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Journal of Basic and Applied Advances in Sciences Vol. 4, No. 1 (2016), 34-38
Effect of memory improvement by a cholinergic agonist on morphine-conditioned place
preference in mice
Department of Biology, Fars Science and Research Branch, Fars, Iran Abstract
This research aims at studying the effect of memory improvement by a cholinergic agonist ,donepezil, on creating
morphine dependence in mice using conditioned place preference(CPP) method.The donepezil was dissolved in
distilled water and was administered subcutaneously at a dose of 2.5 mg/kg. The mice received 10 mg/kg morphine
subcutaneously. In the conditioning stage that lasted for 8 days, different groups of mice, after receiving the
treatment were randomly placed in compartment for 30 minutes. The post-conditioning stage included the fourth,
the ninth days ( that is a 24h after the last session), the twelfth day ( that is 96h after the last session) and the
sixteenth day ( that is a 192h after the last session).The results showed a significant decrease (p≤0.05) in the place
preference of morphine+donepezil group compared with the morphine group.This last for eight days after quitting
the use of this drug.According to the results of study, donepezil through Ach increase ,inhibits the activity of
dopaminergic neurons and reduces the morphine-addiction.

Keywords:
Memory, cholinergic agonist, morphine, donepezil, CPP, mice
amygdala and prefrontal cortex (PFC) play critical roles in the establishment of reward-associated Nowadays, it is believed that addiction caused by memories (Everitt et al 2005).within the PFC, the drugs such as morphine is the most dangerous type olfactory cortex (OFC) integrates emotional and of addiction in developed and developing countries. motivational information and keeps them in the The most significant pathophysiologic procedure in working memory(Schultz et al 2006). the addiction process is the interference and For the first time Carew et al. in 1973(Arnold et al interaction of the drugs with the nerve system (Kelley 2002)demonstrated that hyoscine , a muscarinic et al 2002). Investigators are trying to discover the receptor inhibitor, impairs cognition and physiological relationships involved between the memory(Bunge et al 2001). Donepezil is a centrally nervous system and the addicting drugs in order to acting reversible acetylcholinesterase justify the quick influence of such compounds on the inhibitor(AChEI). It̉s main therapeutic use is in the nervous system and also the frequent long-term need treatment of Alzheimer̉ disease(Xiong et al 2005). to use the drugs even after withdrawal. Investigations have shown that the AChEI, Opioids are potential drugs for creating addictive donepezil, microinjected into the NAc or states. Morphine, as a member of this group, systemically administered suppresses cocaine- activates the opioid receptors, which in turn modulate seeking behavior (Zhou et al 2007). systemic various cellular functions(Bailey et al 2005). administration of donepezil can be attenuated, Researchers consider opioid drugs rewarding, metamphetamine(MAP)- CPP (Corrigall et al 2002). because they cause an increase dopamine release in CPP is a technique commonly used in animal studies the nucleus accumbens (NAc) (Robinson et al 2004). to evaluate preferences for environmental stimuli that Furthermore, administration of morphine results in a have been associated with a positive or negative synaptic plasticity in the mesolimbic dopaminergic reward. The technique is often used to determine the synapses(Saal et al 2003). In addition to the NAc, the addiction potential of drugs (Derea et al 2010). Journal of Basic and Applied Advances in Sciences Vol. 4, No. 1 (2016), 34-38
Finding indicate that, opioids administration usually In the conditioning stage that lasted for 8 days, after cause various social and behavioral damage in receiving the treatment, different groups of mice addicts , and they will have difficulties recognizing were randomly placed in either the black or the their roles in the society (Weiss et al 2002). white compartment for 30 minutes. The guillotine Consequently finding an effective way to treat windows were closed and the mice were not able to addiction is one of the most important concerns in move freely in the whole box. This procedure was every society. Considering the aforementioned facts, repeated for 8 consecutive days exactly the same the aim of the present study was to find the effect of time. The animals were placed in the compartment memory improvement, by a cholinergic agonist on that they had been on the first day, so they could morphine-CPP in mice . become conditioned, and could be tested on the postconditioning day as to their tendency to choose MATERIALS AND METHODS
the same compartment(Adam et al 2009). Animals and drugs
During the conditioning period, prior to be placed Fifty mature male mice, weighing about 30-35 g randomly in either the white or the black with an average age of 90 days, were used in the compartment, the mice in the control group did not study. The mice were divided into 5 groups of 10 receive any treatment, while the witness group including the control group, the witness group and received 1ml of distilled water and the morphine three experimental groups which were treated either group received 10mg/kg morphine, subcutaneously. by morphine, donepezil or morphine + donepezil. This amount of morphine is supposed to induce the ̉protocol was made based on the best CPP (Vargas-Perez et al 2009). The donepezil international laws protecting lab animals and was group had a subcutaneous injection of 2.5 mg/kg of confirmed by the ethics committee of the Fars the drug (Knol et al 2008), After 15 minutes, when Science and Research Branch, Islamic Azad the maximum effect of the drug was obtained University. Morphine was bought from Iran Daru (Ramesh et al 2007), the mice were placed in one of and donepezil was obtained from Sobhan Daru drug the compartments. The morphine + donepezil group companies (Tehran, Iran).The acetylcholinesterase received 2.5 mg/kg donepezil subcutaneously, and inhibitor, donepezil at the dose of 2.5 mg/kg was after 15min, they were injected with 10 mg/kg of dissolved into 1ml of distilled water and administered morphine through the same route and were randomly subcutaneously.The mice receive 10mg/kg morphine placed in one of the two compartments. at the volume of 1mg/kg subcutaneously. The post-conditioning stage included the fourth, the ninth days ( that is a day after the last day of the Conditioned place preference
injection), the twelfth day ( that is four days after To do the CPP experiment, a plexiglass box (15cm × the last day of the injection) and the sixteenth day ( 37cm × 15cm) was used. The box was divided into that is eight days after the last day of the injection). two separate compartments by a central section The purpose of post conditioning stage on the 12th which were connected by a guillotine window. The and 16th days was to find out if the effects of the two compartments had equal dimensions of various injections had lasted after discontinuing the 15×15×15 cm (Funada et al 2002 ). The walls and the floor of one of the compartments were white, while In the post conditioning stage, each animal was the walls and the floor of the other were black. placed for 10 minute in the compartment that had To study the effect of each drug, a group of mice was been during the preconditioning day(Kenneth et al chosen. Then through an eighy-day period the 2009). The mice were free to move to either following stages were followed . compartment in the box as in the pre-conditioning The Preconditioning day which is called the day of day. The time each animal spent in a given the animal ̉familiarity with the environment lasted compartment was measured by a chronometer to find one day. At this stage, after removing the guillotine out, if donepezil, as a cholinergic agonist and a window, each animal was placed in either the white memory improvement drug, has any effect on or the black compartment for 10 minute. This was morphine dependency in the animals. recorded in order to place the mice in the same compartment on the day of the test. The length of Statistical analysis
time that each animal spent in each of the The data were analyzed by one way ANOVA, compartments was recorded separately. The mice Duncan test and Student's t-test, using SPSS usually did not show any preferences at this stage, software. A P value of < 0.05 was considered as and spent 50% of the time in either compartment.If statistically significant. any of the mice stayed in a compartment more than 90% of the time, it was eliminated from the study. Journal of Basic and Applied Advances in Sciences Vol. 4, No. 1 (2016), 34-38
The results revealed that the mice did not have any inhibition by donepezil in these regions might thus preferences to any special compartment on the pre- activate auto-inhibitory mAchRs and nAchRs, conditioning day of the total time spent in the box thereby inhibiting the activity of these cells resulting ,the Mice spent an average of 50% in the white in a decrease in the release of Ach in the VTA and compartment and 50% in the black on(Table 1). SNc (Zheng et al 2003). It can be concluded that The statistics shows that there is no significant donepezil decreases morphine-CPP through an difference between the mean of the time spent in inhibitory effect on the dopaminergic neurons of stimuli paired chamber on the test days and the pre- VTA (Kalivas et al 2005). conditioning day within witness,donepezil and the The results show no significant difference between control groups and between them separately. the means of the time spent in stimuli paired chamber However, there was a significant increase (p≤0.05) in on the4th, 9th , 12th and 16th test days by the the mean of the time spent in stimuli paired chamber morphine + donepezil group and the morphine group. by the morphine group on the test days compared In the morphine withdrawal test, 10mg/kg morphine with pre-conditioning day. In addition, a significant administration subcutaneously, increased self- increase (p≤0.05) is also evident in the mean of the stimulation responses until the end of the 5th week time spent during the test days by the morphine group (Frenois et al 2005). compared with the control group̉. Comparing the The expression of PSD-95 protein, in hippocampal mean of the time spent in stimuli paired chamber on CA1 region, influences addiction memory (Han et al the test days, no significant difference is observed in 2008). PSD-95 increases the plasticity of the the morphine group . dendritic spine and the synapse(Zarrindast et al Statistical analysis of the data reveals that the mean 2006). After morphine withdrawal, the expression of of the time spent in the stimuli paired chamber on PSD-95 decreases, but the plasticity of the dendritic the test days compared with the pre-conditioning spine and the synapse induced by morphine had been day by the morphine + donepezil group has also steady, therefore, the addiction memory induced by increased significantly (p≤0.05). Similar increases are morphine and craving for drugs are expressed obtained when comparing the mean of the time significantly(Zhu et al 2006).In addition when opiate spent in the stimuli paired chamber by the morphine withdrawal occurs, c-fos mRNA expression also + donepezil group and the control group on the same increases specially in the extended amygdala,VTA days. However, the mean of the time spent in the and hippocampus(De Vries et al 2002).This results in stimuli paired chamber by the morphine + donepezil an activation of dopaminergic neurons during group showed a significant decrease (p≤0.05) withdrawal(Bissiere et al 2004). compared to that of the morphine group. There was Thus,donepezil might decrease the negative effects of no significant difference between the mean of the opiate withdrawal and thus reduce compulsive time spent by the morphine + donepezil group in the morphine-seeking behaviors and relapse to opiate stimuli paired chamber on different test days (Table CONCLUSION
DISCUSSION
According to the results of the study, donepezil The results of this study show that donepezil, causes seems to diminish addiction. Hence, it is the reduction of Morphine-CPP. Investigations have recommended to do similar research on other shown that lesions of NAc cholinergic neurons acetylcholine estrase inhibitors using animal and prevent the inhibitory effects of donepezil on morphine-CPP. studies show that increased Ach released in NAc by donepezil, decreases the release of dopamine from dopaminergic neurons in this region (Kume et al 2005). The authors of the article would like to extend their Data suggests that both Muscarinic acetylcholine special thanks to the Islamic Azad University, Fars receptors (mAchRs) and nicotinic acetylcholine Science and Research Branch, and Islamic Azad receptors (nAchRs) are placed on the dopaminergic University, Kazerun Branch for their cooperation in neurons and regulate these neurons in VTA and substantia nigra pars compacta(SNc) (Fiorillo et al 2000). Increased levels of Ach due to AchE Journal of Basic and Applied Advances in Sciences Vol. 4, No. 1 (2016), 34-38
Table 1: mean and standard deviation of the time animals spent in either compartments on the preconditioning day(in seconds) ( N=10) Animal presence in the box(in seconds) Table 2: Mean and standard deviation of the time spent on different experimental days by different test groups (in seconds) ( N=10) Mean and standard deviation of the time spent on different experimental days by different test groups(in seconds) *The mean difference is significant at the .05 level reinforcement and emotions. Behavioural Brain Research. 215:209–220 REFERENCES:
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