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3j'����3������������������������������������������������������������������������%:���������T	]:	Electrical stimulation of the Ventral Tegmental Area effects the acquisition and expression of morphine�induced conditional placed preference
Ali-akbar Kargari-Chaharborji�, Hojjat-allah Alaei*b� Maryam Radahmadib, Hamid Azizi-Malekabadic

� Department of Biology, Faculty of Science, Payame Noor University, Isfahan, Iran
*b Department of Physiology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
cDepartment of Biology, Faculty of Basic Science, Islamic Azad University, Khorasgan Branch, Isfahan, Iran
Abstract
Ventral tegmental area (VTA) is an important section of reward pathway involved in opiate reinforcement. Herein, we studied the effect of electrical stimulation with varying levels of current intensities from 50 �A to 10�A at constant frequency of 100 Hz, on VTA with effective and ineffective doses of morphine (5 mg/kg and 0.5 mg/kg, respectively), during conditioning and post-conditioning phases of Conditioned Placed Preference. Our results demonstrate that subcutaneous administration of 5 mg/kg of morphine produced significant CPP in comparison with that of the saline group. Electrical stimulation of VTA blocked the effect of both acquisition and expression of morphine-induced CPP and stimulation of VTA at the highest current intensity (50 �A) at the ineffective low dose of morphine significantly enhanced the acquisition phase of CPP. Our findings suggest that the electrical stimulation of VTA has a notable effect on memory and learning formation during the conditioning induced process by morphine.
Keywords: Ventral tegmental area (VTA), Morphine; Conditioned place preference (CPP); Electrical stimulation
" Corresponding author: Tel.:+98 03117922407; fax: +98 0311 6687898. E-mail address: HYPERLINK "mailto:alaei@med.mui.ac.ir"alaei@med.mui.ac.ir.

Introduction
VTA is an important area of the mesolimbic dopamine system (MLDS) and contributes to the reward pathway. VTA dopaminergic neurons branch out to other parts of the brain reward system including medial prefrontal cortex (mPFC) and nucleus Accumbens (NAc). Dopaminergic neurons in the VTA mediate the addictive behavior in animals and humans such that chronic administration of opiates including morphine and codeine produces a number of changes in MLDS and can lead to dependence behavior [1-3]. These drugs enhance the brain reward circuit and are multi-functional in the core of dopamine (DA) reward system [1, 3, 4]. Diverse rewards, such as eating food, sweets, sexual encounters, electrical brain stimulation, and consumption of addictive drugs, have been implicated in conditional placed preferences (CPP) in a number of animals including rats, monkeys and rabbits [4-6]. CPP is a standard pre-clinical behavioral model applied on animals in order to study the rewarding effects of a candidate drugs. The basic principal of CPP involves the association of a particular environment with drug treatment, followed by the association of a different environment with the absence of the drug treatment commonly in the form of a vehicle. As the candidate drug has potentially rewarding properties it raises motivational significance for the animal to spend more time in the environment associated with the drug treatment. However, if the animal spends more time in the environment associated with the absence of the drug (i.e. vehicle) then the animals show no CPP to the candidate drug studied [7-9]. 
There is a growing body of literature reporting the effect of  electrical or chemical stimulation on different parts of the animal brains and the link to changes in the animals� behavioral characteristics [3, 5-16]. For example, Liu et al. (2008) has reported that chronic high-frequency stimulation (HFS) of the rat NAc can block morhine-induced CPP. Other studies point to the effects of electrical stimulation on CPP induced by addictive drugs that increase release of neurotransmitters in some areas of the brain [17-22]. Herein, we examined the responses to inter-cranial VTA electrical stimulation through a range of current intensities to determine whether stimulation of VTA can block CPP induced by morphine. We addressed this key question taking into consideration the acquisition and expression phases of CPP and determining the range and optimal electrical stimulation. 

2. Materials and methods
2.1 Animals
Male Wistar rats weighing approximately 300� 350 g were used in the study. The animals were housed in an animal house with a controlled (20�22 �C) free access to food and water under 12-h light and 12-h dark illumination cycle beginning at 6:30 AM. The subjects were allowed to adapt to the laboratory conditions for at least 1 week before surgery and were handled for 5 minutes per day. Each animal was exposed to the test once. Each group consists of 6-8 rats per experiment. The Ethics Committee for Animal Experiments at Isfahan Medical University approved the study and all procedures were carried out in accordance with institutional guidelines for animal care and experimental use. 
2.2. Drugs
Morphine sulfate (Temad Iran Co.) was dissolved in 0.9% saline, freshly prior to each experiment. Morphine as the candidate drug was injected subcutaneously (SC), whereas control animals received saline (vehicle).

2.3. Surgical procedures
The animals were anesthetized with chloral hydrates (350 mg/kg, i.p) [1, 6] and placed in a stereotaxic apparatus. Dipolar electrode (Teflon�coated stainless steel, 250�m diameter) was stereotaxically inserted unilaterally into the right VTA of each animal. The electrodes were connected to sockets. In this approach rats can freely move around during stimulation. Coordinates for the electrode implantation according to the atlas of Paxinos and Watson were Anterioposterior (AP), 5.8; mediolateral (ML), 0.6; dorsoventral (DV), 8.6 relative to bregma and the skull surface [23]. The implantation is secured to the skull through screws and dental acrylic cement. Following surgery, animals are housed in Plexiglas cages in groups of 4 for 5-6 days before behavioral testing begins.

2.4. Apparatus
The CPP apparatus consisted of three compartments. Two compartments were identical in size (30cm�30cm�30cm) but each had a different color pattern on the wall and different textures on the floor. Compartment A had black walls and a smooth floor. Compartment B had white walls and a meshed floor. The third compartment C was gray (30cm�10 cm�30 cm) and connected to A and B through two separate opening. The apparatus had a digital chronometer with electronic sensors which were installed under the floor of both A and B compartments. A stimulations time recording and animal activity was controlled and displayed by electronic devices produced by KBBR Engineering Co.  A closed circuit camera with a computer [USB camera (Shark Professional, China) and Ulead.V.S software, Version 9] monitored the animals' behavior in apparatus during behavioral tests.

2.5. Behavioral testing
The CPP paradigm took place on 9 consecutive days by using a biased procedure. The experiment consisted of the following three phases [4, 5].
2.5.1. Pre-conditioning phase
This phase consisted of two days: during the first day the animals were placed in the compartment C with the open guillotine door and were allowed to explore the three compartments for 15 minutes. The second day was the repeat of the first day but here the stay in compartment A and B was measured in seconds [4, 5]. Here the individual rat spent an average of 77% and 11% of time in each chamber.

2.5.2. Conditional place Phase
Conditioning place phase began immediately after the pre-conditioning phase. This phase consisted of six 45-min sessions (three saline and three drug pairing). These sessions were conducted once every day (from day 3 to day 8). On days 3, 5 and 7, animals received morphine (SC) and on the days 4, 6 and 8, animals received saline (SC) of equivalent volume. During these sessions, the animals with morphine are confined in the compartment where they spent less time in the second day of the test and the animals with saline are confined in the compartment where they spent more time in the second day of the test. The confinement  period was 45 minutes [4, 5].  

2.5.3. Post- conditioning phase (testing phase)
This phase was carried out on day 9, one day after the last conditioning session, in a morphine-free state. On day 9, rats are placed in the compartment C of the apparatus and are free to explore both compartments for 15 min. The time spent in each compartment is measured in seconds. Each animal was tested once. The time spent and animal behavior in paired-drug compartment was recorded and displayed at the end of each test; the preference change was calculated based on the difference (in seconds) between the time spent in the paired- drug compartment in the final step and the time spent in this compartment in the pre-conditioning day. In fact the scores for the paired �drug place were calculated by subtracting the pre-conditioning score from post-conditioning score [5].

2.6. Experimental design
2.6.1. Morphine dose-response on conditioned place preference paradigm
The different doses of morphine 0.5 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg and 10 mg/kg, on CPP were studied. Rats were divided into 6 groups: 5 groups received different doses of morphine and one group (control) received saline. Morphine or saline (SC) were injected during 6 days according to conditioning schedule (behavioral testing). The change of preference was calculated with the difference (in seconds) between the time spent in the paired- drug compartment on the final test day and the time spent in this compartment in the pre-conditioning day. This procedure is applied to assess the CPP induction. 

2.6.2. Effects of electrical stimulation with different current intensities on VTA in combination with effective doses of morphine on expression phase of CPP
According to the experiment design the animals were divided into four groups as follows: three groups were test groups that were stimulated and the fourth the sham group is not stimulated.  After surgery and electrode implantation the rats were housed in cages in the animal house for one week for recovery from surgery. After this period, morphine (5 mg/kg) or saline (1 ml/kg) was injected for a 6 day period according to conditioning schedule (Conditioning Phase). In the three test groups each animal is stimulated with electrical stimulation pulses for 15 min before the testing phase begins in post conditioning phase. The test groups were divided into three current intensities. Group one wasat50 �A group only, group two was at 25 �A and the third was at 13 �A and each group was kept to a constant frequency of 100 Hz for 1s every 5s without stimulation in the conditioning phase. This was done by a Data Acquisition D3111 stimulator produced by Science Beam. Conditioning scores were calculated for each animal on the test day.

2.6.3 Effects of electrical stimulation with different current intensities on VTA in combination with ineffective doses of morphine on expression phase of CPP
 In this experiment rats were divided into five groups (four groups for different electrical stimulations and one group for sham). This experiment is preformed similarly to the above section 2.6.2, however here an ineffective dose of morphine is used (0.5, mg/kg). Animals are stimulated with electrical stimulation pulses for 10 min total at 50 �A (for 50 �A group only), 25 �A (for 25 �A group only) and 13 �A (for 13 �A group only) current intensities and constant frequency 100 Hz during 1s every 5s] for 15 minutes before the testing phase begins (post conditioning phase) without stimulation in the conditioning phase. Conditioning scores are calculated for each animal on the test day and these groups were compared with the sham group. 

2.6.4. Effects of VTA electrical stimulation with different current intensities in combination with effective doses of morphine on acquisition phase of CPP
Here, rats were divided into 6 groups. The first group was the control group which received normal saline (1ml/kg). The second group was the sham group that was the same as other experiments however; they were not stimulated by electrical stimulation.  In the remaining four groups each animal was stimulated once a day 15 minutes prior to administration of morphine at days 3, 5 and 7 of conditioning phase. The stimulation of these four groups is based on: 50 �A, 25 �A, and 13 �A and 10�A current intensities respectively with constant frequency of (100 Hz) during 1s every 5s for all. The rats of these four groups were not stimulated on final test day. The procedure to calculate the preference change was based on the difference between the time spent in the test day and the time spent in the pre-conditioning day.

2.6.5. Effects of VTA electrical stimulation with different current intensities in combination with ineffective doses of morphine on acquisition phase of CPP
For this purpose the rats were divided into six groups: five groups for electrical stimulation and one group for sham. This experiment is performed like part 2.6.4 but ineffective dose of morphine is used (0.5, mg/kg). The five groups are stimulated by electrical stimulation pulses [10 min total at 50 (for 50 �A group only), 25 (for 25 �A group only), 13 (for 13 �A group only) and 10 (for 10�A group only) current intensities and constant frequency 100 Hz during 1s every 5s] for 15 minutes prior to administration of morphine during the days 3, 5 and 7 of conditioning phase. Conditioning score are calculated for each animal on the test day and these groups are compared with the sham group.

2.6.6 Effects of VTA electrical stimulation with 50 �A current intensity in combination with different doses of morphine on expression and acquisition phases of CPP paradigm.
In this part of experiments we chose three groups of animals for each dose of morphine (0.5, 2.5, 5, 7.5 and 10 mg/kg). The first group is sham group, the second group is stimulation group in expression phase and the last group is stimulation group in acquisition phase of CPP. All of the stimulation groups are stimulated with 50 �A current intensity. 

2.7. Histology
After completion of behavioral test, the subjects were anesthetized by 350 mg/kg chloral hydrate.  Next, the animals were transcardially perfused with 0.9% saline, followed by 10% buffered formalin and sacrificed. The brain was removed and placed in 10% formalin for at least 3 days before sectioning. Sections were examined to determine location of the electrode aimed for the VTA. The electrode placements were verified using the atlas of Paxinos and Watson [23]. Data from animals� with injection sites located outside the VTA region were detected in this analysis (Fig. 1).

Insert Figure 1.

2.8. Statistical analysis
Comparisons between groups were made with one way ANOVA followed by Post Hoc Tukey and Dennett�s post-test. All results were expressed as mean � S.E.M and difference with P<0.05 between experimental groups were considered statistically significant. Calculations were performed through SPSS statistical software.

3. Results
3.1. Determination of morphine dose-response on CPP paradigm
To determine the effective and ineffective doses of morphine on CPP, we injected a range of doses of morphine in rats and examined the consequent time spent in selected testing compartments. Firstly, we observed that  with the increasing doses of morphine from lowest point to highest (0.5, 2.5, 5, 7.5,10 mg/kg) correlated positively with a normal distribution profile  for the time spent in the paired-drug compartment A compared with saline compartment B which was unresponsive to saline (vehicle).ANOVA statistical analysis confirmed a significant response to morphine induced P<0.01]. Moreover, Dennett�s post-test demonstrated that at doses such as 2.5 mg/Kg and 5 mg/Kg injection of morphine we observed the optimal times spent in the paired-drug compartment compared to time spent in the saline-paired compartment (*P<0.05, ***P<0.01) and the other doses of morphine had not significant effect on CPP (P>0.05). Based on this data we selected the highest (5 mg/kg) and lowest (0.5 mg/kg) doses for subsequent experiments (Fig.  2).   
Insert Figure. 2.

3.2. Effects of electrical stimulation of VTA at different current intensities in the presence of effective and ineffective doses of morphine during expression phase of CPP
To determine whether current intensity plays a role on VTA stimulation under different doses of morphine in expression phase of CPP, our experimental groups were subjected to electrical stimulation with pulses for 10 minutes at 50 �A, 25 �A and 13 �A current intensities at a constant frequency (100 Hz) for 1s every 5s and compared with one another as well as with the saline and sham group which did not receive electrical stimulation. As illustrated in Figure 3. Statistical analysis of ANOVA showed notable effects of electrical stimulation of VTA on the expression phase of CPP induced at 5 mg/kg of morphine [F (4, 29) = 4.106, P= 0.009]. A Tukey post-hoc-test confirmed that at 13 �A current intensity significantly reduced the time spent in drug-paired Compartment A (*P< 0.05) to levels observed at the ineffective dose response of 0.5 mg/kg in the Sham group. However, it should be stated that although other current intensities gradually decreased the time spent in drug-paired Compartment A the results were not statistically significant P>0.05). As expected ANOVA analysis showed no response to VTA current stimulation at ineffective doses of morphine (0.5mg/kg) in expression phase of CPP paradigm compared with 5 mg/kg morphine dose response. 

Insert Fig.3.
3.3. Effects of electrical stimulation of VTA at different current intensities in the presence of effective and ineffective doses of morphine during acquisition phase of CPP
To determine whether current intensities plays a role on VTA stimulation under different doses of morphine in acquired phase of CPP, our VTA experimental groups were stimulated once a day for 15 minutes prior to administration of morphine during the 6 conditioning phase days and compared with one another as well as with the saline and sham group that did not receive electrical stimulation. Our results as illustrated in Fig. 4, expresses the mean � S.E.M of 6-8 animals per group. In comparison with the sham group the stimulation groups showed that electrical stimulation of VTA gradually decreased the time spent in acquisition phase of CPP induced by 5 mg/kg morphine , however, Tukey post-hoc-test revealed that the lowest current stimulation of 10 �A resulted in the most significant decrease in time spent in CPP (*P< 0.01). In the shams group we observed no response to CPP during low dose of morphine at 0.5 mg/kg except at the highest currency stimulation where data observed were similar to levels observed at 5 mg/kg for the same currency stimulation.

Insert Fig. 4.
3.4. Effects of VTA stimulation at 50 �A current intensity in different doses of morphine during expression and acquisition phases of CPP 
One-way ANOVA with Tukey post-hoc-test showed that electrical stimulation of VTA with 50 �A current intensity significantly increased the length of time spent in the acquisition phase of CPP at 0.5 mg/kg dose of morphine  compared with sham group [F (2, 21) = 7.001, P = 0.005]. In contrast, the same level of current intensity at the same dose showed no increase in the time spent in the expression phase of CPP with results similar to those in the sham group, Interestingly, electrical stimulation of VTA with the current intensity of 50 �A at a range of higher doses of morphine did not produce significant changes in acquisition nor expression phases of CPP (P> 0.05) (see Fig. 5). 

Insert Fig. 5.
3.5. The effect of VTA electrical stimulation on morphine- induced CPP
The statistical analysis of ANOVA showed significant differences among sham, saline and VTA electrical stimulation groups in expression and acquisition phase of CPP [F (2, 18) = 10.773, P = 0.001] and in expression phase of CPP [F (2, 19) = 9.129, P = 0.002] in combination with 5 mg/kg injection of morphine. A Tukey post-hoc-test revealed that the electrical stimulation of VTA suppressed acquisition and expression of morphine induced-CPP in comparison with sham-morphine group (*P<0.01). Morphine in the sham group without any stimulation induced strong CPP in comparison with saline control group (**P= 0.002 at Expression phase and***P= 0.004 at Acquisition phase) (see Fig. 6)
Insert Fig. 6.
4. Discussion
The results of our study demonstrate that administration of morphine at 2.5 mg/kg and 5 mg/kg induced CPP, and that at the lower (0.5 mg/kg) and higher (10 mg/kg) doses of morphine alone did not induce CPP. This is consistent with others in the literature reporting similar findings [24, 25]; [13, 26]. Moreover, the optimal dose of morphine that could induce CPP was shown to be 5 mg/kg. Morphine in this dose can affect the reward circuit and learning substructure in the central region of the brain. Our results are in agreement with the results obtained in previous studies showing that morphine at these doses did not enhance or reduce locomotors  activity in comparison with the saline control groups [8, 27]. We next set out to establish a relationship between electric stimulation and rewarding effects of morphine on VTA and demonstrated that at low current intensity of 13 �A we were able to reverse the rewarding effects of morphine induced CPP. Conversely, VTA stimulation at 50 �A current intensity at the low ineffective morphine dose of 0.5 mg/kg induced acquisition of morphine-CPP mimicking the rewarding effect of higher dose morphine on VTA. Moreover, our results here show that the electrical stimulation with 13 �A and 50 �A had no significant effect on ineffective morphine dose (0.5 mg/kg) in the expression phase. Shi et al (2004) have found that 100 Hz peripheral electrical stimulation (PES) can suppress CPP-induced by morphine and reverse the cell size reduction in VTA while, chronic stimulation of NAc with high frequency blocked CPP induced by morphine and attenuated morphine reinforcement [7]. Our findings here also indicate that with 100 Hz ICES in combination with low current intensities of VTA may suppress CPP induced at 5 mg/kg morphine. Electrical stimulation has been shown to enhance DA neuron activity with more efficiency than morphine [1, 28] therefore, it is plausible that the phasic activation of dopaminergic neuron in the VTA by electric stimulation may lead to behavioral conditioning. One possible mechanism for this may be the enhancement of DA neuron activity at lowest electric current intensity which can evoke pleasure centers and lack of desire in animals to prefer morphine chambers, while increasing the electric current intensity may directly affect VTA interneurons and provide inhibition to DA cells and control DA release in the VTA circuits that underlie addictive behaviors. The outcome of this study along  with others suggest that VTA plays an important role in the reward circuit, memory and learning process [6, 29, 30]. Moreover, this study showed that different combinations of current intensities with 100 Hz frequency can vary in the degree of effecting morphine-induced CPP in expression and acquisition phases. Our study suggests that examining the optimum combination of current intensity and frequency of electrical stimulation may contribute significantly to changes in memory, learning and behavioral characteristics in the brain. In addition, we showed that during VTA stimulation the rats became active with no behavioral abnormalities without any significance in their locomotors function.
Our findings support the hypothesis introduced by Fibiger et al (1987) who proposed that VTA electrical stimulation increases dopaminergic neurons activity and VTA electrical stimulation increases morphine enhanced effect on dopamine volume  in VTA where the CPP effect on rats are enhanced [21, 24, 31-35]. 

5. Conclusion
CPP is a learning model which requires the formation of associations between reward and particular location on the brain. This study indicates that low intensity electrical stimulation of the VTA can reverse the effects of morphine induced-CPP. On the contrary, using high current intensity causes an increase in the acquisition phase of CPP in otherwise ineffective low doses of 0.5 mg/Kg doses of morphine. Lack of morphine-induced CPP in this study with low intensity may be due to a reduction in the reward signal or in adequate response to the rewarding stimuli which impairs learning and memory formation in the process of conditioning; while stimulation of VTA with 50 �A leads to reward system activation and pleasure, mimicking the effect of morphine at optimal doses alone.
 


Authorship and Acknowledgements
Author�s contribution: 
A.K., M.R., H.A. and H.M. were responsible were for the study concept and design. A.K., H.A. contributed to do experiment, the acquisition of animal data and assisted with data analysis and interpretation of findings. M.R. performed the proteomics analysis drafted the manuscript. H.M. and H.A. provided critical revision of the manuscript for important intellectual content. All authors critically reviewed content and approved final version for publication. 

Acknowledgements: A special thanks go to Drs; A. Nasimi, P. Reisi, MR.Sharifi, and N. Hosseini for their many helpful assistance during this study. We also thank Dr. Shafiei our collaborator in Australia who has reviewed and edited the scientific language and writing style of the manuscript.


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Figure Legends

Fig. 1 Photomicrograph scan of a coronal section (50 �m). The arrow indicates the stimulating electrode site in the VTA.





Fig.  2 CPP produced by different doses of morphine. Different doses of morphine (0.5 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg and 10 mg/kg) and saline (1 ml/kg) were administered in a 3-day schedule of conditioning. On the test day, the animals were tested for a 15-min period. The change of preference was calculated based on the difference between the time spent on the day of testing and the time spent on the day of the pre-conditioning session. Data are expressed as mean � S.E.M of 6- 9 animals per group. The data were analyzed using one-way ANOVA followed by Dennett post-test, *P <0.05;***P<0.01 different from the saline control group.




Fig 3. The effects of different current intensities stimulation of VTA on morphine-induced CPP. At the effective dose (5 mg/kg) and ineffective dose (0.5 mg/kg) of morphine before initiation of post-conditioning of expression phase. The most effective current intensity at 13 �A at effective dose of morphine can suppress morphine induced CPP in expression phase. Other current intensities had no significant effect. Data are expressed as mean � S.E.M. of 6-8 animals per group and were analyzed using one-way ANOVA followed by Tukey post-hoc-test (*p < 0 .05).




Fig.4. The different current electrical stimulation of VTA on the acquisition phases of morphine�induced CPP. The VTA were stimulated once a day for 15 min prior to receiving morphine (5 or 0.5 mg/kg) in a 6-day schedule of conditioning. On the test day animals were tested for a 15 min period. The change of preference was calculated with the difference between the time spent on the day of testing and the time spent on the day of pre-conditioning session. Electrical stimulation of VTA with 50 �A current intensity in combination with ineffective doses of morphine can reinforce morphine induced-CPP and electrical stimulation of VTA with 10 �A current intensity in combination with effective d����������C	D	E	�	�	


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��b���c���_�*�/�0�1�3�4�6�7�9�:�<�?�@�A�����������������ܵ������������h~jh~Uh�� he�h!�CJOJQJ^JaJ&h�y�h!�5�CJOJQJ\�^JaJU h�y�h!�CJOJQJ^JaJ#h�y�h!�5�CJOJQJ^JaJ������������������+�,�-�.�/�0�2�3�����������������������$��d��$��$d��A$gd��$d��A$a$gd��$d��7$8$A$H$a$gd��oses of morphine can suppress morphine induced -CPP. Data are expressed as mean � S.E.M of (6-8) animals per group and were analyzed using one-way ANOVA followed by Tukey post-hoc-test (**p<0.05,*p<0.01). Other current intensity had no significant effects.



Fig. 5 Effect of electrical stimulation of VTA at 50�A on CPP in different doses of morphine. Stimulation (50 �A) at expression or acquisition phases or no stimulation (sham) was tested in different amount of morphine. Only the lowest dose of morphine at this current intensity increased the acquisition phase of CPP. The data were analyzed using one-way ANOVA followed by Tukey post-hoc-test,*p<0.05 compared with sham-morphine group. Each dose of morphine was compared with its control group. 



Fig. 6. Effect of electrical stimulation of VTA on morphine-induced expression and acquisition phases of CPP. The VTA were stimulated with at the effective morphine (5 mg/kg) dose in a 6-day schedule of conditioning and before starting post-conditioning phase for a 10 min period. The change of preference was calculated with the difference between the time spent on the day of testing and the time spent on the day of the preconditioning and post-conditioning sessions. The data were analyzed using one-way ANOVA followed by Tukey post-hoc-test, *P < 0.01 compared with morphine group. Morphine in the sham group without any stimulation increased both expression and acquisition phases of CPP compared with saline control group (**P < 0.01).

Supplementary Figure
Fig.7. The effect of VTA electrical stimulation on locomotors activity is unchanged in morphine induced CPP. This figure shows that the numbers of compartment crossing do not significant change during electrical stimulation of VTA at 50 �A, 25 �A, 13 �A and10 �A in compare with only morphine group (P > 0.05).


















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