Extinction and reinstatement of methamphetamine‐induced conditioned place preference in zebrafish

Conditioned place preference (CPP) paradigm in zebrafish has been used to measure drug reward, but there is limited research on CPP reinstatement to determine relapse vulnerability. The present study aimed to investigate extinction and reinstatement of methamphetamine (MA)‐induced CPP in zebrafish and evaluate the model's predictive validity. Zebrafish received different doses of MA (0–60 mg/kg) during CPP training. The preferred dose of MA at 40 mg/kg was used for extinction via either confined or nonconfined procedures. The extinguished CPP was reinstated by administering a priming dose of MA (20 mg/kg) or various stressors. To assess persistent susceptibility to reinstatement, MA CPP and reinstatement were retested following 14 days of abstinence. In addition, the effects of SCH23390, naltrexone, and clonidine on MA CPP during acquisition, expression, or reinstatement phases were monitored. MA induced CPP in a dose‐dependent manner. Both nonconfined and confined extinction procedures time‐dependently reduced the time spent on the MA‐paired side. A priming dose of MA, chasing stress, or yohimbine reinstated the extinguished CPP. After 14 days of abstinence, the MA CPP remained extinguished and was significantly reinstated by MA priming or chasing stress. Similar to the observations in rodents, SCH23390 suppressed the acquisition of MA CPP, naltrexone reduced the expression and MA priming‐induced reinstatement, while clonidine prevented stress‐induced reinstatement of MA CPP. This work expanded the zebrafish CPP paradigm to include extinction and reinstatement phases, demonstrating predictive validity and highlighting its potential as a valuable tool for exploring drug relapse.

environmental cues.If animals show increased preference for the drug-paired environment, it indicates positive-reinforcing properties of the drug. 1 Addiction is a chronic and relapsing disorder, with over half of addicted individuals experiencing relapse even after extended periods of withdrawal.Thus, animal models for relapse are important in addiction research.Because CPP memory is liable to extinction and reinstatement, the reinstatement of drug-seeking behaviour in CPP is also a commonly used model of craving that mimics the relapse stage of addiction in humans.The ability to reinstate CPP is indicative of persistent drug-associated memories that contribute to the propensity to elicit drug cravings. 2fore CPP reinstatement, animals undergo extinction training, either by nonconfined extinction procedure with repeated testing of the animals or by confined extinction procedure, pairing the vehicle with the previously drug-paired compartment of the apparatus for several sessions, until loss of preference for the drugpaired compartment. 3Reinstatement of CPP can be robustly reinstated by a priming dose of drug or by different stressors, such as footshock, immobilization, social defeat, and forced swim stress in rodents. 4,5mpared with rodents, zebrafish (Danio rerio) husbandry and behavioural research facilities are relatively simple and economical.
Zebrafish exhibit a high degree of genetic homology and conservation of neurotransmitter and neuroendocrine systems with humans.Additionally, zebrafish are sensitive to all major classes of psychotropic drugs, which highlight the utility of zebrafish models in drug addiction studies. 6The current available genetic tools and tracking techniques further offer the zebrafish an excellent organism for a deeper understanding of the genes implicated in drug addiction and large-scale drug screenings.
CPP has been adopted in zebrafish to study drug reward.][9][10] However, studies on the extinction and reinstatement of CPP in zebrafish are limited.
MA increases the release of monoamine neurotransmitters such as serotonin, dopamine, and norepinephrine. 11As a result, MA use triggers elevated mood, alertness, increase concentration, energy, decreased appetite, and causes weight loss and increased sexual libido.MA is a highly addictive stimulant with potent reinforcing effects and a high propensity to relapse.However, there are currently no Food and Drug Administration-approved pharmacotherapies for MA use disorder, highlighting the urgent need to search for more effective agents specifically designed to achieve abstinence and prevent relapse in MA users.
In rodent models, MA CPP extinction-reinstatement has been extensively employed to investigate relapse. 12In addition to drug priming, both acute and chronic physical stress have been shown to substantially induce the reinstatement of MA CPP. 13 Although MA CPP has been introduced in zebrafish, 7 it has been primarily limited to the stages of acquisition and expression.Further exploration into the extinction-reinstatement phases of MA CPP in zebrafish could yield valuable insights into the mechanisms underlying craving and relapse behaviour.
The present study aimed to establish a comprehensive MA CPP model, including acquisition, expression, extinction, and reinstatement, in adult zebrafish and determine whether MA CPP in zebrafish is comparable with rodents in modelling reward and relapse.Initially, a biased CPP procedure paired different doses of MA with a nonpreferred compartment for zebrafish was used to test the dosedependent rewarding effect of oral administration of MA.We then assessed whether confined and nonconfined extinction procedures could successfully extinguish the MA CPP.After extinction, a priming dose of MA or a stressor was given prior to the test session to observe the reinstatement of CPP.Two standardized physical stressors were employed in zebrafish: net stress 14,15 and chasing stress, [16][17][18] both of which provoke cortisol release.Additionally, an anxiogenic drug yohimbine, which increases norepinephrine release by blocking alpha-2 noradrenergic receptors 19 and has been shown to effectively reinstate MA CPP in rodents, 20 was used to induce reinstatement of MA CPP in zebrafish.
We further investigated whether MA CPP in zebrafish could be a suitable laboratory setting for elucidating the neuropharmacological mechanisms of MA reward and relapse.Prior studies have demonstrated that the D1 receptor antagonist SCH23390 21 can suppress MA-induced CPP in rats, while the opioid antagonist naltrexone can attenuate MA CPP expression and MA priming-induced reinstatement of MA CPP in mice. 22Additionally, alpha-2 adrenergic agonists like clonidine have been shown to block stress-induced reinstatement of heroin, 23 cocaine, 24 and nicotine 25 seeking in rodents.As such, we evaluated whether SCH23390, naltrexone, and clonidine have similar effects on MA CPP in zebrafish.

| Animals and housing
The wild-type AB strain of zebrafish (D. rerio) 3 to 6 months old was obtained from Taiwan Zebrafish Core Facility at the National Health Research Institutes.The zebrafish were housed in a water circulation system at 28 C with a 14 h light and 10 h dark cycle following standard protocol for zebrafish care.Common white light (36 W, TLD light system) was provided in the facility, and water quality was monitored to maintain a temperature of 27 ± 1 C, a pH range of 6.5-7.8, the conductivity of 200-500 μs, and dissolved oxygen levels of ≥5 mg/mL.Fish were fed with zebrafish powder food (Zeigler, Pentair Aquatic Eco-Systems, Inc., Cary, NC, USA) twice a day.Zebrafish weighing approximately 0.5 g were selected.A total of 191 zebrafish were utilized in this study.
All experimental procedures on zebrafish were carried out following the approved guidelines and with the approval of the Institutional Animal Care and Use Committee of the National Health Research Institutes, Taiwan (NHRI-IACUC-109049-M1-A-S01).

| Drugs
MA hydrochloride (>99%) was obtained from the Taiwan Food and Drug Administration (Taipei, Taiwan).Yohimbine hydrochloride (19869) was purchased from Cayman Chemical.SCH23390 hydrochloride (D054), clonidine hydrochloride (C7897), and ethyl 3-aminobenzoate methanesulfonate salt (Tricaine) (A5040) were purchased from Sigma-Aldrich, while naltrexone was obtained from Lotus Pharmaceutical CO.Doses of chemicals were prepared based on salt weight.All drugs were dissolved in system water.SCH23390

| Oral gavage
A gavage procedure was utilized to administer precise quantities of drugs, following previously established methods with minor adjustments. 26,27Prior to the procedure, each zebrafish was anaesthetized using Tricaine (200 mg/L) and placed within a damp sponge submerged in water with its dorsal side facing upwards.Administering the drug solution or system water was achieved using a 10 μL syringe (Hamilton, MA, USA) outfitted with a plastic feeding tube.
Specifically, each zebrafish weighing approximately 0.5 g received 5 μL (10 μL/g) of the drug solution or water.Subsequent to the oral gavage procedure, the fish were promptly reintroduced into their housing tanks or CPP experimental tank.Typically, the fish resumed free swimming within 1-2 min.During the experiments to evaluate the effects of SCH23390 and naltrexone, the fish were anaesthetized twice for oral gavage of these compounds and MA, with 5 min intervals between administrations to allow the fish to regain consciousness.

| CPP apparatus design
For the CPP experiment, a rectangular tank measuring 44 cm (L) Â 8 cm (W) Â 14 cm (H) and filled with 2 L of fresh water was used.
The tank was divided into two equally sized compartments by a divider.Each compartment had a different colour on its three walls and bottom; one was white and the other had vertical navy blue and yellow stripes measuring 2 cm in width (Figure 1A).The choice of colour combination was based on the reliable and stable preference behaviour observed in the zebrafish during our preliminary study.A biased CPP procedure was used, with the white compartment designated as the preferred side, as the fish spent over 60% of their exploration time in this area.The remaining compartment (vertical navy blue and yellow stripes) was labelled as the nonpreferred side (Figure 1B).

| CPP paradigm
Two days prior to the initiation of the experiments, zebrafish were weighed.Those fish weighing approximately 0.5 g were selected and individually housed to ensure accurate identification in plastic tanks 28 measuring 16 cm (L) Â 11 cm (W) Â 10 cm (H).These tanks, holding 500 mL of water, were situated in the housing room of the zebrafish core facility.This housing arrangement was maintained throughout the entire experiment because individually housed zebrafish have lower basal cortisol levels than group housed zebrafish. 29The zebrafish underwent a 1 h habituation period after being moved to the experimental room, which was located next to the housing room.
The preference of zebrafish in two-compartment conditioned place preference (CPP) apparatus.(A) The CPP apparatus consists of two compartments, coloured, either white or vertical 2 cm wide navy blue and yellow stripes.The zebrafish were allowed to roam freely in the CPP apparatus (with no divider) for 10 min habituation, and the percentage of time spent on each side of the tank was determined for the next 10 min.(B) Mean percentage (±SEM) of the time zebrafish spent in each compartment of the CPP apparatus (n = 100).*** P < 0.001 significant difference between two compartments

| Preconditioning test
In the preconditioning test phase, each zebrafish was placed in the CPP experimental tank with no divider separating the two compartments and allowed to habituate to the whole apparatus for the initial 10 min.Digital videos of zebrafish behaviour were captured using a side-mounted camera and subsequently analysed using Ethovision 13.0 software (Noldus Infor Tech).The percentage of time spent on each side of the tank was then determined for the next 10 min.

| Conditioning phase
On the day following the preconditioning phase, zebrafish underwent conditioning phase using a two-session-per-day regimen (at 09:00 and 15:00) for 2 days.The experimental apparatus was partitioned into two equally sized compartments by a divider.In the morning session, the zebrafish initially received system water through oral gavage and were then immediately placed in the preferred compartment (white) for 20 min.In the afternoon session, they were orally administered MA and placed in the nonpreferred compartment (navy blue and yellow stripes) for 20 min.This conditioning order was applied to mitigate the residual effects of MA.Zebrafish were individually placed in their housing tanks between conditioning sessions.For the control group, system water was administered before each conditioning session.

| CPP test phase (expression)
On the day following conditioning, each zebrafish was placed into the CPP experimental tank with no divider and allowed to explore for 15 min.After a 5 min habituation period, the percentage of time spent on each side of the tank was recorded for 10 min.

| Extinction
Two extinction procedures, nonconfined and confined extinction, were applied.During the confined extinction training, a divider was still located in the middle of the CPP apparatus.Zebrafish received water by oral gavage and then placed in the nonpreferred side (previous drug-paired side) of the CPP apparatus for 20 min daily for three or seven consecutive days.
In the case of nonconfined extinction, zebrafish continued to receive CPP tests for 15 min each day.The time spent by each fish on each side of the tank was recorded for a duration of 10 min.This process was repeated every day until each zebrafish individually met the extinction criteria, defined as when the zebrafish spent equal or lesser amount time in the drug-paired side for three consecutive days, to ensure that the animal extinguished the memory consolidated during conditioning and to avoid spontaneous recovery of the extinguished memory.

| Drug priming-induced reinstatement of MA CPP
After extinction, each zebrafish received system water or a priming dose of MA via oral gavage before being placed into the apparatus to evaluate CPP reinstatement.Similar to the test phase, after a 5 min habituation period, the percentage of time spent on each side of the tank was measured for 10 min.

| Stress-induced reinstatement of drug CPP
In this phase, two stressful conditions and yohimbine were used as stressors to induce CPP reinstatement.Zebrafish were subjected to either 30 s or 1 min of suspension in a net above the water, 15,30 chased by a stainless steel long handle spoon (26 cm) for 1 min, or orally administered with yohimbine (20 mg/kg) before being placed into the CPP experimental tank.After an initial habituation period of 5 min, the percentage of time spent on each side of the tank was measured for 10 min.

| CPP retest
As CPP test, each zebrafish was placed into the CPP experimental tank with no divider and allowed to explore for 15 min.After a 5 min habituation period, the percentage of time spent on each side of the tank was recorded for 10 min.

| Statistical analyses
Data were obtained and expressed as mean ± standard error of the mean (SEM).Two-way mixed analysis of variance (ANOVA) was used to analyse the dose-dependent effect of MA.Paired t-test or one-way repeated ANOVA was used when the same animals were repeatedly tested in different phases.Post hoc Tukey's comparison test was used.P < 0.05 was considered statistically significant.

| Dose-dependent effects of MA on CPP
After the pretest, zebrafish received various doses of MA (0, 20, 40, or 60 mg/kg) and were placed in the initial nonpreferred side during the conditioning phase.Each dose group comprised six fish, except for MA (20 mg/kg)-treated group which had five fish.
The data were analysed by a two-way mixed designed ANOVA with time (pretest and test) as the within-subject factor (MA dose:

| Confined and nonconfined extinction followed by reinstatement induced by MA priming
After the pretest, MA (40 mg/kg) was used to induce CPP.Following the MA CPP test, confined extinction for 3 (n = 8) or 7 (n = 8) days was conducted prior to the post-extinction test.As shown in Figure 3A, there is a significant difference between three phases (F 2,14 = 114.6,P < 0.001).Zebrafish displayed MA CPP after training, while the 3 day confined extinction did not reduce the MA CPP.
Figure 3B showed that the 7 day confined extinction procedure significantly extinguished MA CPP.Thereafter, all the fish received water first, followed by a priming dose of MA (20 mg/kg) on the subsequent day, to evaluate reinstatement.There is a significant difference between five phases (F 4,28 = 172.7,P < 0.001).MA priming, but not the administration of water, robustly reinstated the previously extinguished MA CPP (Figure 3B).
In the case of nonconfined extinction, after the conditioning with MA (40 mg/kg) and initial CPP test, repeated CPP tests were conducted daily (n = 8).On the seventh session of nonconfined extinction, the extinction criterion was met for each zebrafish individually.
The next day, reinstatement was induced in all fish by administering system water, followed by the application of a priming dose of MA (20 mg/kg) on the subsequent day.The data were analysed using a one-way repeated measures ANOVA, which showed a significant difference between phases (F 10,70 = 26.61,P < 0.001).Post hoc tests revealed no significant differences between the pretest and extinction sessions 4-7.The administration of MA (20 mg/kg), but not water, resulted in the reinstatement of the previously extinguished MA CPP (Figure 3C).

| Stress-induced reinstatement of MA CPP
To explore the possibility of reinstating MA CPP with different stressors, the effects of a net stressor, chasing stressor, and yohimbine were assessed.After the pretest, MA (40 mg/kg)-induced CPP was followed by nonconfined extinction.Zebrafish were then subjected to either being suspended in a net above the water for 30 s 15,31   After a 14 day abstinence period, a CPP retest was conducted.At this time, the zebrafish did not display a preference for the MA-paired site.Following this, a second reinstatement of CPP was induced by MA (20 mg/kg) priming or chasing stress.The data were analysed using one-way repeated measures ANOVA (MA priming: F 11,77 = 26.22,P < 0.001; stress: F 11,77 = 32.88,P < 0.001), revealing differences across various phases.As shown in Figure 5A,B, both MA priming and chasing stress resulted in zebrafish spending significantly more time in the MA-paired side after a 14 day abstinence period, reflecting a persistent susceptibility to reinstatement of MA CPP.

| Effects of naltrexone on the expression of MA CPP in zebrafish
A total of 24 zebrafish were separated as three groups.After MA (40 mg/kg) CPP conditioning, naltrexone (0, 100, or 200 mg/kg) was

| DISCUSSION
In this study, we successfully established a comprehensive MA CPP model in zebrafish, encompassing all phases of CPP, which are acquisition, expression, extinction, and reinstatement.3][34] However, it can be challenging to precisely control the concentration of the drug that the zebrafish absorb through their gills.The present study demonstrates that MA-induced CPP in zebrafish occurs in a dosedependent manner through oral gavage.This administration method allows for accurate dosing, minimizes the drug consumption, and mimics the way drugs are taken by humans.
Oral gavage was performed with the zebrafish under anaesthesia to ensure successful administration and minimize stress. 26,27Typically, F I G U R E 4 Effects of different stressors on reinstatement of extinguished methamphetamine conditioned place preference.After methamphetamine conditioned place preference and nonconfined extinction, reinstatement was induced by air exposure in the net for (A) 30 s and (B) 1 min, (C) spoon chasing for 1 min, and (D) vehicle or yohimbine (20 mg/kg).The percentage of the time that zebrafish spent in the drug-paired side was expressed as mean ± SEM (n = 8).** P < 0.01 and P < 0.001 compared with last extinction or pretest the zebrafish became anaesthetized within 1 min.The oral gavage procedure was conducted swiftly, taking only a few seconds.The zebrafish regained consciousness within 1-2 min after gavage and swim constantly without displaying any signs of abnormalities.Moreover, upon analysing the data from the pretest (which involved no anaesthesia and oral gavage) and the CPP test of vehicle control group (where anaesthesia and oral gavage were performed using water during conditioning), no significant differences were observed.These observations indicate that the oral gavage procedure does not have a discernible impact on the zebrafish behaviour and is well-tolerated by the zebrafish.
A biased CPP design was employed in the present study.Zebrafish displayed a clear preference for the compartment with white colour, spending more than 60% of their time in the preferred compartment, with high repeatability.However, it is important to note that biased designs for CPP have faced criticism, particularly when the treatment exhibits anxiolytic properties.To distinguish between aversion reduction and preference development after MA conditioning, we conducted a CPP training with the drug paired with the initially preferred side.The results indicated that even when MA paired with the initially preferred side of the chamber, the fish further increased their time spent on the MA-paired side.This observation strongly suggests that the preference for MA is the driving factor behind the MA CPP observed in our biased design.A previous study by Parmar et al. 34 demonstrated that extinguished ethanol CPP could be reinstated by low-dose ethanol exposure in zebrafish.In the present study, in addition to applying a priming dose MA, we introduced three acute stressors: net handling stress, chasing stress, and yohimbine to induce reinstatement.Our results revealed that acute chasing stress and yohimbine, but not acute net handling stress, robustly induced reinstatement of MA CPP.
Net chasing has been shown to provoke escaping responses, aversive behaviours, and cortisol release in zebrafish. 17,18To prevent habituation to stress caused by repeated net chasing during handling, 37 we replaced it with spoon chasing.This method might simulate a predator threatening the zebrafish and disturbing the water, potentially inducing both intense psychogenic and physical stress, to reinstate MA CPP.Similar to what was observed in rodents, 20 yohimbine effectively induced MA CPP reinstatement in zebrafish, providing a standardized chemical stressor for both species.
The net handling stress failed to reinstate MA CPP.Although it has been shown to induce whole-body cortisol levels, net handling stress does not alter the whole-brain monoamines in zebrafish. 15ven the critical role of noradrenergic neurotransmission in the stress-induced reinstatement of drug CPP in rats, 38 it is noteworthy that chasing stress 39  To further validate whether our zebrafish CPP protocol is eligible to investigate the molecular and cellular basis of drug rewardassociated long-term memory and relapse, several pharmacological inhibitors, known for their capability to modulate various stages of MA CPP shown in rodents, were assessed in zebrafish.Dopamine D1 receptor antagonist SCH23390 disrupted the CPP induction of MA, and cocaine 21 and the opioid receptor antagonist naltrexone blocked the expression and reinstatement of extinguished MA CPP in rats. 22nsistent with the observations in rodents, SCH23390 blocked the induction of MA CPP and naltrexone disrupted the expression and MA priming-induced reinstatement in zebrafish.Our results revealed the critical roles of receptors in the reward-based memory formation and opioid receptors in the expression and reinstatement of memory in zebrafish.Furthermore, clonidine, an alpha 2-adrenoceptor agonist, which inhibits noradrenaline cell firing and release and has been shown to effectively reduce the stress-induced reinstatement of cocaine CPP in mice, 38 suppressed chasing stress-induced MA CPP in zebrafish.These results support that MA CPP in zebrafish is a suitable laboratory setting and comparable with rodents for revealing the neuropharmacological mechanisms involved in the MA reward and relapse.

| CONCLUSIONS
In summary, our study has successfully established a biased apparatus and protocol for modelling the different phases of MA CPP in zebrafish.By replicating findings observed in rodents, our study underscores the potential of the zebrafish CPP model as a valuable tool for uncovering the neurobiological mechanisms underlying reward, extinction, and relapse in drug addiction.Additionally, it could offer a rapid and cost-effective method for assessing the abuse potential of various new psychoactive substances and developing novel pharmacological interventions to prevent drug relapse.

AUTHOR CONTRIBUTIONS
Liao-Chen Chen contributed to the acquisition and analysis of the behavioural data.Ming-Huan Chan and Hwei-Hsien Chen provided the study concept and critical revision of the manuscript.All authors have seen and approved the manuscript being submitted.

( 3
or 10 mg/kg) and clonidine (1.5, 3, or 10 mg/kg) were administered to determine their effects on the induction of MA CPP and stressinduced reinstatement of MA CPP, respectively.The effects of naltrexone (100 or 200 mg/kg) on MA CPP expression and MA priminginduced reinstatement were evaluated.

F 3 ,
19 = 18.42,P < 0.001; time: F 1,19 = 63.68,P < 0.001; MA dose Â time: F 3,19 = 9.36, P < 0.001), revealing significant differences among MA dose groups, between pretest and test, and interaction between MA dose and time.As shown in Figure2, MA dosedependently produced a preference for the drug-paired side after conditioning.Because there is no significant difference between MA doses at 40 and 60 mg/kg, the dose of 40 mg/kg MA was used to induce CPP for the following experiments.To rule out the possibility of aversion reduction by conditioning, MA (40 mg/kg) conditioning in the preferred side of the chamber was also examined (n = 8).Paired t-test was used to compare the time spent in drug-paired side between pretest and test for MA CPP (t = 4.094, P < 0.01).Although the zebrafish already spent over 60% of the pretest time in the MA-paired side, MA conditioning significantly increased the time spent in the MA-paired side to 80% (Figure2B).

F I G U R E 2 3 . 4 |
Effects of methamphetamine (MA) conditioned place preference (CPP) using a bias procedure.(A) After the pretest conducted, zebrafish were individually administered with MA (0, 20, 40, or 60 mg/kg) (n = 5-6) before placement in the nonpreferred compartment (navy blue and yellow stripes) during CPP conditioning.(B) After the pretest, zebrafish were administered with MA (40 mg/kg) and then placed in the preferred compartment (white) for CPP conditioning (n = 8).The percentage of time zebrafish spent in the MA-paired side was expressed as mean ± SEM. ** P < 0.01 and *** P < 0.001, between the pretest and CPP test at the same dose of MA Effects of long abstinence on MA CPP expression and reinstatement Zebrafish underwent MA-induced CPP and nonconfined extinction.A priming dose of MA (20 mg/kg) (n = 8) or chasing stress (n = 8) was used to induce reinstatement.
The extinction of MA CPP was conducted following CPP training.During CPP training, associations were formed between contextual cues and the rewarding properties of MA.Extinction involves new learning that serves to suppress the original behaviour.We employed both confined and nonconfined extinction procedures.Similar to the results observed in MA CPP in mice,36 both procedures successfully extinguished the MA CPP response in zebrafish.Specifically, confined extinction over a period of 7 days, but not 3 days, resulted in a significant reduction in MA CPP.The nonconfined extinction procedure also required 7 days to meet the extinction criteria.It appears that both F I G U R E 5 Persistent susceptibility to reinstatement of methamphetamine (MA) conditioned place preference (CPP) induced by (A) MA priming and (B) stress.The treatment timeline is shown in the upper panel.MA CPP was retested 14 days after the first reinstatement induced by MA priming.Following the retest, a priming dose of MA or chasing stress (n = 8) was applied to induce the second reinstatement of MA CPP.The percentage of time zebrafish spent in drug-paired side was expressed as mean ± SEM (n = 8).*** P < 0.001 between the indicated phases F I G U R E 6 Effects of Scheme 23390 on methamphetamine (MA) conditioned place preference in zebrafish.Scheme 23390 (0, 3, or 10 mg/kg) was administered prior to each MA-paired session during conditioned place preference conditioning.The percentage of the time zebrafish spent in MA-paired side was expressed as mean ± SEM (n = 8).*** P < 0.001 between pretest and test extinction procedures gradually diminished the contextual memory associated with MA over time.After either of the extinction procedures, oral administration of a priming dose of MA, but not water, reinstated the previously extinguished drug-seeking behaviour in zebrafish.
and the administration of yohimbine19 are recognized for their ability to increase catecholamine secretion.This discrepancy might provide insight into why acute net handling stress failed to reinstate MA CPP in zebrafish.This is the first study to demonstrate the robust reinstatement of extinguished CPP in zebrafish through exposure to stressful stimuli.In addition to the stressors used in this research, it would be valuable to assess the potential of other stressors for zebrafish, such as conspecific alarm substance or electrical shock, to induce reinstatement of MA CPP behaviour in future studies.Relapse to drug use during abstinence is a defining feature of addiction.In the present study, MA CPP was retested after 14 days of abstinence following the first reinstatement.There was no MA CPP behaviour observed during this time.However, the MA CPP could be robustly reinstated again by MA priming or chasing stress.This demonstrates the persistent susceptibility of zebrafish to reinstatement in our MA CPP paradigm.Although the CPP model has limitations, particularly noncontingent and minimal drug exposure, the CPP F I G U R E 7 Effects of naltrexone on expression and methamphetamine (MA) priming-induced reinstatement of MA conditioned place preference (CPP).(A) After MA CPP conditioning, zebrafish received naltrexone (0, 100, or 200 mg/kg) prior to CPP test.(B) After MA CPP conditioning and extinction, naltrexone (0, 100, or 200 mg/kg) was administered before MA priming-induced reinstatement of MA CPP.The percentage of time zebrafish spent in the MA-paired side was expressed as mean ± SEM (n = 8).*** P < 0.001 compared with pretest or last extinction F I G U R E 8 Effects of clonidine on stress-induced reinstatement of methamphetamine (MA) conditioned place preference (CPP).After MA CPP conditioning and extinction, zebrafish received clonidine (0, 1.5, 3, or 10 mg/kg) prior to chasing stress-induced reinstatement of MA CPP.The percentage of the time zebrafish spent in the MApaired side was expressed as mean ± SEM (n = 8).*** P < 0.001 between last extinction and reinstatement reinstatement procedure has good validity in evaluating vulnerability to relapse and allows the study of different environmental manipulations, such as stress, which can modulate relapse.Our results provide empirical evidence that the zebrafish CPP model is suitable for studying drug relapse, whether elicited by drug priming or stress.