The study of the effects of high dose and low dose of morphine , and morphine addiction on the acquisition , consolidation and retrieval of the memory in passive avoidance test on mice.

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Author: Amin Ataee, Masoome Sabetkasaii, Ramin Ataee

The study of the effects of high dose and low dose of morphine , and morphine addiction on the acquisition , consolidation and retrieval of the memory in passive avoidance test on mice.

Amin.ataie* , Reza Lashgary, Abbas Hghparast , Sabet Kasaie , Ramin Ataee Department of pharmacology,Shahid beheshty Medical University . Tehran .IRAN * Dr.Amin Ataie ,Pharm D PhD candidate in Pharmacology: ataieamin@yahoo.com

Introduction :

It is important to know that increasing or decreasing the memory in some clinical circumstances is needed. In the treatment of major depression or in general anesthesia,decreasing the memory and in the treatment of Alzheimer,s diseas, dementia and pathological amnesia memory reinforcement has been needed. Many drugs reinforces and some of them diminishes the memory. For example, it is found that scopolamin , benzodiazepine ,barbiturate, phenytoine , digoxine ,general anesthetics, decreases the memory and many drugs ; piracetam, cholinomimetic drugs( in AD treatment), neuropeptide, vasopressin, vasodilators,naloxon and..... increases the memory.

Dependence and impairment of learning and memory are two well-established features caused by abused drugs such as opioids. The hippocampus is an important region associated with both drug dependence and learning and memory. It is found that chronic exposure of mice to morphine for 10 days produced robust morphine withdrawal jumping and memory impairment, and also resulted in a significant downregulation of hippocampal protein levels of three metabolic enzymes, including Fe-S protein 1 of NADH dehydrogenase, dihydrolipoamide acetyltransferase or E2 component of the pyruvate dehydrogenase complex and lactate dehydrogenase[1].

Further real-time quantitative PCR analyses confirmed that the levels of the corresponding mRNAs were also remarkably reduced. Consistent with these findings, lower ATP levels and an impaired ability to convert glucose into ATP were also observed in the hippocampus of chronically treated mice[1]. Opioid antagonist naltrexone administrated concomitantly with morphine significantly suppressed morphine withdrawal jumping and reversed the downregulation of these proteins.

Acute exposure to morphine also produced robust morphine withdrawal jumping and significant memory impairment, but failed to decrease the expression of these three proteins[1]. The possible involvement of N-methyl-D-aspartate (NMDA) receptors in the nucleus accumbens (NAc) in nicotine's effect on impairment of memory by morphine was investigated[2]. It is found that Pretest administration of the same doses of morphine reversed impairment of memory because of post-training administration of the opioid. Moreover, administration of nicotine (0.2 and 0.4 mg/kg, s.c.) before the test prevented impairment of memory by morphine (5 mg/kg) given after training[2]. The results suggest a possible role for NMDA receptors of the NAc in the improving effect of nicotine on the morphine-induced amnesia[2]. It has recently emerged that there is a commonality in the molecular mechanisms underlying long-term neuronal changes in drug addiction and those mediating synaptic plasticity associated with learning and memory. Several lines of evidence indicate that the brain regions involved in learning and memory and those underlying drug addiction may overlap.

Learning and memory processes and drug addiction also share intracellular signaling cascades and are associated with similar changes in synaptic plasticity. the hypothesis was approved that learning and memory are essential for the development of addiction. The data suggest that hippocampal calcineurin signaling is involved in the long-term adaptation after chronic drug treatment in a way that may parallel its role during memory formation. Such an effect is consistent with the common molecular mechanisms shared by addiction and memory processes.

Given the well-established role of the hippocampus in associative learning and memory[3]. It is found that Kappa opioids selectively control dopaminergic neurons projecting to the prefrontal cortex [4]. Dopaminergic afferents arising from the ventral tegmental area (VTA) are crucial elements in the neural circuits that mediate arousal, motivation, and reinforcement. Two major targets of these afferents are the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAc). Whereas dopamine (DA) in the mPFC has been implicated in working memory and attentional processes [4]. On the other hand a variant of the sigma receptor type-1 gene is a protective factor for Alzheimer disease [5]. Some preclinical evidence suggests that the sigma receptor type 1, which plays several roles in learning and memory, may also be involved in the pathogenesis of Alzheimer disease (AD). The authors provide here genetic evidence that the sigma receptor type 1 (SIGMAR1) gene is involved in susceptibility to AD[5]. . In this paper we try to distinguish the effects of three doses of morphine and morphine addiction on learning and short term memory in mice. ,we can describe aquisition , consolidation and retrieval of the memory in mice.

Methods:

Male albino mice (25-30g) housed in clear cages in a room maintained at constant temperature 23 -25 c and kept on a 12/12 h light/dark cycle .Food and water were provided ad libitum.Mice was injected (ip) with three doses of morphine (1,3,9 mg/kg) respectively and distilled water as solvent of morphine, and some mice addicted with Marshal methods . In acquisition test ,drugs (morphine) was injected (i.p.) 1 h before animals training. In consolidation test, drugs was injected (i.p.) immediately after teraining. In retrieval test,23.5 after training ,drugs wae injected and 0.5h later,mice was tested. All of the animals was tested 24 h later.In this behavior study we used passive avoidance apparatus (shuttle box) and mice has to be learned to avoid from stepping throw illuminated room to dark room. if they go to dark room they received electrical shok.

Induction of dependence(Marshall-Smith method) :

Some of mice in our study was addicted with morphine in Marshal method and then the effect of addiction on learning and memory was studied. Morphine sulfate was injected subcutaneously 3 times daily at 9:30, 13:30 and 17:30 on the following dosage schedule. The first three doses were ,50-50-75mg/kg ;the second three doses were,75-75-100mg/kg in second days;and 100-100-125mg/kg in third day.A dose of 50mg/kg of morphine sulfate also was injected on the 4th day (2h before naloxone injection). Hyperactivity and the straub tail effect were seen after morphine injection. With chronic administration of morphine sulfate, loss of weight (8-12%)and death (5-10%)also were observed. Jumping Groups of mice were tested for the occurance of jumping after their tenth injection of morphin on day 4.Two hours after the last dose of morphine (50mg/kg),abstinence was precipated by an intraperitoneal (i.p.)injection of naloxone ;then animals were placed individually on filter paper in a cylindrical glass (25 cm in diameter, 40 cm height)and the number of jumps was recorded over a 30 min period.

Behavioral studies :

Passive avoidance (PA)learning :

Male mice, weighing 25-39 g, of the ddY strain were used. They were housed in 42 x 26 x 15 cm plastic cages with free access to food and water under a natural day/night regime. The step-through type of one-trial passive avoidance task was used. The apparatus consisted of an illumi nated and a dark compartment (each 4 x 13 x 10 cm) adjoining each other through a small gate (3 cm in diameter) with a grid floor, 2.5mm stainless steel rods set 7 mm apart. At the training trial, the animal was placed in the illuminated compartment facing away from the dark compartment. When the animal entered into the dark compartment, an electric shock (ES)( 50hz square wave , 1.2 mA constant current shock was applied for 1.5s) was delivered through the grid floor until the animal returned to the illuminated compartment. At the test trial,24 hr after the training trial, the animal was again placed in the illuminated compartmentand its latency to enter the dark compartment, maximum 600 sec, was measured. Data were expressed as medians and interquartile ranges, and they were analyzed by Kruskal Wallis non-parametric one way analysis followed by the 2-tailed Mann-Whitney U test for the paired comparisons. In all statis tical evaluations, P •

PASSIVE AVOIDANCE APPARATUS (STEP THROUGH )

In acquisition test ,drugs (morphine) was injected (i.p.) 1 h before training.and 24h after training,mice was testsd.In consolidation test, drugs was injected (i.p.)after teraining ,and 24h later,,mice was tested. In retrieval test,23.5 after training ,drugs wae injected, 0.5h later,mice was tested.

Memory acquisition test :

In our study 3 groups of mice(test,blank,control) were considered.In blank and test group saline and morphine was injected respectively 1h before training and 24h after training mice was tested.morphine in three doses,1—3—9mg/Kg and in volum maximum 0.3 cc was injected (i.p.) respectively.

Results :

With seeing to statistic results(pPASSIVE AVOIDANCE APPARATUS (STEP THROUGH )

Figure 1- latency times pretraining and posttraing in acquisition test , (morphineinjected9mg/kgip.)

With considering statistical results in sham group(salin injected ip.) we see a significant different between latency times in first and second days. It means that mice was learned to avoide steping through from light to dark parts in passive avoidance tests.

Figure 2-Latency times pretraining and post training in acquisition test (saline was injected ip in sham group) X = NUMBER OF MICE Y= LATENCY TIME ( step therouh )

With comparision between latency times of sham (saline)group and test group (morphie 9mg/kg) in second day (24h after training)and analyzes the results ,we can find that there is no significant different between test and sham groups that means ,morphine in high doses (9mg/kg ) has not significant effect on learning and memory in passive avoidance test.

Figure 3-latency times post training in test group(morphine inj.ip.9mg/kg) and sham group (salin inj.ip.)

We tested mice with low dose( 1mg/kg ) and middle dose(3mg/kg) of morphine and did not see any significant difference between test and shsm groups , therefore morphine ih these doses has no effect on learning and memory.

Effect of addiction on memory :

Mice was addicted with Marshal method ,and after repeated injections of high doses of morphine, in 4th day , jumping was seen after 2mg/kg naloxon injection.

Addicted mice was trained in passive avoidance trial and retention of memory (acquisition)was tested in passive avoidance test; test results has been shown figure bellow . As we see in below baradigram a significant rising in latency time in second day (24 after shock) is occurred.that means ,addiction has no decreasing effect on memory retention.

Figure 4- Latency times pre traning and post training in addicted mice with Marshal method.

The study of retrieval of memory in mice:

In the study of retrieval of memory in mice the mice was trained in the first day and when mice was learned to avoide stepping through the hole ,(from light to dark part)in passive avoidance apparatus; 24 h later, they was tested for retrieval of memory. morphine in three doses of 1,3,9mg/kg injected respectively, 0.5 h before testing (in the second day ).latency times in pretraining and post training was measured.

Figue 5- latency times pretrainning and post training in retrieval test ( morphine inj. Ip. 1 mg/kg)

Figure 6-Latency times in pre training and post training in retrieval test (morphine inj.ip.3 mg/kg)

Figure 7- Latency times in pre training and post training in retrieval test (morphine inj. Ip. 9mg/kg)

The study of these baradiagrams ,we understand that morphine in three dose 1,3,9 mg/kg ,dosn't decrease retrival of memory and dosn't affect the mice avoidance learning ,compare with saline group .and morphine addiction has no decreasing effect on learning in passiove avoidance test.

Discussion:

By looking at the results in this study, It is found that morphine in low and high dose has no decreasing effects on learning and memory and may reinforced it. On the other hand addiction has no decreasing effect on the memory.By attention to these data ,we suppose that morphine with mu-receptor or sigma-1 or kappa opiod receptors may have positive effect on memory. The data suggested that hippocampal calcineurin signaling is involved in the long-term adaptation after chronic drug treatment in a way that may parallel its role during memory formation[3]. Such an effect is consistent with the common molecular mechanisms shared by addiction and memory processes. Given the well-established role of the hippocampus in associative learning and memory[3]. It is reported that kappa-opioid receptor agonists improve impairment of learning and memory in mice and/or rats. In some study, the effects of KT-95 were investigated in an acetic acid-induced writhing test and scopolamine-induced memory impairment test using spontaneous alternation performance in a Y-maze and a step-down type passive avoidance test[6]. The demonstration of the multiplicity of opiate receptor types has led to the understanding that,depending on their site of action, opioid peptides as well as opiate alkaloids may bind to more than one opiate receptor subtype. Impairment of memory performance in the animals because of post-training administration of morphine (5 mg/kg) was also prevented by pretest administration of a noncompetitive NMDA receptor antagonist, MK-801 (0.75 and 1 microg/rat)[2]. Interestingly, an ineffective dose of MK-801 (0.5 microg/rat) in combination with low doses (0.075 and 0.1 mg/kg) of nicotine, which had no effects alone, synergistically improved memory performance impaired by morphine given after training. On the other hand, pretest administration of NMDA (0.1 and 0.5 microg/rat), which had no effect alone, in combination with an effective dose (0.4 mg/kg, s.c.) of nicotine prevented the improving effect of nicotine on memory impaired by pretreatment morphine.

In addition to the two main µ opiate receptor subtypes, µ1 and µ2, it has been demonstrated a third µ opiate receptor (µ3) that is selective for opiate alkaloids but insensitive to opioid peptides. Recently, the µ3 opiate receptor subtype has been cloned from human immune, vascular and neural tissues. This µ3 story complements many biochemical reports; demonstrating morphine is an endogenous signaling molecule, functioning in the capacity of a neurotransmitter and hormone[7]. It is found that facilitation of memory retrieval by pretest morphine mediated by mu but not delta and kappa opioid receptors[8]. Mice were trained to avoid electric shock (0.6 mA) in a step-through type passive avoidance learning task, retention being measured 24 h after the training trial. Morphine 10 mg/kg administered 30 min before the test trial (pretest) facilitated memory retrieval, and the effect was completely antagonized by 1 mg/kg naloxone, a selective mu-opioid receptor antagonist. On the other hand, pretest administration of 0.01-10 mg/kg DTLET, a selective delta-opioid receptor agonist, did not produce the same effect as morphine. Nor-binaltorphimine, a kappa-opioid receptor antagonist, did not antagonize the effect of pretest morphine, at doses of 1 and 2 mg/kg. These results suggest that the facilitation of memory retrieval by pretest morphine is mediated through mu- but not delta- or kappa-opioid receptors[8].

Reference:

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2- Nicotine improves morphine-induced impairment of memory: possible involvement of N-methyl-D-aspartate receptors in the nucleus accumbens. Ahmadi S, Zarrindast MR, Haeri-Rohani A, Rezayof A, Nouri M. Department of Animal biology, School of Biology, University College of Science, University of Tehran, Tehran, IranDev Neurobiol. 2007 Jul;67(8):1118-27

3-Memory processes and addiction: involvement of the calcineurin signaling pathway. Katedra i Zakład Farmakologii z Farmakodynamika Akademii Medycznej im. prof. F. Skubiszewskiego w Lublinie, Poland

grazyna.biala@am.lublin.pl Postepy Hig Med Dosw (Online). 2007;61:199-203 .

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5- A variant of the sigma receptor type-1 gene is a protective factor for Alzheimer disease. Am J Geriatr Psychiatry. 2005 Dec;13(12):1062-

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