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Interaction between dorsal hippocampal NMDA receptors and lithium on spatial learning consolidation in rats
Accepted Manuscript Title: Interaction between dorsal hippocampal NMDA receptors and lithium on spatial learning consolidation in rats Author: Leila Parsaeia Anahita Torkaman-Boutorabi Fereshteh Asadi Mohammad-Reza Zarrindast PII: DOI: Reference:
S0361-9230(16)30159-9 http://dx.doi.org/doi:10.1016/j.brainresbull.2016.07.007 BRB 9056
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
19-3-2016 4-7-2016 18-7-2016
Please cite this article as: Leila Parsaeia, Anahita Torkaman-Boutorabi, Fereshteh Asadi, Mohammad-Reza Zarrindast, Interaction between dorsal hippocampal NMDA receptors and lithium on spatial learning consolidation in rats, Brain Research Bulletin http://dx.doi.org/10.1016/j.brainresbull.2016.07.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Interaction between dorsal hippocampal NMDA receptors and lithium on spatial learning consolidation in rats Leila Parsaeiaa,b, AnahitaTorkaman-Boutorabi*b, Fereshteh Asadia, MohammadReza Zarrindast*a,b, c,d,e a
Department of Pharmacology, School of Medicine, Tehran University of Medical
Sciences, Tehran, Iran b
Department of Neuroscience, School of Advanced Technologies in Medicine,
Tehran University of Medical Sciences, Tehran, Iran c
Institute for Studies in Theoretical Physics and Mathematics School of Cognitive
Sciences (IPM), Tehran, Iran d
Institute of Cognitive Sciences, Tehran, Iran
e
Iranian National Center for Addiction Studies, Tehran, Iran
Co-corresponding Authors: Mohammad-Reza Zarrindast, [email protected], AnahitaTorkaman-Boutorabi, [email protected]
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Highlights Intra-CA1 injection of lithium impaired spatial learning. Intra-CA1 injection of NMDA increased spatial learning. Intra-CA1 injection of D-AP5 decreased spatial learning. D-AP5 potentiated, while NMDA restored lithium-induced spatial memory deficit.
Abstract Previous investigations have shown that NMDA receptors play an important role in learning and memory process. Lithium is a primary drug for management and prophylaxis of bipolar disorder. It can regulate signal transduction pathways in several regions of the brain and alter the function of several neurotransmitter systems involved in memory processes. The present study aimed to test the interaction of NMDA glutamatergic system of the CA1 region of dorsal hippocampus and lithium on spatial learning. Spatial memory was assessed in Morris water maze task by a single training session of eight trials followed by a probe trial and visible test 24 h later. All drugs were injected into CA1 regions, 5 min after training. Our data indicated that post- training administration of lithium (20 µg/rat, intra-CA1) significantly impaired memory consolidation. Intra2
CA1administration of NMDA, a glutamate receptor agonist (0.001 and 0.01 μg/rat) showed spatial learning facilitation. Infusion of D-AP5, a glutamate receptor antagonist (0.05 and 0.1 μg/rat) showed impairment of spatial memory. Our data also indicated that post- training administration of ineffective dose of NMDA (0.0001 μg/rat) significantly decreased amnesia induced by lithium in spatial memory consolidation. In addition, post-training intra-CA1 injection of ineffective dose of D-AP5 (0.01 μg/rat) could significantly increase lithium induced amnesia. It seems probable that signaling cascades of NMDA receptors that regulates synaptic plasticity are targets of anti-manic agents such as lithium. Our results suggest that NMDA receptors of the dorsal hippocampus may be involved in lithium-induced spatial learning impairment in the MWM task. Key words: D-AP5; Lithium; Morris Water Maze; NMDA; Rat 1. Introduction Lithium is an important mood stabilizing drug which is used for the treatment of manic-depressive illness. It can potentiate the effects of antidepressant drugs (Schou, 1968). There are several reports showing that lithium treatment decreased learning, memory, and speed of information processing in bipolar patients and in control subjects (Pachet and Wisniewski, 2003; Stip et al., 2000). It seems that lithium effects are due to neuroplastic alterations involving intracellular signaling 3
pathways, transcription factors, and regulation of gene expression (Al Banchaabouchi et al., 2004).The data concerning the effect of lithium on learning and memory are controversial. Some reports introduce lithium as a neuroprotective agent (Creson et al., 2003). However, a number of investigation indicate the memory impairing effect of lithium in both human and animal (Al Banchaabouchi et al., 2004; Parsaei et al., 2011). It has been shown that lithium can regulate signal transduction pathways in several regions of the brain. The drug may also change the function of different neurotransmitter systems (Manji et al., 1995), including acetylcholine (Ach), serotonin (5HT), dopamine (DA), N-methyl-D-aspartic acid (NMDA), nitric oxide and neurotrophic factors (Ghasemi and Dehpour, 2011). In particular, the N-methyl-D-Aspartate (NMDA) subclass of glutamate receptors was obviously involved in conditioned taste aversion paradigm, an action that was modulated by lithium (Ferreira et al., 2002) .There is suggestion that NMDA receptor/nitric oxide (NO) signaling may mediate some responses of lithium in the brain and peripheral tissues (Ghasemi and Dehpour, 2011). NMDA signaling has been shown to be involved in the antidepressant-like effects of lithium (Ghasemi et al., 2009). Acute lithium administration stimulated glutamate release, which was accompanied by an increase in inositol 1,4,5-trisphosphate (IP3) accumulation (Hokin et al., 1996).
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It has also been reported that the increase in IP3 accumulation was a result of selective activation of the NMDA receptors by glutamate (Hokin et al., 1996) . Hippocampal glutamate levels have been documented to be elevated in stably remitted bipolar patients receiving chronic lithium maintenance therapy for an average of 10 years (Colla et al., 2009). Considering the above-mentioned points and findings, the present study was designed with the following aims: (i) to determine the effect of acute intra-CA1 administration of lithium on spatial memory consolidation using Morris Water Maze task;(ii) to evaluate whether NMDA glutamatergic receptors of the CA1 region of the hippocampus lead changes of lithium effect on spatial memory consolidation. 2. Materials and methods 2.1. Animals and substances Adult male albino Wistar rats (200–250 g, aged 10–12 weeks) were obtained from Pasteur Institute of Iran. They were housed in a temperature (25±2 ◦C) and humidity-controlled room. The animals were maintained under a 12:12-h light/dark cycle, with lights off at 7:00 p.m. Food and water provided ad libitum except for the periods of behavioral testing in Morris water maze (MWM). Each animal was used once only. All experimental procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 5
2.2. Drugs The drugs used in the present study were lithium chloride (LiCl; Merck, Germany), NMDA and D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5; Tocris Cookson Ltd, UK). All drugs were dissolved in sterile 0.9% saline just before using. All drugs were bilaterally injected into the hippocampal CA1 region (intra-CA1), 5 min after training. Control animals received 0.9% physiological saline. 2.3. Surgical procedure The animals were anaesthetized with intraperitoneal injection of ketamine hydrochloride (50 mg/kg) plus xylazine (5 mg/kg) and placed in a stereotaxic apparatus, while maintaining the incisor bar at approximately 3.3 mm below horizontal zero to achieve a flat skull position. A mid-saggital incision was made to expose the rat skull. Two stainless steel, 22-gauge guide cannulae were placed (bilaterally) 1 mm above the intended site of injection according to the atlas of Paxinos and Watson (Paxinos and Watson, 1997). Stereotaxic coordinates for the CA1 regions of the dorsal hippocampus were: −3 to −3.5 mm (depending on body weight) posterior to bregma, ±1.8 to 2 mm lateral to the midline, and −2.8 to −3 mm ventral to the dorsal surface of the skull. The guide cannula was anchored by a jeweler's screw, and the incision was closed with dental cement. After completing the surgery, two stainless steel stylets (27 gauge) were inserted into the guide
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cannulae, and left in places until injections were made. All animals were allowed to recover for 1 week after surgery. 2.4. Injection into the CA1 region of dorsal hippocampus The animals were gently restrained by hand; the stylets were removed from the guide cannulae. For intra-hippocampal CA1 injections of drugs, a 1.0-μl glass Hamilton syringe was used. The injection (inner) cannulae (27-gauge), which projected a further 1 mm ventral to the tip of the guides, were attached with polyethylene tubing to the Hamilton syringe. The injection volume of drugs was 1.0 μl (0.5 μl per side) for all groups. Each dose of drug used/rat was dissolved in 1.0 μl. The injections were made over a 60-s period, and the injection cannulae were left in the guide cannulae for an additional 60 s to facilitate diffusion of the drugs. 2.5. Apparatus The water maze was a black circular pool with a diameter of 136cm and a height of 60 cm, filled with 20±1 ◦C water to a depth of 20 cm. The maze was divided geographically into four equal quadrants and release points that were designed at each quadrant as N, E, S, and W. A hidden circular platform (10cm in diameter), made of Plexiglas, was located in the center of the southwest quadrant, submerged 1.5cm beneath the surface of the water. Fixed, extra maze visual cues were present at various locations around the maze (i.e., computer, MWM hard wares, posters). 7
A camera was mounted above the center of the maze and animal motion can be recorded and sent to the computer. A tracking system was used to measure the escape latency, traveled distance and swimming speed. 2.6. Behavioral procedure Spatial memory was assessed using a 2-day Morris water maze task. The single training session consisted of eight trials with four different starting positions that were equally distributed around the perimeter of the maze (Moosavi et al., 2007). The task requires rats to swim to the hidden platform guided by distal spatial cues. After mounting the platform, the rats were allowed to remain there for 20 s, and were then placed in a holding cage for 30 s until the start of the next trial. Rats were given a maximum of 60 s to find the platform and if it failed to find the platform in 60 s, it was placed on the platform and allowed to rest for 20s. Latency to platform and distance traveled were collected and analyzed later. After completion of the training, the animals were returned to their home cages until retention testing (probe trial) 24 h later. The probe trial consisted of 60s free swim period without a platform and the time swum in the target quadrant was recorded. After the probe trial, non-spatial visual discrimination task was done. In this session, the platform was elevated above the water surface and placed in the different positions in the four quadrants. This procedure is believed to provide
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information on the possible nonspecific effects involving motor, visual, or motivational abilities for learning and memory. 2.7. Drug treatment Eight animals were used in each experimental group. In the experiments where the animals received one or two injections, the control groups also received one or two saline injections. The intervals of drug administration were based on our previous studies in order to obtain a maximum response (Fig.1). 2.8. Experimental design 2.8.1. Experiment 1: Effect of lithium on spatial memory consolidation. The effect of post-training administration of lithium on memory consolidation was examined. Four groups of animals received bilateral intra-CA1 infusions of saline (1 μl/rat) or different doses of lithium (5, 10 and 20 μg/rat) 5 min after training trial. 2.8.2. Experiment 2: Effect of NMDA on spatial memory consolidation. The effect of post-training administration of NMDA on memory consolidation was examined. Four groups of animals received bilateral intra-CA1 infusions of saline (1 μl/rat) or different doses of NMDA (0.0001, 0.001 and 0.01 μg/rat) 5 min after training trial.
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2.8.3. Experiment 3: Effect of DAP-5 on spatial memory consolidation. The effect of post-training administration of DAP-5 on memory consolidation was examined. Four groups of animals received bilateral intra-CA1 infusions of saline (1 μl/rat) or different doses of DAP-5 (0.01, 0.05, 0.1, 0.25 μg/rat) 5 min after training trial. 2.8.4. Experiment 4: The effects of an ineffective dose of NMDA on the response to different doses of lithium on the spatial memory consolidation. In this experiment, eight groups of animals were used. Four groups received posttraining intra-hippocampal administration of saline (1 μl/rat) or lithium (5, 10 and 20 μg/rat), 5 minutes after training. In the other four groups the possible interaction between NMDA and lithium was examined. In this regard, an ineffective dose of NMDA (0.0001 μg/rat) was administered in combination with different doses of lithium (5, 10 and 20 μg/rat). 2.8.5. Experiment 5: The effects of DAP-5 on the response to different doses of lithium on the spatial memory consolidation. In this experiment, eight groups of animals were used. Four groups received posttraining intra-hippocampal administration of saline (1 μl/rat) or lithium (5, 10 and 20 μg/rat), 5 minutes after training. In the other four groups the possible interaction between DAP-5 and lithium was examined. In this regard, an ineffective dose of
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DAP-5 (0.01μg/rat) was administered in combination with different doses of lithium (5, 10 and 20 μg/rat). 2.9. Verification of cannulae placements Following behavioral testing, the animals were decapitated and receive microinjection of methylene blue (1%) in the same volume as drug microinjections, in order to mark the site of the drug injection. The brains were then removed and fixed in a 10% formalin solution for 10 days before sectioning. To check the cannulae track in the CA1 area, histological examinations were done (Fig. 2). Data from animals with injection sites located outside the CA1 regions were not analyzed.
2.10. Statistical analysis Data are expressed, as means±S.E.M. The statistical analysis of the data was carried out by one-way and two-way ANOVA. Post-hoc comparison of means was carried out with the Tukey test for multiple comparisons, when appropriate. In all comparisons, P < 0.05 was considered significant. 3. Results 3.1. Effects of lithium on spatial memory consolidation To investigate the effects of post-training intra-hippocampal administration of lithium on spatial memory consolidation, different doses of lithium (5, 10 and 20 11
μg/rat) or saline (1 μg/rat) were administered 5 min after training followed by a probe test trial 24h later. One-way ANOVA of the time spent in the target quadrant on the test day revealed significant differences between groups [F (3,28)= 6.66, P<0.001]. Fig. 3(A) shows that post-training intra-hippocampal administration of lithium at the doses of 10 and 20 μg/rat significantly decreased the time animals spent in the target quadrant. Similarly, post-training administration of lithium (20 μg/rat) decreased traveled distance in the target quadrant on the test day [F (3, 28)=4.09, P<0.05; Fig. 3(B)] which showed lithium impairment on memory consolidation. Post-training administration of lithium (20 μg/rat) significantly decreased the number of entrances by the animals to the target quadrant as shown in Fig. 3(C) [F(3,28)=2.73, P<0.05]. As Fig.3 (D) shows, post-training administration of lithium did not have any significant effect on the animals' swimming speeds on the test day [F(3, 28)=0.24, P>0.05]. 3.2. Effects of NMDA on spatial memory consolidation To investigate the effects of post-training intra-hippocampal administration of NMDA on spatial memory consolidation, different doses of NMDA (0.0001, 0.001 and 0.01 μg/rat) or saline (1 μg/rat) were administered 5 min after training followed by a probe test trial 24h later. One-way ANOVA of the time spent in the target quadrant on the test day revealed significant differences between groups [F (3,28)= 8.55, P<0.001]. Fig. 4 (A) shows that post-training intra-hippocampal 12
administration of NMDA at the doses of 0.001 and 0.01μg/rat significantly increased the time animals spent in the target quadrant. Similarly, post-training administration of NMDA (0.001 and 0.01 μg/rat) increased traveled distance in the target quadrant on the test day [F (3, 28)=7.24, P<0.01; Fig. 4(B)] which showed NMDA improvement on memory consolidation. Post-training administration of NMDA (0.001 μg/rat) significantly increased the number of entrances by the animals to the target quadrant as shown in Fig. 4(C) [F(3,28)=3.27, P<0.05]. As Fig.4 (D) shows, post-training administration of lithium did not have any significant effect on the animals' swimming speeds on the test day [F(3, 28)=0.07, P>0.05]. 3.3. Effects of DAP-5 on spatial memory consolidation To investigate the effects of post-training intra-hippocampal administration of D-AP5 on spatial memory consolidation, different doses of D-AP5 (0.01, 0.05 and 0.1 μg/rat) or saline (1 μg/rat) were administered 5 min after training followed by a probe test trial 24h later. One-way ANOVA of the time spent in the target quadrant on the test day revealed significant differences between groups [F (3,28)= 9.82, P<0.001]. Fig. 5(A) shows that post-training intra-hippocampal administration of D-AP5 at the doses of 0.05 and 0.1 μg/rat significantly decreased time that the animals spent in the target quadrant. Similarly, post-training administration of D-AP5 (0.1 μg/rat) decreased traveled distance in the target quadrant on the test 13
day [F (3, 28)=6.07, P<0.01; Fig. 5(B)] which showed D-AP5 impairment on memory consolidation. Post-training administration of D-AP5 (0.1 μg/rat) significantly decreased the number of entrances by the animals to the target quadrant as shown in Fig. 5(C) [F(3,28)=3.75, P<0.05]. As Fig.5 (D) shows, posttraining administration of lithium did not have any significant effect on the animals' swimming speeds on the test day [F(3, 28)=0.20, P>0.05]. 3.4. The effect of NMDA on the impairment of spatial memory consolidation induced by acute lithium In these experiments, the effect of post-training administration of different doses of lithium (5, 10 and 20 μg/rat) in the presence or absence of an ineffective dose of NMDA (0.0001 μg/rat) was assessed on spatial memory consolidation (Fig. 6). Two-way ANOVA indicated a significant difference between the effects of lithium alone and lithium plus NMDA (0.0001 μg/rat) on time spent in target quadrant [for treatment, F(1,56)=64.86, P<0.0001; Dose, F(3,56)=0.06, P>0.05; and treatment×dose interaction, F(3,56)=13.86, P<0.0001; Fig. 6(A)]. In addition, twoway ANOVA and post-hoc analyses revealed that there is a significant difference between the effects of lithium alone and lithium plus NMDA (0.0001 μg/rat) on distance traveled in target quadrant [for treatment, F(1,56)=33.43, P<0.001; Dose, F(3,56)=0.17, P>0.05;and treatment×dose interaction, F(3,56)=9.21, P<0.0001; Fig. 6(B)]. Two- way ANOVA of entrance to target quadrant also showed a 14
significant difference between the effects of lithium alone and lithium plus NMDA (0.0001 μg/rat) [for treatment, F(1,56)=15.3, P<0.001; Dose, F(3,56)=0.47, P>0.05; and treatment×dose inter-action, F(3,56)=6.45, P<0.001; Fig. 6(C)]. 3.5. The effect of D-AP5 on the impairment of spatial memory consolidation induced by acute lithium Fig. 7 shows the effect of post-training intra-hippocampal administration of different doses of lithium (5, 10 and 20 μg/rat) alone or in combination with intrahippocampal administration of an ineffective dose of D-AP5 (0.01 μg/rat) on spatial learning consolidation. Two-way ANOVA indicated a significant difference between the effects of lithium alone and lithium plus D-AP5 (0.01 μg/rat) on time spent in the target quadrant [for treatment, F(1,56)=31.1, P<0.001; Dose, F(3,56)=19.91, P<0.001; and treatment×dose interaction, F(3,56)=3.09, P<0.05; Fig. 7(A)]. In addition, two-way ANOVA and post-hoc analyses revealed that there is a significant difference between the effects of lithium alone and lithium plus D-AP5 on distance traveled in the target quadrant [for treatment, F(1,56)=26.51, P<0.001; Dose, F(3,56)=11.72, P<0.001; and treatment×dose interaction, F(3,56)=2.8, P<0.05; Fig. 7 (B)]. Two- way ANOVA of entrance to target quadrant did not show a significant difference between the effects of lithium alone and lithium plus D-AP5 [for treatment, F(1,56)=11.47, P<0.01; Dose,
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F(3,56)=9.80, P<0.001; and treatment×dose inter-action, F(3,56)=1.53, P>0.05; Fig. 7(C)]. Discussion The present data indicated that lithium and D-AP5 impaired while NMDA improved spatial learning. Moreover, ineffective dose of D-AP5 potentiated lithium induced spatial memory impairment, but ineffective dose of NMDA reversed the lithium effect. This study addressed the possible involvement of the NMDA receptors in the effect of lithium on spatial learning memory in rats. The results of experiment 1 indicated that post-training intra-CA1 injections of lithium (10 and 20 μg/rat) decreased the percent of time spent in the target quadrant, distance traveled in target quadrant and entrance to target quadrant (20 μg/rat), indicating a spatial learning impairment. To the best of our knowledge there are a few studies concerning the acute effect of lithium on learning and memory. In agreement with our present results, we have previously shown that post-training intrapritonealy (i.p.) administration of lithium decreased step-down latency of inhibitory avoidance task in mice (Parsaei et al., 2011; Rezayat et al., 2009; Zarrindast et al., 2007). However, it has been reported that i.p. acute administration of lithium (50 and 100 mg/kg) had no significant effect in four-arm maze task in mice (Furusawa, 1991). Moreover, i.p. administration of lithium (200 or 300 mg/kg) before and after learning did not affect memory acquisition and 16
consolidation (Roussinov and Yonkov, 1974). It has been shown that lithium may increase GABA signaling (Manji et al., 2001), which can mediate lithium induced memory impairment. Some studies have suggested that acute lithium administration increased accumulation of IP3 through glutamate release and subsequently the activation of NMDA receptors (Dixon and Hokin, 1998). Other investigators reported that chronic and subchronic lithium administration delayed passive avoidance acquisition (Cappeliez and Moore, 1988), while effects on active avoidance were controversial (Richter‐Levin et al., 1992). A possible interpretation for our findings could be the fact that lithium administration after training might enhance stress mechanisms due to high glutamate release and memory impairment. In contrast, some studies indicate significant improvements in the long-term retention of passive avoidance under subchronic or chronic lithium administration at clinically relevant doses (Pascual and Gonzalez, 1995; Tsaltas et al., 2007). An electrophysiological study indicates that chronic lithium treatment increases long-term potentiation (LTP) in neurons of the hippocampus (Shim et al., 2007; Son et al., 2003). Another study found that chronic lithium administration can improve spatial learning and memory impairment (Yan et al., 2007). Chronic lithium is able to decrease anxiety in humans (Dorrego et al., 2002) and also reduce an animal’s response to stress, shock, and novelty (Masi et al., 2000; Roybal et al., 2007). Lithium can also upregulate the expression and 17
production of neurotrophic factors such as p-CREB and BDNF which are involved in learning and memory formation and LTP generation (Heldt et al., 2007; Yu et al., 2003). Glycogen synthase kinase-3 (GSK-3) is also one of the targets which may be directly and indirectly inhibited by lithium(Klein and Melton, 1996; Stambolic et al., 1996). There are evidences that indicate lithium elicits its neuroprotective/neurotrophic effects through inhibition of GSK-3 (Gould et al., 2004; Liang and Chuang, 2007). GSK-3 inhibition is probably involved in the antidepressant and anti-manic effects of lithium observed in rodent models (Gould et al., 2004; Kaidanovich-Beilin et al., 2004; O'Brien et al., 2004). Omatta et al. (2008) revealed that lithium was neuroprotective against hypoxia only after chronic treatment but not subacute and acute treatment only in specific brain regions, and that CREB and BDNF might contribute to this effect (Omata et al., 2008). It seems probable that lithium affects memory by inhibiting the enzyme inositol monophosphatase (Belmaker et al., 1996) and/or its effect on cAMP (Newman et al., 1991) and/or G-proteins (Friedman and Wang, 1996). Lithium modulates the 3’,5’-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway. Generally, lithium increases basal levels, although inhibits stimulusinduced increase in cAMP and PKA activity. The biochemical basis underlying
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anti-depressant and anti-manic efficacy of lithium has been proposed to be due to the bimodal action of the drug (Jope, 1999; Liang et al., 2008). Taken together it seems that lithium affects learning, memory and the speed of information processing in patients and in preclinical tests. The discrepancies between results of our study and those of others can be related to the injection site of the lithium, type of task employed, duration of lithium exposure, various effects of lithium on different areas of the brain and time and concentration dependent effects of lithium on a variety of neurotransmitters, and neuronal pathways (Karimfar et al., 2009). The results of experiment 2 and 3 showed that post-training intra-CA1 administration of NMDA, a glutamate receptor agonist increased the percent of time spent in the target quadrant (0.001 and 0.01 μg/rat), distance traveled in the target quadrant (0.001 and 0.01 μg/rat) and entrance to target quadrant (0.001 μg/rat ), suggesting a spatial learning facilitation. Furthermore, infusion of D-AP5, a glutamate NMDA receptor antagonist, decreased the percent of time spent in the target quadrant (0.05 and 0.1 μg/rat), distance traveled in target quadrant (0.1 μg/rat) and entrance to target quadrant (0.1 μg/rat), indicating an impairment of spatial memory consolidation. NMDA is or seems to be involved in the modification of synaptic plasticity and certain types of learning and memory (Rebola et al., 2010; Wang et al., 2014). Some studies showed that NMDA 19
receptors, via activity-dependent modifications of CA1 synapses, play an important role in the acquisition of spatial memories (Tsien et al., 1996). Blockade of NMDA receptors impair the acquisition of spatial reference memory (Bannerman et al., 2006; Morris et al., 2013). The results of experiment 4 showed that microinjection of the ineffective dose of NMDA (0.0001 μg/rat) can significantly reverse the lithium effect on the percent of time spent in target quadrant (10 and 20 μg/rat), distance traveled in the target quadrant (10 and 20 μg/rat) and entrance to target quadrant (20 μg/rat). We have previously shown that NMDA receptors reverse the memory impairing effect of lithium in the state-dependent learning in mice (Rezayat et al., 2009). Moreover, the ineffective dose of D-AP5 could significantly potentiate the memory impairing effect of lithium on the percent of time spent in the target quadrant (5 and 10 μg/rat), distance traveled in target quadrant (5 μg/rat) and entrance to target quadrant (10 μg/rat). In agreement with this finding, some investigators also suggested a possible role for glutamatergic transmission in the therapeutic effects of lithium (Dixon and Hokin, 1998; Ghasemi and Dehpour, 2011). We have previously shown that NMDA receptors to be involved, at least partly, in the statedependent learning induced by lithium (Rezayat et al., 2009). Furthermore, the effects of lithium treatment on the cholinergic potentiation of responses to NMDA in the rat hippocampal slice have been tested. It has been proposed that 20
acetylcholine potentiated the NMDA receptor through the activation of IP3 branch of phosphoinositide pathway by increasing Ca2+ which can be a common pathway for these receptors to initiate an amplification of NMDA responses and thereby enhance the probability of LTP generation at a given synapse (Markram and Segal, 1992). It is also proposed that the effects of lithium on the PI pathway may not be sufficient to explain the behavioral consequences of chronic lithium treatment (Richter‐Levin et al., 1992). It is suggested that NMDA receptor/NO signaling mediates some of the lithium effects in the mRNA expression of NMDA receptors. Lithium may change glutamatergic/ NMDAR functioning through different mechanisms such as glutamate system , regulation of NMDA receptor phosphorylation, NMDA receptor mRNA level, or indirectly via regulating GABAergic transmission or BDNF levels in the CNS (Ghasemi and Dehpour, 2011). It has been shown that the requisite calcium influx through N-methyl-D-aspartate receptors (NMDARs) couples to the cAMP signaling pathway, though via different intermediaries (Chay et al., 2016). The calcium (bound to calmodulin) stimulates adenylyl cyclase types 1 and 8 (AC1 and AC8) which catalyze cAMP production (Chetkovich et al., 1991; Chetkovich and Sweatt, 1993) in CA1 pyramidal neurons (Chetkovich and Sweatt, 1993; Nicol et al., 2005) which is required for LTP induction and long-term memory (Wong et al., 1999). Considering the fact that 21
cAMP and PKA activity can be altered by lithium, it can be assumed that the interaction between lithium and NMDA receptors can be mediated by cAMP signaling pathway. Conclusion: Our results suggest that NMDA receptors of the dorsal hippocampus may be involved in lithium-induced spatial learning impairment in the MWM task. Conflict of interest: The authors declare that they have no conflict of interest. Acknowledgement The authors thank the IRAN National Science Foundation (INSF) for providing the financial support for the project.
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Jope, R., 1999. A bimodal model of the mechanism of action of lithium. Molecular psychiatry. 4, 21-25. Kaidanovich-Beilin, O., et al., 2004. Rapid antidepressive-like activity of specific glycogen synthase kinase-3 inhibitor and its effect on β-catenin in mouse hippocampus. Biological psychiatry. 55, 781-784. Karimfar, M., et al., 2009. Time course effects of lithium administration on spatial memory acquisition and cholinergic marker expression in rats. DARU Journal of Pharmaceutical Sciences. 17, 113123. Klein, P.S., Melton, D.A., 1996. A molecular mechanism for the effect of lithium on development. Proceedings of the National Academy of Sciences. 93, 8455-8459. Liang, M.-H., Chuang, D.-M., 2007. Regulation and function of glycogen synthase kinase-3 isoforms in neuronal survival. Journal of Biological Chemistry. 282, 3904-3917. Liang, M.-H., Wendland, J.R., Chuang, D.-M., 2008. Lithium inhibits Smad3/4 transactivation via increased CREB activity induced by enhanced PKA and AKT signaling. Molecular and Cellular Neuroscience. 37, 440-453. Manji, H.K., Potter, W.Z., Lenox, R.H., 1995. Signal transduction pathways: molecular targets for lithium's actions. Archives of General Psychiatry. 52, 531-543. Manji, H.K., et al., 2001. The cellular neurobiology of depression. from Nature Medicine online. Markram, H., Segal, M., 1992. The inositol 1, 4, 5-trisphosphate pathway mediates cholinergic potentiation of rat hippocampal neuronal responses to NMDA. The Journal of physiology. 447, 513. Masi, F., et al., 2000. Acquisition of an appetitive behavior reverses the effects of long-term treatment with lithium in rats. Neuroscience. 100, 805-810. Moosavi, M., et al., 2007. Insulin protects against stress-induced impairments in water maze performance. Behavioural brain research. 176, 230-236. Morris, R., et al., 2013. N‐methyl‐d‐aspartate receptors, learning and memory: chronic intraventricular infusion of the NMDA receptor antagonist d‐AP5 interacts directly with the neural mechanisms of spatial learning. European Journal of Neuroscience. 37, 700-717. Newman, M.E., Ben-Zeev, A., Lerer, B., 1991. Chloroamphetamine did not prevent the effects of chronic antidepressants on 5-hydroxytryptamine inhibition of forskolin-stimulated adenylate cyclase in rat hippocampus. European Journal of Pharmacology: Molecular Pharmacology. 207, 209-213. Nicol, X., et al., 2005. Spatiotemporal localization of the calcium‐stimulated adenylate cyclases, AC1 and AC8, during mouse brain development. Journal of Comparative Neurology. 486, 281-294. O'Brien, W.T., et al., 2004. Glycogen synthase kinase-3β haploinsufficiency mimics the behavioral and molecular effects of lithium. The Journal of neuroscience. 24, 6791-6798. Omata, N., et al., 2008. Neuroprotective effect of chronic lithium treatment against hypoxia in specific brain regions with upregulation of cAMP response element binding protein and brain‐derived neurotrophic factor but not nerve growth factor: comparison with acute lithium treatment. Bipolar disorders. 10, 360-368. Pachet, A.K., Wisniewski, A.M., 2003. The effects of lithium on cognition: an updated review. Psychopharmacology. 170, 225-234. Parsaei, L., et al., 2011. GABA A Receptors in the Dorsal Hippocampus are Involved in Sate-dependent Learning Induced by Lithium in Mice. Iranian journal of pharmaceutical research: IJPR. 10, 127. Pascual, T., Gonzalez, J.-L., 1995. A protective effect of lithium on rat behaviour altered by ibotenic acid lesions of the basal forebrain cholinergic system. Brain research. 695, 289-292. Paxinos, G., Watson, C., 1997. The rat brain in stereotaxic coordinates: computer graphics files. San Diego, Academic. Rebola, N., Srikumar, B.N., Mulle, C., 2010. Activity‐dependent synaptic plasticity of NMDA receptors. The Journal of physiology. 588, 93-99. 24
Rezayat, M., et al., 2009. N-methyl-D-aspartate receptors are involved in lithium-induced statedependent learning in mice. Journal of Psychopharmacology. Richter‐Levin, G., Markram, H., Segal, M., 1992. Spontaneous recovery of deficits in spatial memory and cholinergic potentiation of NMDA in CA1 neurons during chronic lithium treatment. Hippocampus. 2, 279-286. Roussinov, K., Yonkov, D., 1974. Comparative studies on the effect of lithium and haloperidol on learning and memory. Acta physiologica et pharmacologica Bulgarica. 3, 51-57. Roybal, K., et al., 2007. Mania-like behavior induced by disruption of CLOCK. Proceedings of the National Academy of Sciences. 104, 6406-6411. Schou, M., 1968. Lithium in psychiatric therapy and prophylaxis. Journal of Psychiatric Research. 6, 6795. Shim, S.S., et al., 2007. Effects of sub-chronic lithium treatment on synaptic plasticity in the dentate gyrus of rat hippocampal slices. Progress in Neuro-Psychopharmacology and Biological Psychiatry. 31, 343-347. Son, H., et al., 2003. Lithium enhances long‐term potentiation independently of hippocampal neurogenesis in the rat dentate gyrus. Journal of neurochemistry. 85, 872-881. Stambolic, V., Ruel, L., Woodgett, J.R., 1996. Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Current Biology. 6, 1664-1669. Stip, E., et al., 2000. A double-blind, placebo-controlled study of the effects of lithium on cognition in healthy subjects: mild and selective effects on learning. Journal of affective disorders. 60, 147157. Tsaltas, E., et al., 2007. Enhancing effects of chronic lithium on memory in the rat. Behavioural brain research. 177, 51-60. Tsien, J.Z., Huerta, P.T., Tonegawa, S., 1996. The essential role of hippocampal CA1 NMDA receptor– dependent synaptic plasticity in spatial memory. Cell. 87, 1327-1338. Wang, H., et al., 2014. The relationship between NMDA receptors and microwave induced learning and memory impairment: a long term observation on Wistar rats. International journal of radiation biology. 1-25. Wong, S.T., et al., 1999. Calcium-stimulated adenylyl cyclase activity is critical for hippocampusdependent long-term memory and late phase LTP. Neuron. 23, 787-798. Yan, X.-B., et al., 2007. Lithium regulates hippocampal neurogenesis by ERK pathway and facilitates recovery of spatial learning and memory in rats after transient global cerebral ischemia. Neuropharmacology. 53, 487-495. Yu, I.T., et al., 2003. Chronic lithium enhances hippocampal long-term potentiation, but not neurogenesis, in the aged rat dentate gyrus. Biochemical and biophysical research communications. 303, 1193-1198. Zarrindast, M.R., Parsaei, L., Ahmadi, S., 2007. Repeated administration of histamine improves memory retrieval of inhibitory avoidance by lithium in mice. Pharmacology. 81, 187-194.
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Fig.1
Fig. 1. Diagram indicating the time line of the experimental procedures: surgery, training and testing in 20 groups of animals. On the 7th day, the drugs have been administered 5 min after training.
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Fig.2
Fig. 2. Approximate location of the injection cannulae tips in the CA1 region.
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Fig. 3
Fig. 3. The effect of post-training acute administration of lithium on spatial memory consolidation. Animals received intra-hippocampal injections of saline (1µL/rat) or different doses of lithium (5, 10 and 20 µg/rat) 5 min after training. The probe trial test was achieved 24h after training. One-way ANOVA with posthoc Tukey's test has shown significant differences among the groups. (A) Time spent in target quadrant. (B) The distance traveled in target quadrant. (C) Number of entries to target quadrant. (D) The animals swimming speed. Data are presented as mean± S.E.M. of eight animals per group. *p<0.05 and **p<0.01 different from the control group.
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Fig. 4
Fig. 4. The effect of post-training acute administration of NMDA on spatial memory consolidation. Animals received intra-hippocampal injections of saline (1 µL/rat) or different doses of NMDA (0.0001, 0.001 and 0.01 µg/rat) 5 min after training. The probe trial test was achieved 24h after training. One-way ANOVA with post-hoc Tukey's test has shown significant differences among the groups. (A) Time spent in target quadrant. (B) The distance traveled in target quadrant. (C) Number of entries to target quadrant. (D) The animals swimming speed. Data are presented as mean± S.E.M. of eight animals per group. *p<0.05, **p<0.01 and ***p<0.001 different from the control group. 29
Fig. 5
Fig. 5. The effect of post-training acute administration of D-AP5 on spatial memory consolidation. Animals received intra-hippocampal injections of saline (1 µL/rat) or different doses of D-AP5 (0.01, 0.05 and 0.1 µg/rat) 5 min after training. The probe trial test was achieved 24h after training. One-way ANOVA with post-hoc Tukey's test has shown significant differences among the groups. (A) Time spent in target quadrant. (B) The distance traveled in target quadrant. (C) Number of entries to target quadrant. (D) The animals swimming speed. Data are presented as mean± S.E.M. of eight animals per group. *p<0.05, **p<0.01 and ***p<0.001 different from the control group.
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Fig. 6
Fig. 6. The effect of post-training administration of lithium on spatial memory consolidation in the presence or absence of NMDA. Animals received post-training co-administration of saline (1 μl/rat, intra-hippocampal) or lithium (5, 10 and 20 μg/rat, intra-hippocampal) with intra-hippocampal injections of ineffective dose of NMDA (0.0001 μg/rat) 5 min after training. The probe trial test was achieved 24 h after training. One-way and two-way ANOVA with post-hoc Tukey's test have
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shown significant differences among the groups. (A)Time spent in target quadrant. (B) The distance traveled in target quadrant. (C) Number of entries to target quadrant. (D) The animals swimming speed. Data are presented as mean ± S.E.M. of eight animals per group. *p<0.05 and ** p <0.01 different from the saline/saline or saline/NMDA groups. ++p<0.01 and +++ p <0.001 different from comparable saline/ lithium group.
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Fig. 7
Fig. 7. The effect of post-training administration of lithium on spatial memory consolidation in the presence or absence of D-AP5. Animals received post-training co-administration of saline (1 μl/rat, intra-hippocampal) or lithium (5, 10 and 20 μg/rat, intra-hippocampal) with intra-hippocampal injections of ineffective dose of D-AP5 (0.01 μg/rat) 5 min after training. The probe trial test was achieved 24 h
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after training. One-way and two-way ANOVA with post-hoc Tukey's test have shown significant differences among the groups. (A)Time spent in target quadrant. (B) The distance traveled in target quadrant. (C) Number of entries to target quadrant. (D) The animals swimming speed. Data are presented as mean ± S.E.M. of eight animals per group. *p<0.05, ** p <0.01 and *** p <0.001 different from the saline/saline or saline/ D-AP5 groups. +p<0.05, ++p<0.01 and +++ p <0.001 different from comparable saline/ lithium group.
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S0361-9230(16)30159-9 http://dx.doi.org/doi:10.1016/j.brainresbull.2016.07.007 BRB 9056
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
19-3-2016 4-7-2016 18-7-2016
Please cite this article as: Leila Parsaeia, Anahita Torkaman-Boutorabi, Fereshteh Asadi, Mohammad-Reza Zarrindast, Interaction between dorsal hippocampal NMDA receptors and lithium on spatial learning consolidation in rats, Brain Research Bulletin http://dx.doi.org/10.1016/j.brainresbull.2016.07.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Interaction between dorsal hippocampal NMDA receptors and lithium on spatial learning consolidation in rats Leila Parsaeiaa,b, AnahitaTorkaman-Boutorabi*b, Fereshteh Asadia, MohammadReza Zarrindast*a,b, c,d,e a
Department of Pharmacology, School of Medicine, Tehran University of Medical
Sciences, Tehran, Iran b
Department of Neuroscience, School of Advanced Technologies in Medicine,
Tehran University of Medical Sciences, Tehran, Iran c
Institute for Studies in Theoretical Physics and Mathematics School of Cognitive
Sciences (IPM), Tehran, Iran d
Institute of Cognitive Sciences, Tehran, Iran
e
Iranian National Center for Addiction Studies, Tehran, Iran
Co-corresponding Authors: Mohammad-Reza Zarrindast, [email protected], AnahitaTorkaman-Boutorabi, [email protected]
1
Highlights Intra-CA1 injection of lithium impaired spatial learning. Intra-CA1 injection of NMDA increased spatial learning. Intra-CA1 injection of D-AP5 decreased spatial learning. D-AP5 potentiated, while NMDA restored lithium-induced spatial memory deficit.
Abstract Previous investigations have shown that NMDA receptors play an important role in learning and memory process. Lithium is a primary drug for management and prophylaxis of bipolar disorder. It can regulate signal transduction pathways in several regions of the brain and alter the function of several neurotransmitter systems involved in memory processes. The present study aimed to test the interaction of NMDA glutamatergic system of the CA1 region of dorsal hippocampus and lithium on spatial learning. Spatial memory was assessed in Morris water maze task by a single training session of eight trials followed by a probe trial and visible test 24 h later. All drugs were injected into CA1 regions, 5 min after training. Our data indicated that post- training administration of lithium (20 µg/rat, intra-CA1) significantly impaired memory consolidation. Intra2
CA1administration of NMDA, a glutamate receptor agonist (0.001 and 0.01 μg/rat) showed spatial learning facilitation. Infusion of D-AP5, a glutamate receptor antagonist (0.05 and 0.1 μg/rat) showed impairment of spatial memory. Our data also indicated that post- training administration of ineffective dose of NMDA (0.0001 μg/rat) significantly decreased amnesia induced by lithium in spatial memory consolidation. In addition, post-training intra-CA1 injection of ineffective dose of D-AP5 (0.01 μg/rat) could significantly increase lithium induced amnesia. It seems probable that signaling cascades of NMDA receptors that regulates synaptic plasticity are targets of anti-manic agents such as lithium. Our results suggest that NMDA receptors of the dorsal hippocampus may be involved in lithium-induced spatial learning impairment in the MWM task. Key words: D-AP5; Lithium; Morris Water Maze; NMDA; Rat 1. Introduction Lithium is an important mood stabilizing drug which is used for the treatment of manic-depressive illness. It can potentiate the effects of antidepressant drugs (Schou, 1968). There are several reports showing that lithium treatment decreased learning, memory, and speed of information processing in bipolar patients and in control subjects (Pachet and Wisniewski, 2003; Stip et al., 2000). It seems that lithium effects are due to neuroplastic alterations involving intracellular signaling 3
pathways, transcription factors, and regulation of gene expression (Al Banchaabouchi et al., 2004).The data concerning the effect of lithium on learning and memory are controversial. Some reports introduce lithium as a neuroprotective agent (Creson et al., 2003). However, a number of investigation indicate the memory impairing effect of lithium in both human and animal (Al Banchaabouchi et al., 2004; Parsaei et al., 2011). It has been shown that lithium can regulate signal transduction pathways in several regions of the brain. The drug may also change the function of different neurotransmitter systems (Manji et al., 1995), including acetylcholine (Ach), serotonin (5HT), dopamine (DA), N-methyl-D-aspartic acid (NMDA), nitric oxide and neurotrophic factors (Ghasemi and Dehpour, 2011). In particular, the N-methyl-D-Aspartate (NMDA) subclass of glutamate receptors was obviously involved in conditioned taste aversion paradigm, an action that was modulated by lithium (Ferreira et al., 2002) .There is suggestion that NMDA receptor/nitric oxide (NO) signaling may mediate some responses of lithium in the brain and peripheral tissues (Ghasemi and Dehpour, 2011). NMDA signaling has been shown to be involved in the antidepressant-like effects of lithium (Ghasemi et al., 2009). Acute lithium administration stimulated glutamate release, which was accompanied by an increase in inositol 1,4,5-trisphosphate (IP3) accumulation (Hokin et al., 1996).
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It has also been reported that the increase in IP3 accumulation was a result of selective activation of the NMDA receptors by glutamate (Hokin et al., 1996) . Hippocampal glutamate levels have been documented to be elevated in stably remitted bipolar patients receiving chronic lithium maintenance therapy for an average of 10 years (Colla et al., 2009). Considering the above-mentioned points and findings, the present study was designed with the following aims: (i) to determine the effect of acute intra-CA1 administration of lithium on spatial memory consolidation using Morris Water Maze task;(ii) to evaluate whether NMDA glutamatergic receptors of the CA1 region of the hippocampus lead changes of lithium effect on spatial memory consolidation. 2. Materials and methods 2.1. Animals and substances Adult male albino Wistar rats (200–250 g, aged 10–12 weeks) were obtained from Pasteur Institute of Iran. They were housed in a temperature (25±2 ◦C) and humidity-controlled room. The animals were maintained under a 12:12-h light/dark cycle, with lights off at 7:00 p.m. Food and water provided ad libitum except for the periods of behavioral testing in Morris water maze (MWM). Each animal was used once only. All experimental procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 5
2.2. Drugs The drugs used in the present study were lithium chloride (LiCl; Merck, Germany), NMDA and D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5; Tocris Cookson Ltd, UK). All drugs were dissolved in sterile 0.9% saline just before using. All drugs were bilaterally injected into the hippocampal CA1 region (intra-CA1), 5 min after training. Control animals received 0.9% physiological saline. 2.3. Surgical procedure The animals were anaesthetized with intraperitoneal injection of ketamine hydrochloride (50 mg/kg) plus xylazine (5 mg/kg) and placed in a stereotaxic apparatus, while maintaining the incisor bar at approximately 3.3 mm below horizontal zero to achieve a flat skull position. A mid-saggital incision was made to expose the rat skull. Two stainless steel, 22-gauge guide cannulae were placed (bilaterally) 1 mm above the intended site of injection according to the atlas of Paxinos and Watson (Paxinos and Watson, 1997). Stereotaxic coordinates for the CA1 regions of the dorsal hippocampus were: −3 to −3.5 mm (depending on body weight) posterior to bregma, ±1.8 to 2 mm lateral to the midline, and −2.8 to −3 mm ventral to the dorsal surface of the skull. The guide cannula was anchored by a jeweler's screw, and the incision was closed with dental cement. After completing the surgery, two stainless steel stylets (27 gauge) were inserted into the guide
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cannulae, and left in places until injections were made. All animals were allowed to recover for 1 week after surgery. 2.4. Injection into the CA1 region of dorsal hippocampus The animals were gently restrained by hand; the stylets were removed from the guide cannulae. For intra-hippocampal CA1 injections of drugs, a 1.0-μl glass Hamilton syringe was used. The injection (inner) cannulae (27-gauge), which projected a further 1 mm ventral to the tip of the guides, were attached with polyethylene tubing to the Hamilton syringe. The injection volume of drugs was 1.0 μl (0.5 μl per side) for all groups. Each dose of drug used/rat was dissolved in 1.0 μl. The injections were made over a 60-s period, and the injection cannulae were left in the guide cannulae for an additional 60 s to facilitate diffusion of the drugs. 2.5. Apparatus The water maze was a black circular pool with a diameter of 136cm and a height of 60 cm, filled with 20±1 ◦C water to a depth of 20 cm. The maze was divided geographically into four equal quadrants and release points that were designed at each quadrant as N, E, S, and W. A hidden circular platform (10cm in diameter), made of Plexiglas, was located in the center of the southwest quadrant, submerged 1.5cm beneath the surface of the water. Fixed, extra maze visual cues were present at various locations around the maze (i.e., computer, MWM hard wares, posters). 7
A camera was mounted above the center of the maze and animal motion can be recorded and sent to the computer. A tracking system was used to measure the escape latency, traveled distance and swimming speed. 2.6. Behavioral procedure Spatial memory was assessed using a 2-day Morris water maze task. The single training session consisted of eight trials with four different starting positions that were equally distributed around the perimeter of the maze (Moosavi et al., 2007). The task requires rats to swim to the hidden platform guided by distal spatial cues. After mounting the platform, the rats were allowed to remain there for 20 s, and were then placed in a holding cage for 30 s until the start of the next trial. Rats were given a maximum of 60 s to find the platform and if it failed to find the platform in 60 s, it was placed on the platform and allowed to rest for 20s. Latency to platform and distance traveled were collected and analyzed later. After completion of the training, the animals were returned to their home cages until retention testing (probe trial) 24 h later. The probe trial consisted of 60s free swim period without a platform and the time swum in the target quadrant was recorded. After the probe trial, non-spatial visual discrimination task was done. In this session, the platform was elevated above the water surface and placed in the different positions in the four quadrants. This procedure is believed to provide
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information on the possible nonspecific effects involving motor, visual, or motivational abilities for learning and memory. 2.7. Drug treatment Eight animals were used in each experimental group. In the experiments where the animals received one or two injections, the control groups also received one or two saline injections. The intervals of drug administration were based on our previous studies in order to obtain a maximum response (Fig.1). 2.8. Experimental design 2.8.1. Experiment 1: Effect of lithium on spatial memory consolidation. The effect of post-training administration of lithium on memory consolidation was examined. Four groups of animals received bilateral intra-CA1 infusions of saline (1 μl/rat) or different doses of lithium (5, 10 and 20 μg/rat) 5 min after training trial. 2.8.2. Experiment 2: Effect of NMDA on spatial memory consolidation. The effect of post-training administration of NMDA on memory consolidation was examined. Four groups of animals received bilateral intra-CA1 infusions of saline (1 μl/rat) or different doses of NMDA (0.0001, 0.001 and 0.01 μg/rat) 5 min after training trial.
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2.8.3. Experiment 3: Effect of DAP-5 on spatial memory consolidation. The effect of post-training administration of DAP-5 on memory consolidation was examined. Four groups of animals received bilateral intra-CA1 infusions of saline (1 μl/rat) or different doses of DAP-5 (0.01, 0.05, 0.1, 0.25 μg/rat) 5 min after training trial. 2.8.4. Experiment 4: The effects of an ineffective dose of NMDA on the response to different doses of lithium on the spatial memory consolidation. In this experiment, eight groups of animals were used. Four groups received posttraining intra-hippocampal administration of saline (1 μl/rat) or lithium (5, 10 and 20 μg/rat), 5 minutes after training. In the other four groups the possible interaction between NMDA and lithium was examined. In this regard, an ineffective dose of NMDA (0.0001 μg/rat) was administered in combination with different doses of lithium (5, 10 and 20 μg/rat). 2.8.5. Experiment 5: The effects of DAP-5 on the response to different doses of lithium on the spatial memory consolidation. In this experiment, eight groups of animals were used. Four groups received posttraining intra-hippocampal administration of saline (1 μl/rat) or lithium (5, 10 and 20 μg/rat), 5 minutes after training. In the other four groups the possible interaction between DAP-5 and lithium was examined. In this regard, an ineffective dose of
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DAP-5 (0.01μg/rat) was administered in combination with different doses of lithium (5, 10 and 20 μg/rat). 2.9. Verification of cannulae placements Following behavioral testing, the animals were decapitated and receive microinjection of methylene blue (1%) in the same volume as drug microinjections, in order to mark the site of the drug injection. The brains were then removed and fixed in a 10% formalin solution for 10 days before sectioning. To check the cannulae track in the CA1 area, histological examinations were done (Fig. 2). Data from animals with injection sites located outside the CA1 regions were not analyzed.
2.10. Statistical analysis Data are expressed, as means±S.E.M. The statistical analysis of the data was carried out by one-way and two-way ANOVA. Post-hoc comparison of means was carried out with the Tukey test for multiple comparisons, when appropriate. In all comparisons, P < 0.05 was considered significant. 3. Results 3.1. Effects of lithium on spatial memory consolidation To investigate the effects of post-training intra-hippocampal administration of lithium on spatial memory consolidation, different doses of lithium (5, 10 and 20 11
μg/rat) or saline (1 μg/rat) were administered 5 min after training followed by a probe test trial 24h later. One-way ANOVA of the time spent in the target quadrant on the test day revealed significant differences between groups [F (3,28)= 6.66, P<0.001]. Fig. 3(A) shows that post-training intra-hippocampal administration of lithium at the doses of 10 and 20 μg/rat significantly decreased the time animals spent in the target quadrant. Similarly, post-training administration of lithium (20 μg/rat) decreased traveled distance in the target quadrant on the test day [F (3, 28)=4.09, P<0.05; Fig. 3(B)] which showed lithium impairment on memory consolidation. Post-training administration of lithium (20 μg/rat) significantly decreased the number of entrances by the animals to the target quadrant as shown in Fig. 3(C) [F(3,28)=2.73, P<0.05]. As Fig.3 (D) shows, post-training administration of lithium did not have any significant effect on the animals' swimming speeds on the test day [F(3, 28)=0.24, P>0.05]. 3.2. Effects of NMDA on spatial memory consolidation To investigate the effects of post-training intra-hippocampal administration of NMDA on spatial memory consolidation, different doses of NMDA (0.0001, 0.001 and 0.01 μg/rat) or saline (1 μg/rat) were administered 5 min after training followed by a probe test trial 24h later. One-way ANOVA of the time spent in the target quadrant on the test day revealed significant differences between groups [F (3,28)= 8.55, P<0.001]. Fig. 4 (A) shows that post-training intra-hippocampal 12
administration of NMDA at the doses of 0.001 and 0.01μg/rat significantly increased the time animals spent in the target quadrant. Similarly, post-training administration of NMDA (0.001 and 0.01 μg/rat) increased traveled distance in the target quadrant on the test day [F (3, 28)=7.24, P<0.01; Fig. 4(B)] which showed NMDA improvement on memory consolidation. Post-training administration of NMDA (0.001 μg/rat) significantly increased the number of entrances by the animals to the target quadrant as shown in Fig. 4(C) [F(3,28)=3.27, P<0.05]. As Fig.4 (D) shows, post-training administration of lithium did not have any significant effect on the animals' swimming speeds on the test day [F(3, 28)=0.07, P>0.05]. 3.3. Effects of DAP-5 on spatial memory consolidation To investigate the effects of post-training intra-hippocampal administration of D-AP5 on spatial memory consolidation, different doses of D-AP5 (0.01, 0.05 and 0.1 μg/rat) or saline (1 μg/rat) were administered 5 min after training followed by a probe test trial 24h later. One-way ANOVA of the time spent in the target quadrant on the test day revealed significant differences between groups [F (3,28)= 9.82, P<0.001]. Fig. 5(A) shows that post-training intra-hippocampal administration of D-AP5 at the doses of 0.05 and 0.1 μg/rat significantly decreased time that the animals spent in the target quadrant. Similarly, post-training administration of D-AP5 (0.1 μg/rat) decreased traveled distance in the target quadrant on the test 13
day [F (3, 28)=6.07, P<0.01; Fig. 5(B)] which showed D-AP5 impairment on memory consolidation. Post-training administration of D-AP5 (0.1 μg/rat) significantly decreased the number of entrances by the animals to the target quadrant as shown in Fig. 5(C) [F(3,28)=3.75, P<0.05]. As Fig.5 (D) shows, posttraining administration of lithium did not have any significant effect on the animals' swimming speeds on the test day [F(3, 28)=0.20, P>0.05]. 3.4. The effect of NMDA on the impairment of spatial memory consolidation induced by acute lithium In these experiments, the effect of post-training administration of different doses of lithium (5, 10 and 20 μg/rat) in the presence or absence of an ineffective dose of NMDA (0.0001 μg/rat) was assessed on spatial memory consolidation (Fig. 6). Two-way ANOVA indicated a significant difference between the effects of lithium alone and lithium plus NMDA (0.0001 μg/rat) on time spent in target quadrant [for treatment, F(1,56)=64.86, P<0.0001; Dose, F(3,56)=0.06, P>0.05; and treatment×dose interaction, F(3,56)=13.86, P<0.0001; Fig. 6(A)]. In addition, twoway ANOVA and post-hoc analyses revealed that there is a significant difference between the effects of lithium alone and lithium plus NMDA (0.0001 μg/rat) on distance traveled in target quadrant [for treatment, F(1,56)=33.43, P<0.001; Dose, F(3,56)=0.17, P>0.05;and treatment×dose interaction, F(3,56)=9.21, P<0.0001; Fig. 6(B)]. Two- way ANOVA of entrance to target quadrant also showed a 14
significant difference between the effects of lithium alone and lithium plus NMDA (0.0001 μg/rat) [for treatment, F(1,56)=15.3, P<0.001; Dose, F(3,56)=0.47, P>0.05; and treatment×dose inter-action, F(3,56)=6.45, P<0.001; Fig. 6(C)]. 3.5. The effect of D-AP5 on the impairment of spatial memory consolidation induced by acute lithium Fig. 7 shows the effect of post-training intra-hippocampal administration of different doses of lithium (5, 10 and 20 μg/rat) alone or in combination with intrahippocampal administration of an ineffective dose of D-AP5 (0.01 μg/rat) on spatial learning consolidation. Two-way ANOVA indicated a significant difference between the effects of lithium alone and lithium plus D-AP5 (0.01 μg/rat) on time spent in the target quadrant [for treatment, F(1,56)=31.1, P<0.001; Dose, F(3,56)=19.91, P<0.001; and treatment×dose interaction, F(3,56)=3.09, P<0.05; Fig. 7(A)]. In addition, two-way ANOVA and post-hoc analyses revealed that there is a significant difference between the effects of lithium alone and lithium plus D-AP5 on distance traveled in the target quadrant [for treatment, F(1,56)=26.51, P<0.001; Dose, F(3,56)=11.72, P<0.001; and treatment×dose interaction, F(3,56)=2.8, P<0.05; Fig. 7 (B)]. Two- way ANOVA of entrance to target quadrant did not show a significant difference between the effects of lithium alone and lithium plus D-AP5 [for treatment, F(1,56)=11.47, P<0.01; Dose,
15
F(3,56)=9.80, P<0.001; and treatment×dose inter-action, F(3,56)=1.53, P>0.05; Fig. 7(C)]. Discussion The present data indicated that lithium and D-AP5 impaired while NMDA improved spatial learning. Moreover, ineffective dose of D-AP5 potentiated lithium induced spatial memory impairment, but ineffective dose of NMDA reversed the lithium effect. This study addressed the possible involvement of the NMDA receptors in the effect of lithium on spatial learning memory in rats. The results of experiment 1 indicated that post-training intra-CA1 injections of lithium (10 and 20 μg/rat) decreased the percent of time spent in the target quadrant, distance traveled in target quadrant and entrance to target quadrant (20 μg/rat), indicating a spatial learning impairment. To the best of our knowledge there are a few studies concerning the acute effect of lithium on learning and memory. In agreement with our present results, we have previously shown that post-training intrapritonealy (i.p.) administration of lithium decreased step-down latency of inhibitory avoidance task in mice (Parsaei et al., 2011; Rezayat et al., 2009; Zarrindast et al., 2007). However, it has been reported that i.p. acute administration of lithium (50 and 100 mg/kg) had no significant effect in four-arm maze task in mice (Furusawa, 1991). Moreover, i.p. administration of lithium (200 or 300 mg/kg) before and after learning did not affect memory acquisition and 16
consolidation (Roussinov and Yonkov, 1974). It has been shown that lithium may increase GABA signaling (Manji et al., 2001), which can mediate lithium induced memory impairment. Some studies have suggested that acute lithium administration increased accumulation of IP3 through glutamate release and subsequently the activation of NMDA receptors (Dixon and Hokin, 1998). Other investigators reported that chronic and subchronic lithium administration delayed passive avoidance acquisition (Cappeliez and Moore, 1988), while effects on active avoidance were controversial (Richter‐Levin et al., 1992). A possible interpretation for our findings could be the fact that lithium administration after training might enhance stress mechanisms due to high glutamate release and memory impairment. In contrast, some studies indicate significant improvements in the long-term retention of passive avoidance under subchronic or chronic lithium administration at clinically relevant doses (Pascual and Gonzalez, 1995; Tsaltas et al., 2007). An electrophysiological study indicates that chronic lithium treatment increases long-term potentiation (LTP) in neurons of the hippocampus (Shim et al., 2007; Son et al., 2003). Another study found that chronic lithium administration can improve spatial learning and memory impairment (Yan et al., 2007). Chronic lithium is able to decrease anxiety in humans (Dorrego et al., 2002) and also reduce an animal’s response to stress, shock, and novelty (Masi et al., 2000; Roybal et al., 2007). Lithium can also upregulate the expression and 17
production of neurotrophic factors such as p-CREB and BDNF which are involved in learning and memory formation and LTP generation (Heldt et al., 2007; Yu et al., 2003). Glycogen synthase kinase-3 (GSK-3) is also one of the targets which may be directly and indirectly inhibited by lithium(Klein and Melton, 1996; Stambolic et al., 1996). There are evidences that indicate lithium elicits its neuroprotective/neurotrophic effects through inhibition of GSK-3 (Gould et al., 2004; Liang and Chuang, 2007). GSK-3 inhibition is probably involved in the antidepressant and anti-manic effects of lithium observed in rodent models (Gould et al., 2004; Kaidanovich-Beilin et al., 2004; O'Brien et al., 2004). Omatta et al. (2008) revealed that lithium was neuroprotective against hypoxia only after chronic treatment but not subacute and acute treatment only in specific brain regions, and that CREB and BDNF might contribute to this effect (Omata et al., 2008). It seems probable that lithium affects memory by inhibiting the enzyme inositol monophosphatase (Belmaker et al., 1996) and/or its effect on cAMP (Newman et al., 1991) and/or G-proteins (Friedman and Wang, 1996). Lithium modulates the 3’,5’-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway. Generally, lithium increases basal levels, although inhibits stimulusinduced increase in cAMP and PKA activity. The biochemical basis underlying
18
anti-depressant and anti-manic efficacy of lithium has been proposed to be due to the bimodal action of the drug (Jope, 1999; Liang et al., 2008). Taken together it seems that lithium affects learning, memory and the speed of information processing in patients and in preclinical tests. The discrepancies between results of our study and those of others can be related to the injection site of the lithium, type of task employed, duration of lithium exposure, various effects of lithium on different areas of the brain and time and concentration dependent effects of lithium on a variety of neurotransmitters, and neuronal pathways (Karimfar et al., 2009). The results of experiment 2 and 3 showed that post-training intra-CA1 administration of NMDA, a glutamate receptor agonist increased the percent of time spent in the target quadrant (0.001 and 0.01 μg/rat), distance traveled in the target quadrant (0.001 and 0.01 μg/rat) and entrance to target quadrant (0.001 μg/rat ), suggesting a spatial learning facilitation. Furthermore, infusion of D-AP5, a glutamate NMDA receptor antagonist, decreased the percent of time spent in the target quadrant (0.05 and 0.1 μg/rat), distance traveled in target quadrant (0.1 μg/rat) and entrance to target quadrant (0.1 μg/rat), indicating an impairment of spatial memory consolidation. NMDA is or seems to be involved in the modification of synaptic plasticity and certain types of learning and memory (Rebola et al., 2010; Wang et al., 2014). Some studies showed that NMDA 19
receptors, via activity-dependent modifications of CA1 synapses, play an important role in the acquisition of spatial memories (Tsien et al., 1996). Blockade of NMDA receptors impair the acquisition of spatial reference memory (Bannerman et al., 2006; Morris et al., 2013). The results of experiment 4 showed that microinjection of the ineffective dose of NMDA (0.0001 μg/rat) can significantly reverse the lithium effect on the percent of time spent in target quadrant (10 and 20 μg/rat), distance traveled in the target quadrant (10 and 20 μg/rat) and entrance to target quadrant (20 μg/rat). We have previously shown that NMDA receptors reverse the memory impairing effect of lithium in the state-dependent learning in mice (Rezayat et al., 2009). Moreover, the ineffective dose of D-AP5 could significantly potentiate the memory impairing effect of lithium on the percent of time spent in the target quadrant (5 and 10 μg/rat), distance traveled in target quadrant (5 μg/rat) and entrance to target quadrant (10 μg/rat). In agreement with this finding, some investigators also suggested a possible role for glutamatergic transmission in the therapeutic effects of lithium (Dixon and Hokin, 1998; Ghasemi and Dehpour, 2011). We have previously shown that NMDA receptors to be involved, at least partly, in the statedependent learning induced by lithium (Rezayat et al., 2009). Furthermore, the effects of lithium treatment on the cholinergic potentiation of responses to NMDA in the rat hippocampal slice have been tested. It has been proposed that 20
acetylcholine potentiated the NMDA receptor through the activation of IP3 branch of phosphoinositide pathway by increasing Ca2+ which can be a common pathway for these receptors to initiate an amplification of NMDA responses and thereby enhance the probability of LTP generation at a given synapse (Markram and Segal, 1992). It is also proposed that the effects of lithium on the PI pathway may not be sufficient to explain the behavioral consequences of chronic lithium treatment (Richter‐Levin et al., 1992). It is suggested that NMDA receptor/NO signaling mediates some of the lithium effects in the mRNA expression of NMDA receptors. Lithium may change glutamatergic/ NMDAR functioning through different mechanisms such as glutamate system , regulation of NMDA receptor phosphorylation, NMDA receptor mRNA level, or indirectly via regulating GABAergic transmission or BDNF levels in the CNS (Ghasemi and Dehpour, 2011). It has been shown that the requisite calcium influx through N-methyl-D-aspartate receptors (NMDARs) couples to the cAMP signaling pathway, though via different intermediaries (Chay et al., 2016). The calcium (bound to calmodulin) stimulates adenylyl cyclase types 1 and 8 (AC1 and AC8) which catalyze cAMP production (Chetkovich et al., 1991; Chetkovich and Sweatt, 1993) in CA1 pyramidal neurons (Chetkovich and Sweatt, 1993; Nicol et al., 2005) which is required for LTP induction and long-term memory (Wong et al., 1999). Considering the fact that 21
cAMP and PKA activity can be altered by lithium, it can be assumed that the interaction between lithium and NMDA receptors can be mediated by cAMP signaling pathway. Conclusion: Our results suggest that NMDA receptors of the dorsal hippocampus may be involved in lithium-induced spatial learning impairment in the MWM task. Conflict of interest: The authors declare that they have no conflict of interest. Acknowledgement The authors thank the IRAN National Science Foundation (INSF) for providing the financial support for the project.
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Fig.1
Fig. 1. Diagram indicating the time line of the experimental procedures: surgery, training and testing in 20 groups of animals. On the 7th day, the drugs have been administered 5 min after training.
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Fig.2
Fig. 2. Approximate location of the injection cannulae tips in the CA1 region.
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Fig. 3
Fig. 3. The effect of post-training acute administration of lithium on spatial memory consolidation. Animals received intra-hippocampal injections of saline (1µL/rat) or different doses of lithium (5, 10 and 20 µg/rat) 5 min after training. The probe trial test was achieved 24h after training. One-way ANOVA with posthoc Tukey's test has shown significant differences among the groups. (A) Time spent in target quadrant. (B) The distance traveled in target quadrant. (C) Number of entries to target quadrant. (D) The animals swimming speed. Data are presented as mean± S.E.M. of eight animals per group. *p<0.05 and **p<0.01 different from the control group.
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Fig. 4
Fig. 4. The effect of post-training acute administration of NMDA on spatial memory consolidation. Animals received intra-hippocampal injections of saline (1 µL/rat) or different doses of NMDA (0.0001, 0.001 and 0.01 µg/rat) 5 min after training. The probe trial test was achieved 24h after training. One-way ANOVA with post-hoc Tukey's test has shown significant differences among the groups. (A) Time spent in target quadrant. (B) The distance traveled in target quadrant. (C) Number of entries to target quadrant. (D) The animals swimming speed. Data are presented as mean± S.E.M. of eight animals per group. *p<0.05, **p<0.01 and ***p<0.001 different from the control group. 29
Fig. 5
Fig. 5. The effect of post-training acute administration of D-AP5 on spatial memory consolidation. Animals received intra-hippocampal injections of saline (1 µL/rat) or different doses of D-AP5 (0.01, 0.05 and 0.1 µg/rat) 5 min after training. The probe trial test was achieved 24h after training. One-way ANOVA with post-hoc Tukey's test has shown significant differences among the groups. (A) Time spent in target quadrant. (B) The distance traveled in target quadrant. (C) Number of entries to target quadrant. (D) The animals swimming speed. Data are presented as mean± S.E.M. of eight animals per group. *p<0.05, **p<0.01 and ***p<0.001 different from the control group.
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Fig. 6
Fig. 6. The effect of post-training administration of lithium on spatial memory consolidation in the presence or absence of NMDA. Animals received post-training co-administration of saline (1 μl/rat, intra-hippocampal) or lithium (5, 10 and 20 μg/rat, intra-hippocampal) with intra-hippocampal injections of ineffective dose of NMDA (0.0001 μg/rat) 5 min after training. The probe trial test was achieved 24 h after training. One-way and two-way ANOVA with post-hoc Tukey's test have
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shown significant differences among the groups. (A)Time spent in target quadrant. (B) The distance traveled in target quadrant. (C) Number of entries to target quadrant. (D) The animals swimming speed. Data are presented as mean ± S.E.M. of eight animals per group. *p<0.05 and ** p <0.01 different from the saline/saline or saline/NMDA groups. ++p<0.01 and +++ p <0.001 different from comparable saline/ lithium group.
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Fig. 7
Fig. 7. The effect of post-training administration of lithium on spatial memory consolidation in the presence or absence of D-AP5. Animals received post-training co-administration of saline (1 μl/rat, intra-hippocampal) or lithium (5, 10 and 20 μg/rat, intra-hippocampal) with intra-hippocampal injections of ineffective dose of D-AP5 (0.01 μg/rat) 5 min after training. The probe trial test was achieved 24 h
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after training. One-way and two-way ANOVA with post-hoc Tukey's test have shown significant differences among the groups. (A)Time spent in target quadrant. (B) The distance traveled in target quadrant. (C) Number of entries to target quadrant. (D) The animals swimming speed. Data are presented as mean ± S.E.M. of eight animals per group. *p<0.05, ** p <0.01 and *** p <0.001 different from the saline/saline or saline/ D-AP5 groups. +p<0.05, ++p<0.01 and +++ p <0.001 different from comparable saline/ lithium group.
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