hippocampus (HIP) is an essential brain site to study reward-related learning tasks, such as conditioning place preference (CPP) that can measure the preference for environmental stimuli related to reward. Furthermore, orexin neurons, situated exclusively in the lateral hypothalamus (LH) and link the rewarding effects of drugs of abuse in the LH and the CA1 region of the HIP. Therefore, in this study adult male rats were conditioned with morphine using a CPP paradigm. After the eighth day of the extinction period, on the reinstatement day, orexin-1 and orexin-2 receptor antagonists were administered bilaterally into the CA1 region prior to acute stress. Using two different types of acute stress, forced swim stress (FSS) and food deprivation (FD), the role of orexin-1 and orexin-2 receptors in the CA1 brain region in FSS and FD induced reinstatement was investigated. Our results showed that application of the orexin-1 and orexin-2 antagonists, SB334867 and TCSOX2 29, respectively, reduced the CPP scores in the reinstatement phase. Moreover, it can be concluded that orexin neurons are activated in acute stress states, such as FSS and FD, as blocking the orexin receptors, decreased the effects of acute stress in triggering the reinstatement of morphine-CPP.