Olanzapine Attenuates the Morphine‐Induced Conditioned Place Preference: The Involvement of the D2‐Like Dopamine Receptors

TL;DR

Olanzapine reduces morphine-induced drug-seeking behavior in mice by affecting dopamine receptors in the brain.

Contribution

Olanzapine's effect on morphine dependence via D2-like dopamine receptors is demonstrated in a CPP model.

Findings

Olanzapine reduced morphine-induced conditioned place preference in a dose-dependent manner.

Olanzapine decreased D2R protein expression in the hippocampus during expression and extinction phases.

Olanzapine accelerated the extinction of morphine-induced CPP without impairing locomotor activity.

Abstract

Opioid use disorder is associated with persistent molecular and cellular changes in the brain, leading to compulsive drug‐seeking behaviors. This study aimed to evaluate the effects of olanzapine (OLZ), a D2‐like dopamine receptor (D2R) antagonist, on morphine dependence using the conditioned place preference (CPP) model.

Morphine‐induced CPP was established by subcutaneous (sc) administration of morphine (5 mg/kg) for three consecutive days. OLZ at doses of 1.5, 3, and 4.5 mg/kg was administered intraperitoneally (ip) during different phases of CPP: acquisition, expression, and extinction. Behavioral assessments included measurement of conditioning scores and locomotor activity. D2R protein expression in the hippocampus (HIP) was evaluated using western blotting.

OLZ reduced both acquisition and expression of morphine‐induced CPP in a dose‐dependent manner. Additionally, OLZ facilitated extinction by shortening the duration of the extinction phase. Western blot analysis showed a significant reduction in D2R protein expression in the expression and extinction phases in the OLZ‐treated groups compared to the morphine‐only group. Importantly, effective doses of OLZ did not impair locomotor activity.

OLZ attenuates morphine‐induced CPP and downregulates D2R expression in the HIP without affecting general locomotor function. These findings suggest a potential therapeutic role for OLZ in the management of opioid‐related behaviors. Further research is needed to determine its clinical relevance.

The results of this study demonstrated that olanzapine can inhibit morphine‐induced CPP behavior during the acquisition, expression, and extinction phases. These effects are likely mediated by the inhibition of dopamine receptors and a reduction in D2R protein expression in the HIP. Additionally, the use of olanzapine during the extinction phase accelerated the extinction process of CPP. These findings suggest that olanzapine may hold therapeutic potential in managing psychological dependence on drugs through D2R‐dependent pathways.

Figures

Experimental protocols for assessing morphine‐induced CPP in rats. (A) On the pre‐test day (Day 1), the time each rat spent in both compartments was recorded. Only animals showing no initial preference for either side were included in the study. Following three consecutive days of morphine administration (5 mg/kg, sc), the conditioning and post‐test session were conducted on Day 5. The CPP score was defined as the difference between the time spent in the drug‐paired compartment and the saline‐paired compartment. The extinction phase began on Day 6, during which animals were exposed to the CPP apparatus for 10 min daily. Testing continued until the CPP score declined by 50% compared to the pre‐conditioning value. (B) To assess the effect of OLZ on morphine‐induced CPP acquisition, treatment groups received OLZ at doses of 1.5, 3, or 4.5 mg/kg via ip injection, once daily, 30 min before the sc morphine administration during the 3‐day conditioning phase. (C) In a separate experiment, to evaluate the effect of OLZ on CPP expression, animals were administered OLZ at the same doses (1.5, 3, and 4.5 mg/kg, ip) 30 min before the post‐conditioning test session in the CPP apparatus. (D) To investigate the effect of OLZ on CPP extinction, two doses of OLZ (1 and 1.5 mg/kg) were administered ip 30 min before daily CPP testing, beginning the day after the post‐conditioning test. This procedure was repeated each day during the extinction phase until the CPP scores stabilized. The extinction latency for each rat was defined as the number of days required to reduce the CPP score by 50% compared to the post‐conditioning level.

The effects of various doses of morphine on CPP to determine the effective dose of morphine. p < 0.01, and * p < 0.001 different from the control group. Mor: morphine.

The effects of saline, vehicle, OLZ alone (3 mg/kg, ip), and different doses of OLZ (1.5, 3, and 4.5 mg/kg, ip) administered 30 min before morphine (5 mg/kg, sc) during the acquisition phase (A). The effects of different doses of OLZ (1.5, 3, or 4.5 mg/kg) on locomotor activity during the acquisition phase (B). *p < 0.05 and p < 0.01 as compared with the saline‐control group. +++p < 0.001 as compared with the morphine group.

The effects of saline, vehicle, OLZ alone (3 mg/kg, ip), and different doses of OLZ (1.5, 3, and 4.5 mg/kg, ip) administered 30 min before morphine (5 mg/kg, sc) during the expression phase (A). The effects of different doses of OLZ (1.5, 3, or 4.5 mg/kg) on locomotor activity during the expression phase (B). p < 0.01, and * p < 0.001 as compared with the saline‐control group. +++p < 0.001 as compared with the morphine group.

The effects of vehicle and different doses of OLZ (1 and 1.5 mg/kg, ip), during the extinction phase (A). The effects of different doses of OLZ (1 and 1.5 mg/kg, ip) on locomotor activity during the extinction phase (B). p < 0.01 as compared with the vehicle‐control group.

The effect of OLZ co‐administration with morphine on D2R protein expression in the HIP during different CPP phases. The western blot images (A) and corresponding densitometric analysis (B) show D2R protein levels in rats treated with morphine alone and those receiving morphine plus the most effective OLZ doses during the acquisition (3 mg/kg), expression (4.5 mg/kg), and extinction (1.5 mg/kg) phases. All values are expressed as mean ± SD (n = 4). Data were analyzed using one‐way ANOVA followed by Tukey's multiple comparisons test. Comparison with the morphine group revealed a significant reduction in D2R protein expression during the expression phase (p < 0.01) and the extinction phase (*p < 0.001).

Full text

Introduction

1

Opioid dependence is considered a significant challenge in public health. Due to complex biological and social factors, this dependence can lead to persistent cravings and relapse into substance abuse, even after prolonged periods of abstinence (Luo et al. 2011). The treatment and control of opioid dependence is one of the most complex and fundamental challenges in the public health system (Moeini et al. 2019). One of the common approaches to treating morphine dependence is the use of oral full opioid agonists like methadone. Methadone is beneficial in controlling craving; however, it may lead to dependence and be linked to various adverse effects, such as withdrawal symptoms, intense drug cravings, and cognitive dysfunction (Javdan et al. 2019). The role of the dopaminergic system is crucial in both the development of opioid dependence and the relapse to substance use (Te 1993). Dopamine plays an important role in essential functions such as emotion, learning, decision‐making, opioid dependence, and reward (Schultz 2015). The mesocorticolimbic pathway, linking the ventral tegmental area (VTA) to the nucleus accumbens and prefrontal cortex, is central to opioid dependence through its role in reward, learning, and memory. Chronic opioid use alters this system, promoting craving and relapse. The mesolimbic pathway, including VTA‐hippocampus (HIP) connections, also contributes to processing reward cues, with the HIP essential for encoding reward‐based learning and preferences (Kutlu and Gould 2016). The HIP is critical for learning and memory and is highly responsive to drug‐induced synaptic changes, especially in regions like the dentate gyrus (DG), CA1, and CA3. Disruption in hippocampal function, particularly through dopaminergic signaling from the VTA to the DG, impairs drug‐associated learning and may contribute to addiction and relapse (Avchalumov and Mandyam 2021).

The conditioned place preference (CPP) paradigm serves as a research tool to investigate substance dependence and its impact on the brain's reward pathways. The CPP test is widely used to evaluate the rewarding effects of substances and the reward memory associated with them (Sun et al. 2017). Previous studies have shown that the injection of D1/D2‐like dopamine receptor antagonists into various regions of the HIP, including CA1 and DG, can inhibit both the CPP learning induced by morphine and its reinstatement (Assar et al. 2016; Khakpour‐Taleghani et al. 2015).

Olanzapine (OLZ), a second‐generation antipsychotic, received approval from the US Food and Drug Administration (FDA) in 1996 for treating schizophrenia and has been offered as a generic medication since 2011 (Meftah et al. 2020). OLZ blocks acetylcholine, dopamine, serotonin, and histamine receptors, and specifically exerts an inhibitory effect on dopamine receptor subtypes (D2/D4) (Torigoe et al. 2012; Go et al. 2018). It has been suggested that OLZ's efficacy in reducing drug cravings is due to its rapid dissociation from the D2‐like dopamine receptor (D2R), a characteristic that may help mitigate dysphoria and dopamine hypersensitivity during withdrawal (Potvin et al. 2003; Akerele and Levin 2007). Research has shown that OLZ can prevent the development of CPP induced by various substances, such as amphetamines (Mechanic et al. 2003) and cocaine (Meil and Schechter 1997; Kosten and Nestler 1994) in rats and reduce addictive behaviors related to opioids (Torigoe et al. 2012). Additionally, OLZ can reduce alcohol cravings and intake in humans (Guardia et al. 2004).

Considering the involvement of the dopaminergic system in regulating reward‐related behaviors and cravings, this animal study aimed to investigate the effects of OLZ on morphine‐CPP and the potential role of D2R. While previous studies, such as Torigoe et al. (2012), have explored OLZ's effects on morphine‐induced reward, the present study provides a comprehensive evaluation of dose‐dependent effects across all CPP phases (acquisition, expression, and extinction) combined with the assessment of D2R expression in the hippocampus. This approach allows for a more detailed understanding of OLZ's modulatory effects on morphine‐induced behaviors and their underlying neural mechanisms.

Materials and Methods

2

Animal

2.1

One hundred and sixty adult male albino Wistar rats (weight: 220–250 g; Basic Science Research Center, Kashan University of Medical Sciences) were kept under standard conditions in a 12‐h light‐dark cycle (lights on from 7:30 a.m. to 7:30 p.m.). Each cage housed two rats in a room with a regulated temperature (25 ± 1°C) and humidity (38 ± 2%). In addition, food and water were provided without restriction. The animals were randomly distributed into various test groups. All experiments were conducted in accordance with the guidelines of Kashan University of Medical Sciences for the care and use of laboratory animals (IR.KAUMS.AEC.1401.004).

Drugs

2.2

Morphine sulfate was provided by the FDA, Tehran, Iran. A preliminary dose–response study was conducted using 2.5, 5, 7.5, and 10 mg/kg subcutaneously (sc) to determine the optimal dose for inducing psychological dependence in CPP. Based on these results and previous reports, 5 mg/kg was selected for the main experiments (Nazari‐Serenjeh et al. 2021). OLZ was provided in powder form by Pharma Chemie. To prepare the solution, the powder was mixed with 2.5–3 drops of DMSO, followed by the addition of 5 mL of normal saline solution and several drops of Tween 80 to create a suspension for intraperitoneal (ip) administration. OLZ doses were selected based on a previous study (Meil and Schechter 1997).

CPP Apparatus

2.3

The device consists of three compartments: A, B, and C. Sections A and B are of the same size, each measuring 30 × 30 × 40 cm, and are connected to section C by guillotine doors. Section A features walls with black and white vertical stripes and a flat floor, while Section B has walls with a white background and black and white horizontal stripes. Section C, located between the other two compartments, measures 40 × 15 × 30 cm and has walls painted black (Modaberi et al. 2023). A camera (Microsoft LifeCam HD‐3000, China) is positioned above the device and starts recording as soon as the rat enters a compartment. It records the duration spent in each compartment and the number of entries, allowing detailed observation of the rat's behavior (Figure 1A).

Experimental protocols for assessing morphine‐induced CPP in rats. (A) On the pre‐test day (Day 1), the time each rat spent in both compartments was recorded. Only animals showing no initial preference for either side were included in the study. Following three consecutive days of morphine administration (5 mg/kg, sc), the conditioning and post‐test session were conducted on Day 5. The CPP score was defined as the difference between the time spent in the drug‐paired compartment and the saline‐paired compartment. The extinction phase began on Day 6, during which animals were exposed to the CPP apparatus for 10 min daily. Testing continued until the CPP score declined by 50% compared to the pre‐conditioning value. (B) To assess the effect of OLZ on morphine‐induced CPP acquisition, treatment groups received OLZ at doses of 1.5, 3, or 4.5 mg/kg via ip injection, once daily, 30 min before the sc morphine administration during the 3‐day conditioning phase. (C) In a separate experiment, to evaluate the effect of OLZ on CPP expression, animals were administered OLZ at the same doses (1.5, 3, and 4.5 mg/kg, ip) 30 min before the post‐conditioning test session in the CPP apparatus. (D) To investigate the effect of OLZ on CPP extinction, two doses of OLZ (1 and 1.5 mg/kg) were administered ip 30 min before daily CPP testing, beginning the day after the post‐conditioning test. This procedure was repeated each day during the extinction phase until the CPP scores stabilized. The extinction latency for each rat was defined as the number of days required to reduce the CPP score by 50% compared to the post‐conditioning level.

Pre‐conditioning (Familiarization) Phase

2.4

On Day 1, rats were allowed to freely explore all compartments of the CPP apparatus for 10 min, and the time spent in each compartment was recorded automatically. Rats showing strong initial bias (>70% time in one compartment) were excluded (Katebi et al. 2018). At this stage, seven rats were excluded based on this criterion.

Conditioning (Acquisition) Phase

2.5

From Day 2 to 4, animals received alternating sc injections of morphine (5 mg/kg) and saline, being confined to the corresponding compartment for 30 min after each injection. The schedule was reversed daily to control for time‐of‐day effects. During this phase, OLZ (1.5, 3, or 4.5 mg/kg, ip) was administered 30 min before morphine injection to assess its effects on CPP acquisition (Figure 1B). Vehicle‐control groups received the drug solvent.

Post‐conditioning (Expression) Phase

2.6

On Day 5, animals were allowed to freely explore the apparatus for 10 min, during which preference scores were recorded to assess morphine‐induced place preference. To evaluate the effects of OLZ on CPP expression, treated groups received a single dose of the drug (1.5, 3, or 4.5 mg/kg, ip) 30 min before testing (Figure 1C).

Extinction Phase

2.7

One day after the post‐conditioning test, OLZ was administered ip at doses of 1 and 1.5 mg/kg, 30 min before the CPP test, followed by assessment of the preference score. This daily procedure continued throughout the extinction phase until complete extinction was achieved (Figure 1D). In this study, “extinction latency” was defined as the number of days required for each animal's CPP score to decrease to half of the value observed on the post‐conditioning day. It is worth noting that due to the daily administration of OLZ and its cumulative effects in the animals, higher doses (3 and 4.5 mg/kg) caused reduced locomotor activity; therefore, lower doses of 1 and 1.5 mg/kg were used during this phase.

Locomotor Activity Measurement

2.8

Locomotor activity can significantly influence CPP outcomes. Therefore, the effects of interventions on locomotion were assessed by measuring and comparing the total distance traveled on Day 5 between experimental and control (vehicle‐treated) groups, as recorded by a Microsoft LifeCam HD‐3000 (China) camera connected to a video tracking system (Noldus Information Technology, Netherlands) and analyzed with EthoVision software (version 7).

Molecular Studies

2.9

To investigate the molecular mechanisms underlying OLZ's effects, hippocampal samples were selectively collected from treatment groups demonstrating the most significant behavioral responses in each experimental phase. Accordingly, acquisition phase samples were obtained from animals treated with 3 mg/kg OLZ, expression phase samples from the 4.5 mg/kg treatment group, and extinction phase samples from animals receiving 1.5 mg/kg OLZ. All samples were collected immediately following the final behavioral test in each respective phase and compared with matched vehicle‐control groups from the same experimental phase.

After completing the experimental sessions and behavioral studies, the rats were euthanized with carbon dioxide under deep anesthesia, and their heads were beheaded with a guillotine. Both the right and left hippocampi were separated and placed in tubes. These microtubes were then dipped in liquid nitrogen and stored in a deep freezer at −80°C. The tissues were kept at −80°C until use and were gradually subjected to molecular tests.

To extract proteins, 100 µL of Pro‐Pre cell lysis solution was added to the cells, and the cells were lysed on ice using a homogenizer. The cell suspension was then incubated at −20°C for 20 min. After centrifuging for 15 min at 4°C and 13,000 rpm, the supernatant was collected. The protein concentration was measured using the Bicinchoninic Acid (BCA) method. Western blotting was performed in two stages using a vertical system with 10 × 10 cm gel units and a Consort EV202 power supply. Electrophoresis was conducted with a 10% separating gel and a 5% stacking gel, prepared with bisacrylamide, Tris base, SDS, ammonium persulfate, and TEMED. After solidification, the gels were placed in an electrophoresis container containing 1.2 L of electrophoresis solution (25 mM Tris base, 190 mM glycine, 0.1% SDS, pH 8). Protein samples (50 µg) were boiled in 2× Laemmli buffer for 5 min and loaded into the gel using a Hamilton syringe. A pre‐colored protein marker was included, and proteins were first tested at 80 V, followed by 180 V. After electrophoresis, the proteins were transferred from the gel to 0.2 µm PVDF membranes (Bio‐Rad). The gel and membrane were stacked in a sandwich format with sponges and filter papers, placed in a transfer tank with a buffer of pH 8.3, and incubated at 300 mA for 75 min. After transfer, the gel was stained with Ponceau‐S solution to check transfer quality and then decolorized. The PVDF membrane was blocked with TBST and BSA or fat‐free dry milk, followed by incubation with primary and secondary antibodies. The bands were detected using ECL solution and x‐ray film, then analyzed using Gel Analyzer software to calculate the relative intensities compared to the beta‐actin control (Babaei et al. 2018; Siavashi et al. 2016; Jabarpour et al. 2018).

Statistical Analysis

2.10

The results are expressed as mean ± standard error of the mean (SEM). Data analysis was conducted using GraphPad Prism software (version 8.0). To compare CPP scores and distances traveled among the experimental groups, a one‐way analysis of variance (ANOVA) with block randomization was performed, followed by post‐hoc analysis using the Newman–Keuls test. A p‐value less than 0.05 was considered statistically significant.

Results

3

CPP Induction by Morphine

3.1

Data analysis revealed a dose–response effect of morphine during the conditioning phase (Figure 2). Among the tested doses (2.5, 5, 7.5, and 10 mg/kg), 5 mg/kg morphine produced the greatest increase in time spent by rats in the morphine‐associated compartment compared to the saline compartment (*** p < 0.001), while 7.5 and 10 mg/kg also induced a significant CPP (** p < 0.01). Based on these results, 5 mg/kg was selected for all subsequent experiments. Effect size (*η^2^ *) has been added to the morphine dose–response ANOVA analysis (η ^2^ = 0.59). Additionally, 95% confidence intervals (CI) saline control: [10.56, 59.77]; morphine 2.5 mg/kg: [26.34, 144.1]; morphine 5 mg/kg: [96.09, 167.6]; morphine 7.5 mg/kg: [91.87, 133.3]; morphine 10 mg/kg: [79.31, 140.0].

*The effects of various doses of morphine on CPP to determine the effective dose of morphine. *p < 0.01, and *** p < 0.001 different from the control group. Mor: morphine.

Olanzapine's Effects on the Acquisition of CPP

3.2

In this series of experiments, the effect of various doses of OLZ (1.5, 3, and 4.5 mg/kg), which acts as a D2R antagonist, on morphine‐induced CPP after ip administration was investigated. Analysis using one‐way ANOVA and the Newman–Keuls multiple comparison test (p < 0.001; n = 6–7; Figure 3A) showed that the ip injection of all three doses of OLZ (1.5, 3, and 4.5 mg/kg) administered 30 min before morphine injection (5 mg/kg, sc) significantly reduced morphine‐induced CPP compared to the vehicle groups. In contrast, the results of the one‐way ANOVA followed by the Newman–Keuls multiple comparison test (p = 0.0531; Figure 3B) showed that the OLZ at the specified dose had no significant effect on the distance traveled during the 10‐min test period (the day after the test) compared to the vehicle‐control groups. The acquisition phase showed a significant effect (F(6,38) = 75.91, p < 0.0001) with a very large effect size (η ^2^ = 0.923). Data are reported as 95% CI: saline [21.35, 57.22], vehicle + morphine [103.6, 160.1], saline + morphine 5 mg/kg [98.15, 159.5], Olanzapine + saline [4.517, 39.48], morphine + Olanzapine 1.5 mg/kg [−216.2, −133.5], morphine + Olanzapine 3 mg/kg [−236.0, −92.02], and morphine + Olanzapine 4.5 mg/kg [−113.0, −52.33].

*The effects of saline, vehicle, OLZ alone (3 mg/kg, ip), and different doses of OLZ (1.5, 3, and 4.5 mg/kg, ip) administered 30 min before morphine (5 mg/kg, sc) during the acquisition phase (A). The effects of different doses of OLZ (1.5, 3, or 4.5 mg/kg) on locomotor activity during the acquisition phase (B). *p < 0.05 and *p < 0.01 as compared with the saline‐control group. +++p < 0.001 as compared with the morphine group.

Olanzapine's Effects on the Expression of CPP

3.3

The day after conditioning, the animals received OLZ as a D2R antagonist via ip injection to investigate the role of this antagonist in the expression of morphine‐induced CPP. The results of the one‐way ANOVA (p < 0.001, n = 6–7; Figure 4A), followed by the Newman–Keuls multiple comparative test, showed that the higher OLZ dose (4.5 mg/kg) markedly reduced the conditioned values compared to the groups of vehicles that received saline solution instead of OLZ on the test day. OLZ at the specified dose had no significant effect on the distance traveled during the 10‐min test period (the day after the test) compared to the vehicle‐control groups (Figure 4B). OLZ significantly reduced morphine‐induced CPP in a dose‐dependent manner during the expression phase (F(6,39) = 78.51, p < 0.0001, η ^2^ = 0.92).  Data are presented as 95% CI: saline, [21.35, 57.22]; vehicle + morphine, [104.8, 211.5]; saline + morphine, [131.4, 219.2]; Olanzapine + saline, [4.517, 39.48]; morphine + Olanzapine 1.5 mg/kg, [99.96, 138.4]; morphine + Olanzapine 3 mg/kg, [−91.80, −18.20]; morphine + Olanzapine 4.5 mg/kg, [−355.6, −201.5].

*The effects of saline, vehicle, OLZ alone (3 mg/kg, ip), and different doses of OLZ (1.5, 3, and 4.5 mg/kg, ip) administered 30 min before morphine (5 mg/kg, sc) during the expression phase (A). The effects of different doses of OLZ (1.5, 3, or 4.5 mg/kg) on locomotor activity during the expression phase (B). *p < 0.01, and *** p < 0.001 as compared with the saline‐control group. +++p < 0.001 as compared with the morphine group.

Olanzapine's Effects on the Extinction of CPP

3.4

To investigate the effect of OLZ on the extinction latency of morphine‐induced CPP starting from the day after the conditioning phase, various doses of the drug were administered daily via ip injection. Based on the results of one‐way ANOVA followed by the Newman–Keuls multiple comparison test (p = 0.0034, n = 6–7; Figure 5A), daily administration of 1.5 mg/kg OLZ during the extinction phase significantly reduced the mean extinction latency. However, the 1 mg/kg dose did not affect the mean extinction latency. Additionally, higher doses (3 and 4.5 mg/kg) decreased the animals’ locomotor activity but did not affect the mean extinction latency. According to the data shown in Figure 5B, neither dose of OLZ (1 and 1.5 mg/kg) produced a significant difference in the mean distance traveled (cm/day) compared to the vehicle‐control group. For the extinction phase, we have reported the 95% confidence intervals for all groups (Vehicle: [5.438, 7.991]; Dose 1: [4.395, 6.748]; Dose 1.5: [0.9240, 5.409]) alongside the effect size (*η^2^

• = 0.487)

*The effects of vehicle and different doses of OLZ (1 and 1.5 mg/kg, ip), during the extinction phase (A). The effects of different doses of OLZ (1 and 1.5 mg/kg, ip) on locomotor activity during the extinction phase (B). *p < 0.01 as compared with the vehicle‐control group.

Olanzapine's Effects on D2R Protein Expression in HIP

3.5

We investigated the expression status of the D2R using a specific anti‐D2 receptor antibody and western blot analysis. The comparison with the morphine group revealed a significant reduction in D2R protein expression during the expression phase (p < 0.0080) and the extinction phase (p < 0.0004). However, in the acquisition phase, no significant difference in the expression of dopamine D2 protein was observed compared to the morphine group with the effective dose of 5 mg/kg (Figure 6B). Due to financial constraints associated with performing western blot experiments, we focused on treatment groups showing the most pronounced behavioral effects.

*The effect of OLZ co‐administration with morphine on D2R protein expression in the HIP during different CPP phases. The western blot images (A) and corresponding densitometric analysis (B) show D2R protein levels in rats treated with morphine alone and those receiving morphine plus the most effective OLZ doses during the acquisition (3 mg/kg), expression (4.5 mg/kg), and extinction (1.5 mg/kg) phases. All values are expressed as mean ± SD (n = 4). Data were analyzed using one‐way ANOVA followed by Tukey's multiple comparisons test. Comparison with the morphine group revealed a significant reduction in D2R protein expression during the expression phase (**p < 0.01) and the extinction phase (**p < 0.001).

Western blot (hippocampal D2R): OLZ had no significant effect on D2R in the acquisition phase (effect size = 0.018; 95% CI: −0.22 to 0.26), but significantly reduced D2R levels in the expression (effect size = 0.322; 95% CI: 0.084–0.561) and extinction phases (effect size = 0.465; 95% CI: 0.227–0.703), showing a phase‐dependent effect.

Discussion

4

This study investigated the effects of OLZ on the acquisition, expression, and extinction phases of morphine‐induced CPP. The key findings are as follows: (1) 5 mg/kg morphine was identified as the optimal dose to induce robust CPP; (2) OLZ effectively suppressed morphine‐induced CPP, likely via modulation of HIP D2R expression; and (3) OLZ administration during the extinction phase accelerated CPP extinction.

Notably, during the acquisition phase, the behavioral and molecular effects of OLZ were dissociated. While OLZ effectively attenuated morphine‐induced CPP behaviorally, it did not affect hippocampal D2R expression. In contrast, during the expression and extinction phases, OLZ reduced both morphine‐induced CPP and hippocampal D2R expression. Our molecular results support the possible link between the behavioral efficacy of OLZ and a phase‐specific modulation of hippocampal D2R expression. During the expression phase, the 4.5 mg/kg dose of OLZ significantly reduced both CPP scores and D2R levels without affecting locomotor activity. In the extinction phase, the 1.5 mg/kg dose specifically facilitated extinction without motor impairment, whereas higher doses (3 and 4.5 mg/kg) reduced locomotion, likely due to sedative effects, without further enhancing extinction. This dose‐dependent profile indicates that the pro‐extinction effect is separable from OLZ's sedative properties.

The facilitated extinction of conditioned behaviors, primarily through D2 receptor modulation during extinction and expression phases, underscores the critical role of dopaminergic signaling in extinction learning (Zbukvic et al. 2016). This process is highly significant because extinction involves the formation of new inhibitory memories while preserving the original drug‐related associations, leaving individuals vulnerable to relapse upon re‐exposure to drug cues. Therefore, interventions capable of modulating the neuroplastic changes induced by chronic drug use are essential for relapse prevention. Pharmacological agents that mitigate these neural adaptations may effectively reduce drug craving and susceptibility to relapse (Hashemizadeh et al. 2024). This is particularly relevant given that most addictive drugs, including morphine, ultimately enhance dopamine activity within reward pathways through diverse mechanisms (Cousins et al. 2002). Morphine binds to μ‐opioid receptors on inhibitory GABAergic neurons in the VTA, leading to neuronal inhibition via multiple mechanisms, including a reduction in cyclic AMP signaling, hyperpolarization mediated by G protein‐gated inwardly rectifying potassium (GIRK) channels, and suppression of presynaptic calcium channel function. This disinhibition of dopaminergic neurons increases dopamine release in the nucleus accumbens, contributing to the reinforcing effects of morphine (Reeves et al. 2022; Listos et al. 2019).

Studies have shown that the HIP, beyond forming drug‐related memories, plays a role in drug‐seeking behaviors and environmental influences. Additionally, the HIP is critical in the acquisition and expression of morphine‐, cocaine‐, and nicotine‐induced CPP (Kramar et al. 2014; Luo et al. 2011). Morphine‐induced CPP is modulated by several neurotransmitter systems, including dopaminergic, adrenergic, muscarinic, and serotonergic systems (Arani et al. 2023). Recent findings highlight the significant role of D2R in enhancing extracellular dopamine levels and initiating and expressing morphine‐induced CPP (Rougé‐Pont et al. 2002). In fact, inhibiting the D2 receptor can significantly reduce the intensity of morphine‐induced CPP (Katebi et al. 2018). Administration of sulpiride, a D2R antagonist, provides valuable evidence for this hypothesis by inhibiting the effect of morphine on reward enhancement and the formation of a CPP (Haghparast et al. 2013). Also, previous research has shown that administration of D1 and/or D2‐like dopamine receptor antagonists in the CA1 region of the HIP prevents morphine‐induced CPP learning (Esmaeili et al. 2012; Assar et al. 2016). The antagonistic effect of OLZ in the induction of morphine‐CPP is consistent with previous results of other atypical antipsychotics, such as risperidone and quetiapine (Arani et al. 2023; Khezri et al. 2022). The effect of OLZ on CPP caused by various substances has been investigated.

The results of Jordan E. A study showed that OLZ (1.5 mg/kg) could prevent the development of a CPP induced by amphetamine (Mechanic et al. 2003). Treated animals at doses of 3.0 or 4.5 mg/kg OLZ showed lower motor activity, and a reduction in cocaine‐induced activity was observed at doses of 4.5 mg/kg. The effects of OLZ in reducing cocaine‐induced hyperactivity and spontaneous motor activity are consistent with the results of a study by Arnett (1995). The effect of OLZ on substance abuse has been studied in patients with dual diagnosis, such as bipolar disorder or schizophrenia with substance use disorder (Harder et al. 2019). It has been suggested that OLZ's ability to reduce cravings may be due to its rapid dissociation from the D2R, which could help reduce the risk of dysphoria and hypersensitivity to dopamine following drug withdrawal (Akerele and Levin 2007; Potvin et al. 2003). According to a study conducted by M. Zhou, administration of OLZ to patients with schizophrenia and opioid use disorder (OUD) can significantly increase the likelihood of recovery from OUD, with these patients having a higher chance of recovery than people who did not receive OLZ treatment (Zhou et al. 2021). Studies have shown that OLZ affects 7 of 19 opioid‐associated genes use disorder, including BDNF, CYP2D6, DRD2, DRD4, HTR1B, POMC, and SLC6A4 (Al‐Hasani and Bruchas 2011; Koob and Volkow 2016). A study showed that OLZ at a dose of 0.3 mg/kg, which had only a minimal effect on motor coordination, effectively suppressed the hyperlocomotion and rewarding effects of morphine (Torigoe et al. 2012). However, the precise role of hippocampal D2R in this effect remained unclear. Therefore, our study aimed to build upon these findings by specifically investigating the involvement of D2R‐mediated signaling in the HIP across all phases of morphine‐induced CPP. Research has shown that muscarinic M1 receptors have a role in improving dopaminergic effects induced by opioids and associated with increased drug dependence (Tanda et al. 2007). Given OLZ's high affinity for muscarinic M1 receptors, it was hypothesized that this drug could increase the potential for opioid abuse. Data have shown that M1 antagonists can increase dopamine release in the nucleus accumbens. In contrast to previous studies, however, OLZ was not only unable to increase hyperlocomotion or morphine‐induced place preference, but its effect on various receptors, such as serotonin, histamine, and dopamine, could also explain the suppressive effects of the drug (Torigoe et al. 2012). Based on these pathophysiological mechanisms, OLZ, as a dopamine receptor antagonist, may effectively manage psychological dependence (Murphy et al. 2017). In addition, this effect may reduce the risk of extrapyramidal symptoms (which make patients more likely to accept and stick to treatment) (Bédard et al. 2013). It may also improve mood and cognitive function, possibly by blocking various 5‐HT and adrenergic receptors (Akerele and Levin 2007). An unblinded comparison has shown that OLZ may be as effective as clonidine in treating heroin withdrawal symptoms (Cha et al. 2013). In a randomized trial, 10 mg of OLZ treated opioid withdrawal symptoms more effectively than clonidine. In addition, in a prospective study, OLZ reduced the frequency of discontinuation of opioid maintenance therapy, which was associated with a reduction in craving (Torigoe et al. 2012).

Conclusion

5

In summary, OLZ attenuated morphine‐induced CPP across acquisition, expression, and extinction phases, likely via dopamine receptor antagonism and reduced hippocampal D2R expression. Notably, OLZ accelerated CPP extinction, suggesting that its inhibitory effects on morphine reward may involve modulation of D2 receptor‐dependent signaling in the hippocampus. These findings provide insights into potential neural mechanisms for future interventions in opioid‐related disorders.

Author Contributions

Farkhondeh Rzazzaghi‐Firozjaei: methodology, software, data curation, investigation, writing – original draft, formal analysis. Amineh Sadat Zahiri‐Pour: methodology, software, data curation, investigation, writing – original draft. Gholamreza Ghavipanjeh: methodology, data curation, writing – original draft. Amir Ghaderi: methodology, software, data curation, writing – original draft. Abbas Haghparast: methodology, writing – original draft. Abolfazl Ardjmand: methodology, writing – original draft. Hamid Reza Banafshe: conceptualization, methodology, software, data curation; supervision, project administration, funding acquisition, investigation, validation, writing – original draft.

Funding

This work was supported by the Vice‐Chancellor for Research and Technology, Kashan University of Medical Sciences, Isfahan, Iran (Grant Number: 40178, 2022). The Vice‐Chancellor for Research and Technology had no role in the design of the study, collection, analysis, and interpretation of data, writing of the report, or the decision to submit the article for publication.

Conflicts of Interest

The authors declare no conflicts of interest.