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Drug addiction is viewed as a maladaptive memory induced by contextual cues even in the abstinent state. However, the variations of hedonia and appetite induced by the context during the abstinence have been neglected. To distinguish the representative behaviors between hedonia and appetite, micro-behaviors in abstinent animal such as psycho-activity and drug seeking behaviors were observed in morphine conditioned place preference (CPP). To confirm the different effects of reward between drug and natural reward, a palatable food CPP paradigm was compared in current work. After a 10-day training in CPP with morphine or food, the preference was tested on day 1, 14, 28, and the changes of micro-behaviors were analyzed further. Our data showed that tree shrews treated with morphine performed more jumps on day 1 and more visits to saline paired side on day 28, which indicated a featured behavioral transition from psycho-activity to seeking behavior during drug abstinence. Meanwhile, food-conditioned animals only displayed obvious seeking behaviors in the three tests. The results suggest that the variations of micro-behaviors could imply such a transition from hedonic response to appetitive behaviors during morphine abstinence, which provided a potential behavioral basis for further neural mechanism studies.
Drug addiction has been viewed as an aberrant learning of the association between drug and the context (
The conditioned place preference (CPP) model was commonly used in preclinical studies to investigate the association between addictive drugs and the contextual stimuli. In CPP, the animal was alternatively confined in one chamber after drug injection or another chamber after saline injection. Following repeated conditionings, the animal was allowed free access to both chambers in a drug-free state, and the time stayed in drug-paired chamber was taken as the index of preference (
In addition to addictive drugs, many natural rewards including food could also form CPP (
In this study, we observed the micro-behaviors in tree shrews to investigate the variations of hedonia and appetite after morphine or palatable food conditioning. Tree shrews were suggested as potential candidates for addiction studies, phylogenetically close to primates (
A total of eight male tree shrews (
The experiments were conducted according to the National Institute of Health, Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Research Ethics committee of Institute of Psychology, Chinese Academy of Sciences.
Morphine hydrochloride (Qinghai Pharmaceutical, China) was dissolved in sterile physiological saline (0.9% NaCl) to its final concentrations.
The CPP apparatus was composed of three stainless steel chambers (395 mm × 300 mm × 595 mm) as chamber A, C, and B in a row. The chamber C was in the middle and could connect with the nest box. The walls of chamber C were removable, separating chamber A and B, respectively. The apparatus were mainly featured with color cues in different sides of chambers according to the well-developed visual system in tree shrews (
The procedures were made, respectively, in the morphine- and food-conditioned group. The procedures of two groups both consisted of three phases: pre-test, conditioning and post-tests on day 1, 14, 28 after last conditioning sessions. The difference in two groups was conditioning phase. The morphine CPP procedure was based on our previous study with minor modifications (
The removable walls of chamber C were opened with a 5 cm width gap, and the tree shrews were placed in the chamber C through their nest boxes. Chamber C was the start for exploring the apparatus. Animals moved freely in the three chambers for 60 min on three consecutive days for habitation and pre-test. When the tree shrew was placed into the apparatus, it might hide in the nest box and did not explore the area for a while because it was sensitive to the change of the environment. Therefore, data acquisition started when the tree shrew first went out from the nest box and lasted for 30 min. Time spent in each chamber, the numbers of visits to each chamber and the numbers of vertical jumps on the third day was recorded as pre-test data. The biased procedure was used in our study, and the disliked chamber for each animal was used as the reward-paired chamber during conditioning training.
After the pre-test, the animals were randomly divided into two groups (
Morphine-conditioned tree shrews were injected with 5 mg/kg (intramuscular injection, IM) morphine and placed in their paired chamber on the first conditioning day. This dose was only used for the first morphine injection and for the following morphine injection the dose increased to 10 mg/kg. This design was based on our previous results which showed that one morphine injection of 5 mg/kg could make tree shrews adapt to the strong pharmacology effect of morphine and avoid the potential harm induced by the increased dose in recent sessions. Twenty-four hours after the morphine injection, the tree shrews were injected with saline (1 ml/kg, IM, the same volume as the morphine injection) and confined to the other chamber. On the subsequent conditioning days, each tree shrew trained for eight consecutive days with alternate injection of morphine (10 mg/kg, IM) and saline. The interval between injection and putting animals into the chamber was 3045 min and the conditioning time was 90 min. The time was based on the previous study to make sure that animals stayed in high locomotor level after morphine injection.
The food CPP was designed to compare with the morphine and the only difference between the procedures was the rewarding event. Since food deprivation might change the motivational state and locomotor activity of animals, the palatable food CPP in the normal feed state of tree shrews was used in our study. Our preliminary study found that apple was their favorite among three kinds of food (dry yellow mealworm, apple and food pellets). Therefore, during the conditioning training (Day -9 to 0), tree shrews had free access to a piece of apple which was placed in the middle of the reward-paired chamber for 30 min and were conditioned with nothing in another chamber for 30 min on alternate day.
After conditioning, the tree shrews freely explored the apparatus with the walls of chamber C opened to test their preference (P1-test). The procedure was similar to the pre-test phase. Moreover, to explore the preference and context induced behaviors during abstinence, tests were taken every 14 days (P14-test, P28-test).
Conditioned place preference score [time in reward-paired chamber/(time in chamber A + time in chamber B)] was the index of preference. All data was shown as mean ± SEM. The statistical package SPSS 19.0 was used for data analysis. Paired
After five alternating conditioning sessions, CPP score in morphine-rewarded tree shrews was increased significantly compared with pre-test (
During the expression of CPP, the behavioral data was further analyzed to distinguish hedonia or appetite induced by the context (
Above results showed that the morphine-conditioned group mainly performed hedonic behaviors, but the palatable food-conditioned group mainly performed appetitive behaviors on the expression of place preference.
The CPP score was an index to reflect the rewarding value, which implied the strength of reward effects associated with the context. More importantly, both hedonia and appetite were induced by this reward effect. Thus, the place preference was examined every 14 days after conditioning (
Above results implied the morphine-induced place preference could maintain for almost 28 days in tree shrews and the palatable food-induced place preference could last 14 days. Thus, the further micro-behavior was analyzed till on day 28.
Beyond the results in the Section “The Rewarding Value of Morphine or Food during Abstinence,” we recorded the micro-behaviors of tree shrews at 10 min intervals during four tests (Supplementary Figure
In the morphine group, although there was no significant test time effect on the number of vertical jumps in morphine paired chamber [
From above results, the increased seeking behaviors with decreased psycho-activity meant a transition from hedonia to appetite induced by context during morphine abstinence. However, food-conditioned group displayed no behavioral change during abstinence.
Our results indicated that morphine-conditioned tree shrews expressed more psycho-activity on short term-abstinence, but more seeking behaviors on long-term abstinence, implying a context-induced transition from hedonia to appetite. As a comparison, the food-conditioned group did not experience this transition during abstinence. There were still some questions to be discussed.
Both morphine and food CPP in tree shrews were established after five alternating sessions. However, the morphine-conditioned group showed higher CPP score than food group, in agreement with studies in rats (
To explore whether the hedonia and appetite were changed or not with the increasing duration of abstinence, the most important thing was to find the appropriate behavioral indexes to represent the hedonic and appetitive responses. We counted the number of jumps and visits in both chambers at 10 min intervals during four tests (Supplementary Figure
During abstinence, the morphine-conditioned group implied a transition from hedonia to appetite. One of the possible reason for this transition was that the negative affective state was elicited because of morphine absence (
As a contrast, the food conditioned group had no transition during abstinence. The stable number of jumps and visits implied that the appetite for food was continuously induced by the context. Although the same behavioral phenomenon was observed on mice study (
The main finding in our study was that the morphine abstinent tree shrew displayed a behavior transition from psycho-activity to seeking behaviors, implying that context induced hedonia after short-term abstinence but appetite after long-term abstinence. It might remind the significance to take the abstinence time as an important factor to make more effective target for treatment on relapse.
YD and FS designed the experiments, preformed the experiments, analyzed data and wrote paper; TG participated in the writing of the article; NS designed the experiments and had primary responsibility for final content.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by grants from the National Basic Research Program Grants (2015CB553501), the National Natural Science Foundation (31170988), the Natural Science Foundation Major Research Plan program (91332115), the Natural Science Foundation Youth program (31500893) and the Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences.
The Supplementary Material for this article can be found online at: