Caspian Journal of Neurological Sciences
http://cjns.gums.ac.ir BDNF Pretreatment Attenuates Morphine-Induced Learning
and Memory Impairment in Rats
Babaei Parvin (PhD) 1,2* , Vahdati Sanaz (MD Stu) 2 , Soltani-Tehrani Bahram (PhD) 1 A B S T R A C T
Article type:
Background: It has been known that Brain-Derived Neurotrophic Factor
Original Article (BDNF) is involved in neural survival and long term memory (LTM). Here we hypothesized that BDNF as a potent neurotrophic factor might modulate amnestic effect induced by morphine. Objectives: The aim of this study was to examine whether infusion of
Article history:
exogenous BDNF in the CA1 regions of the dorsal hippocampi could ameliorate Received: 19 January 2014 Accepted: 23 May 2014 memory impairment induced by morphine. Available online: 20 March 2015 Materials and Methods: Forty rats were divided into 5 groups for dose
CJNS 2015; 1 (1): 12-18 response study of morphine (2.5, 5, 7.5 and 10 mg/kg morphine, and saline, intraperitoneal) on memory retention. For second part of the experiment 24 animals were divided into three groups: (morphine +BDNF, morphine + saline and saline + saline). Two weeks after stereotaxic surgery, animals received 0.5 μl bilateral infusion of either saline or BDNF (5 µg/rat) intrahippocampally, 30 1. Cellular & Molecular Research Center, Faculty of Medicine, Guilan University minutes before morphine treatment (7.5 mg/kg, i.p.). Step-through inhibitory of Medical Sciences, Rasht, Iran avoidance task has been used to examine retrieval of memory formation, 1.5 and 24 h after the training. 2. Physiology Department, Faculty of Medicine, Guilan University of Medical Results: The results showed that systemic administration of 7.5 and 10 mg/kg
Sciences, Rasht, Iran morphine compared with saline immediately after the training impairs long- term retention of memory for passive avoidance task in rats tested 24 hours later (p < 0.01). Surprisingly intra-CA1 microinjection of BDNF 30 minutes
prior to injection of morphine significantly prevented amnesia (p < 0.001).
Conclusions: These findings suggested that increase the level of BDNF in the
CA1 region of the hippocampus during 30 minutes time window before
morphine administration might modulate morphine-induced amnesia.
Brain-Derived Neurotrophic Factor; Amnesia; Morphine; Rats

*Corresponding author:
Physiology Department, Faculty of Copyright 2015 Caspian Journal of Neurological Sciences. All rights reserved.
Medicine, Guilan University of Medical Sciences, Rasht, Iran  Please cite this paper as:
Email: [email protected] Babaei P, Vahdati S, Soltani-Tehrani B. BDNF Pretreatment Attenuates Morphine-Induced Learning and Memory Impairment in Rats. Caspian J Neurol Sci 2015; 1(1):12-18.

orphine has been widely used in influence on the processes of learning and pain treatment, but its strong causes memory impairment. Impairment of M dependency potential is a serious memory has been reported after both chronic challenge to its clinical usage. It is reported and acute morphine administration (1-4). Pre- that opioid peptides, especially morphine or post-training administration of morphine BDNF and Morphine -Induced Amnesia Babaei P et al. impairs specifically performance in the xylazine (100 and 10 mg/kg, respectively), passive avoidance task (3, 5). and fixed in the flat –skull position using Memory is critically depended on the hippocampus region, and can be divided into Instruments, USA).The rats' scalp were cut, a short-term and long-term forms (6, 7). small craniotomy was drilled and cannulas Consolidation of short term (STM) to long- term (LTM) memory takes place immediately implanted into the CA1 region of the following the training experience (8, 9). This hippocampus at coordinates: AP − 3mm, L ± critical period is influenced by different 2mm and V − 2.8mm (19). neurotransmitters Micro infusions: Moreover, LTM strongly depends on protein Morphine sulphate (Darupakhsh, Iran) and synthesis cascades and neurotropic factors human recombinant BDNF (R&D, USA) were particularly BDNF (14-16). According to the dissolved in sterile 0.9% saline. First, animals previous reports hippocampal BDNF appears were divided into five groups (saline, to be necessary for LTM formation in the morphine 2.5, 5, 7.5 and 10 mg/kg.) for dose different discrete periods, immediately after, response study of morphine. Secondly, 24 rats 1.4 hour and 3.6 hours after training (17, 18). were divided into three experimental groups Although many studies proposed that BDNF is (morphine + BDNF, morphine + saline and a key molecule mediating persistence and saline + saline, n = 8 each) and underwent maintenance LTM, it is still unclear whether stereotaxic surgery. Two weeks later, animals received a 0.5 μl bilateral infusion of saline or BDNF pretreatment is capable of ameliorating memory (2.5µg/0.5μl/side) conducted to answer to this question. hippocampally 30 minutes before morphine treatment (7.5 mg/kg, i.p.). Materials and Methods
Inhibitory avoidance apparatus: The apparatus consisted of two equal size of Sixty four male wistar rats weighing 200- compartments, one light and one dark 250g were used in this study. They had free (20×20×30 cm high), connecting via a access to food and water, and kept at 24 ± 2 C guillotine door (7×9 cm). The floor of the dark under a 12h/12h light dark cycle. Each group compartment was made of stainless steel rods consisted of 8 animals and each animal was (2.5 mm in diameter) with a distance of 1 cm. tested once. All experiments were conducted For the acquisition trial, rat was placed in the in accordance with the Guide for Care and Use light compartment and the door between the of Laboratory Animals (National Institute of two compartments was opened 20 seconds Health Publication No.80-23, revised 1996) later. When the rat entered the dark approved by the Research and Ethics compartment, the door closed and an electric Committee of Guilan University of Medical foot shock (1 mA, 50 Hz, 5 seconds) was Sciences. delivered through the grid floor. For the retention trial, the rat was again placed in the light compartment 1.5 and 24 hours following The animals were anaesthetized via the the acquisition trial. The latency time intraperitoneal (i.p.) injection of ketamine and

Caspian J Neurol Sci 2015 March; 1(1): 12-18 (seconds) for entering the dark compartment Upon completion of the inhibitory avoidance test, each rat was deeply anesthetized and 1 ml of a 4% methylene-blue solution was bilaterally infused through the cannula into the decapitated and the brains were removed and placed in formaldehyde for two days (10%). Diagram 1. The effect of post-training administration of morphine on
step-through latency. The rats (n = 8 per group) received post-training
Then, the brains were sliced and the injection saline (1 ml/kg, i.p.) or varying doses of morphine (mor) (2.5, 5, 7.5 and 10 mg/kg, i.p.) and were tested after 1.5 h and 24 h. **p < 0.01 site was verified according to the Paxinos compared with the saline. &Watson, brain atlas 2005(19), (Figure1). Following infusion of BDNF or saline, a 30 minutes wait-time and then an i.p. injection of morphine or saline, the rats performed the acquisition trial of the inhibitory avoidance test. Memory was assessed during the retention trial by measuring step-through latency in the passive avoidance task observed 1.5 and 24 hours after the acquisition trial Figure 1. Photomicrograph from rat brain section showing the
extension of the area reached by infusions into the hippocampus.
Data analysis: Each value represents the mean ± standard error of the mean (S.E.M.). After assaying the normality of data with Kolmogorov - Smirnov test, comparison of data among groups was BDNF + mor (1.5h) BDNF + mor (24h) performed using one-way analysis of variance Diagram 2. The effect of acute BDNF pretreatment on memory
with Tukey's post-test , when the p-values was consolidation prior to morphine injection. The latencies for the rat (n = 8 per group) to enter to the dark room 1.5 h and 24 h after the < 0.05, the difference was considered to be training were expressed as mean ± S.E.M. *p < 0.01 , ***p < 0.001 significant. Calculations were performed using compared to morphine. the SPSS statistical package version 19. There was a strong significant group effect (F(2, 21) = 37; p = 0.001, one way ANOVA) showing that the group receiving BDNF 30
During the training trial, there was no
minutes prior to morphine administration significant difference among groups (p > 0.05, (BDNF + morphine) took longer to enter the one way ANOVA). Systemic post-training dark chamber compared to either control administration of morphine (7.5 and 10 group, saline + morphine or saline + saline. significantly decreased latency to enter to the dark compartment compared to the control receiving saline (p < 0.01; Diagram 1). BDNF and Morphine -Induced Amnesia Babaei P et al. Discussion
memory by promoting neural plasticity (29, 30). Additionally, BDNF is required for the In the current study, we showed that consolidation of short-term and long-term administration of morphine immediately after memory, especially in glutamatergic and training impairs memory retention in the GABAergic synapses (29, 31-33). Previous studies report an increase in BDNF mRNA in the dentate gyrus of the hippocampus hippocampal CA1 region, 30 minutes before following the acquisition trial (17, 34). morphine administration, ameliorated the Conversely, Alonso et al. (2002) showed that morphine-induced memory impairment. infusion of an anti-BDNF antibody impaired The impairment of memory induced by LTM, when given 15 minutes before or 1 and morphine is consistent with previous studies 4 hours after training (17). Our results were in (3, 20, 21). Based on the immediate post- agreement with the Johnston et al. study training administration of morphine and its showing that injection of recombinant BDNF short half-life in the brain (approximately 1 before training enhanced memory recall in day hour), it is likely that morphine is affecting the old chicks (35). Our findings confirmed that early phase of memory consolidation (22). 30 minutes before the induction of amnesia Opioids produce their principal effects on was a sensitive time window which was memory by binding to at least three different types of receptors: μ, δ and κ opioid receptors critical for LTM formation, so it is more likely to relate this result to acute effects of BDNF (3). All opioid receptor subtypes inhibit on synaptic transmission, rather than protein adenylyl cyclase and Ca2+ channels and synthesis due to inadequate time. Binding of stimulate K+ channels. These effects are BDNF to tropomyosin receptor kinase B required for morphine-induced amnesia in the (TrkB) triggers a number of intra-cellular passive avoidance test (23). Alternately, signalling pathways from long lasting effects neuropharmacological studies have revealed to enhancing early long term potentiation that activation of opioid receptors may (LTP) and phosphorylation of synaptic decrease the function of the cholinergic proteins (36-39). Rapid Ca2+ influx through system (24-26). However, one cannot exclude the possibility that morphine interferes with subsequent protein phosphorylation events other neurotransmitter systems, such as modify pre-existing synapses and trigger early adrenergic or dopaminergic, to induce amnesia LTP, an important mechanism mediating memory formation (40, 41). A recent Interestingly, we also showed that infusion of surprising study revealed suppressive effect of BDNF into the dorsal hippocampus 30 BDNF as a negative modulator on morphine minutes before training was sufficient to reward (42). Some of the neurotransmitters prevent the amnestic effect of morphine as regulate BDNF synthesis, and in turn they are well as enhance memory consolidation regulated by BDNF. For example BDNF compared to the saline control group. To our knowledge, this study was the first to glutamate release in the hippocampus, demonstrate that BDNF attenuates morphine- depolarizes neurons and interferes in Ca induced memory impairment. signaling (43-51). The significance of these Animal and human studies suggest that reciprocal regulations was intriguing and hippocampal BDNF plays a major role in Caspian J Neurol Sci 2015 March; 1(1): 12-18 could represent a novel framework into the 6. Milner B, Squire LR, Kandel ER. Cognitive molecular basis of morphine–induced amnesia. Neuroscience Review and the Study of Memory. Neuron 1998; 20:445-68. For future studies we propose administration 7. Kumaran D, Maguire EA. The Human of antibody against BDNF and also different Hippocampus: Cognitive Maps or Relational doses of BDNF prior to morphine injection. Memory?. J Neurosci 2005; 25(31):7254-9. 8. Bekinschtein P, Cammarota M, Igaz LM, Conclusion
Persistence of Long-Term Memory Storage The present study shows that morphine Requires a Late Protein Synthesis-and BDNF- impairs the consolidation phase of long-term dependent Phase in the Hippocampus. Neuron recognition memory, possibly by preventing a 2007; 53(2):261-77. 9. Castellano C, Cestari V, Ciamei A. NMDA learning-induced increase in BDNF levels in the hippocampus. This study suggests that Processes. Curr Drug Targets 2001; 2(3):273- intra-hippocampal prevents the deficit in memory consolidation 10. Camera K, Mello C, Ceretta A, Rubin MA. caused by morphine. Systemic Administration of Polyaminergic Agents Modulate Fear Conditioning in Rats. Psychopharmacology 2007; 192(4):457-64. Conflict of Interest
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