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Contribution of the basolateral amygdala NMDA and muscarinic receptors in rat's memory retrieval
Accepted Manuscript Contribution of the basolateral amygdala NMDA and muscarinic receptors in rat's memory retrieval Efat Nazarinia, Ameneh Rezayof, Maryam Sardari, Nima Yazdanbakhsh PII: DOI: Reference:
S1074-7427(16)30389-6 http://dx.doi.org/10.1016/j.nlm.2016.12.008 YNLME 6596
To appear in:
Neurobiology of Learning and Memory
Received Date: Revised Date: Accepted Date:
1 June 2016 6 December 2016 13 December 2016
Please cite this article as: Nazarinia, E., Rezayof, A., Sardari, M., Yazdanbakhsh, N., Contribution of the basolateral amygdala NMDA and muscarinic receptors in rat's memory retrieval, Neurobiology of Learning and Memory (2016), doi: http://dx.doi.org/10.1016/j.nlm.2016.12.008
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Contribution of the basolateral amygdala NMDA and muscarinic receptors in rat's memory retrieval Efat Nazarinia a, Ameneh Rezayofa,b, Maryam Sardaria, Nima Yazdanbakhshc a
Department of Animal Biology, School of Biology and Center of Excellence in Phylogeny of
Living Organisms, College of Science, University of Tehran, Tehran, Iran. b
School of Cognitive Sciences, Institute for Research in Fundamental Sciences (IPM), Tehran,
Iran c
School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of
Science, University of Tehran, Tehran, Iran
Correspondence to: A. Rezayof, PhD. Professor, Department of Animal Biology, School of Biology, College of Science, University of Tehran, P. O. Box 4155-6455, Tehran, Iran Fax: (+9821)-66405141 Tel: (+9821)-61112483 e-mail: [email protected]
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Abstract The present study was designed to investigate the involvement of the muscarinic cholinergic receptors in the basolateral amygdala (BLA) in memory retrieval. Also, the possible relationship between the BLA muscarinic cholinergic and the NMDA receptor systems was evaluated in the inhibitory avoidance learning. Male Wistar rats were bilaterally cannulated into the BLAs and memory retrieval was measured in a step-through type inhibitory avoidance apparatus. IntraBLA microinjection of different doses of a non-selective muscarinic receptor antagonist, scopolamine (0.5-1 μg/rat, intra-BLA), 5 min before the testing phase dose-dependently induced amnesia. Pre-test intra-BLA microinjection of different doses of NMDA (0.005-0.05 μg/rat) reversed scopolamine-induced amnesia and improved memory retrieval. In addition, different doses of a selective NMDA receptor antagonist, D-AP5 (0.001-0.005 μg/rat, intra-BLA) potentiated the response of an ineffective dose of scopolamine (0.5 µg/rat) to inhibit memory retrieval. It should be considered that pre-test intra-BLA microinjection of the same doses of NMDA or D-AP5 by themselves had no effect on memory retrieval. Similar to ANOVA analysis, our cubic interpolation analysis also predicted that the activation or inactivation of the NMDA receptors by different doses of drugs can affect the scopolamine response. On the other hand, pre-test intra-BLA microinjection of D-AP5 inhibited the reversal effect of NMDA on scopolamine-induced amnesia. It can be concluded that the BLA cholinergic system, via muscarinic receptors, has a critical role in memory retrieval. Our results also suggest that a cooperative interaction between the BLA NMDA and muscarinic acetylcholine receptors modulates memory formation of inhibitory avoidance task in rats. Keywords: Basolateral amygdala; Muscarinic receptors; NMDA receptors; Passive avoidance learning; Rat(s)
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1. Introduction Cholinergic system, via nicotinic and muscarinic receptors, seems to be a major neuropharmacological target for the treatment of Alzheimer diseases (AD)-related memory deficits because these receptors’ expressions may be decreased in AD (Kihara and Shimohama, 2004). Behavioral studies confirm that scopolamine as a non-selective muscarinic receptor antagonist induces amnesic effects in different animal models which can be used as valuable tools for human dementia and AD researches (Biradar et al., 2012). From the molecular point of view, five different muscarinic receptors (M1–M5) which belong to the large family of receptors coupled to G proteins (Haga, 2013) have been identified to mediate learning and memory processes in different brain sites including the hippocampus and the amygdala (Shinoe et al., 2005; van der Zee and Luiten, 1995). Ingles and coworkers (1993) have shown that intraamygdala microinjection of scopolamine impairs working and reference memory in the double Y-maze. Extensive evidence suggests that there is a high density of cholinergic inputs to the amygdala (Emre et al., 1993) which plays critical roles in long-term potentiation (LTP; Mansvelder et al., 2009) and memory formation (Boccia et al., 2009). Using the combined in vivo/in vitro electrophysiological and behavioural studies, it has been shown that long-term fear memory formation may be associated with increased activity of the amygdala cholinergic system (Pape et al, 2005). Furthermore, the blockade of the amygdala muscarinic receptors inhibited the induction of LTP in the medial and lateral amygdala (Watanabe, et al., 1995). A growing body of evidence suggests the involvement of the basolateral amygdala (BLA) in different animal models for learning and memory (Ferry and McGough, 2008; Roesler et al., 2000, Roozendaal et al., 2004). Interestingly, lesion of the BLA impaired memory consolidation or retrieval of the passive avoidance learning in rats (Tomaz et al., 1992). Recently, using
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optogenetic methods and in vitro patch-clamp recordings, Unal et al. (2015) reported that basal forebrain cholinergic inputs on principal BLA neurons may, via the muscarinic receptors, facilitate memory formation in BLA-dependent tasks. Moreover, it is important to note that pyramidal glutamatergic neurons constitute the majority of the BLA neuronal population (for review see Spampanato et al., 2011) which express the metabotropic and ionotropic glutamate receptors to mediate cognitive functions (Ji et al., 2010; Davis, 2011). A study conducted by LaLumiere and coworkers (2004) has shown that blockade of the BLA N-methyl-D-aspartate (NMDA; heteromeric ion channels) subtype of glutamate receptors inhibits the passive avoidance memory formation. Additionally, the BLA NMDA receptors have critical role in the acquisition and storage of fear-motivated learning in rats (Delaney et al., 2013). It seems that the basolateral amygdala activation may, via NMDA receptor mechanism, be able to modulate hippocampal long-term potentiation (LTP) which is a cellular model of learning and memory (Maren and Fanselow, 1995; Li and Richter-Levin, 2013). On the other hand, a functional interaction between glutamatergic and cholinergic systems in the BLA is suggested to modulate memory formation (Sugita et al., 1991; Pidoplichko et al., 2013). The activation of the BLA NMDA receptors potentiated the improving effect of physostigmine, as an anticholinesterase agent, on memory retrieval in an inhibitory avoidance learning task (Jafari-sabet, 2006). Moreover, using in situ hybridization studies, it has been reported that the cholinergic projections to the BLA contain vesicular glutamate transporters which may increase the capacity for the corelease of glutamate and acetylcholine to mediate emotionally motivated learning (Poulin et al., 2006). Considering that the BLA has an important role in passive avoidance learning task (Khajehpour et al., 2011) and that there is a high distribution of NMDA and muscarinic receptors in the BLA (Monaghan and Cotman, 1985 ; McDonald and Mascagni, 2010), the aim of the
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present study was to investigate (i) whether the blockade of the BLA muscarinic receptors by scopolamine affects memory retrieval in a passive avoidance learning task, (ii) and the role of the activation or inactivation of the BLA NMDA receptors in scopolamine effects on passive avoidance memory retrieval.
2. Materials and methods 2.1. Animals Male Wistar rats bred in an animal house, weighing 200-250 g at the time of surgery, were used. Animals were housed four per cage in a room with a 12:12 h light / dark cycle (lights on 07:00 hour and controlled temperature (23 ± 1°C). Animals had access to food and water ad libitum and were allowed to adapt to the laboratory conditions for at least 1 week before surgery. Rats were handled for about 5 min each day prior to behavioral testing. All experiments were performed between 9:00 and 14:00 and each rat was tested only once. All procedures for the treatment of animals were approved by the Research and Ethics Committee of the School of Biology, University of Tehran and were done in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH publications no. 80-23).
2.2. Surgery
Animals were anesthetized with intraperitoneal microinjection of ketamine hydrochloride (50 mg/kg) plus xylazine (5 mg/kg). Stainless steel, 22-gauge guide cannulas were implanted (bilaterally) 1 mm above the intended site of the microinjection according to the atlas of Paxinos and Watson (2007). Stereotaxic coordinates for the BLA were incisor bar (-3.3 mm), -2.4 mm posterior to the bregma, 5 mm lateral to the sagital suture and −8.5 mm from the top of the skull. 5
Cannulas were secured to anchor jewelers' screws with dental acrylic. To prevent clogging, stainless steel stylets (27 gauge) were placed in the guide cannulas until the animals were given the BLA microinjection. All animals were allowed 1 week to recover from surgery and clear from anesthetic drugs. For drug infusion, the animals were gently restrained by hand; the stylets were removed from the guide cannulas and replaced with 27-gauge microinjection needles (1 mm below the tip of the guide cannulas). Each microinjection unit was connected by polyethylene tubing to 2 µl Hamilton syringe. The left and right BLA were injected with a 0.3 µl solution on each side (0.6 µl/rat) over a 60-s period. The microinjection needles were left in place for an additional 60 s to allow diffusion and the stylets were then reinserted into the guide cannulas.
2.3. Drugs The drugs included scopolamine hydrochloride, NMDA (N-methyl-D-aspartic acid) and D-AP5 [D-(-)-2-amino-5-phosphonopentanoic acid] (Tocris, Bristol, UK). All drugs were dissolved in sterile 0.9% saline just before the experiments. Control animals received saline. All drugs were injected into the BLA.
2.4. Passive avoidance apparatus The learning box consisted of two compartments, one light (white compartment, 20 cm × 20 cm × 30 cm) and the other dark (black compartment, 20 cm × 20 cm × 30 cm). The guillotine door opening (7 cm × 9 cm) was made on the floor in the center of the partition between the two compartments. Stainless steel grids (2.5 mm in diameter) were placed at 1-cm intervals (distance between the centers of grids) on the floor of the dark compartment to produce foot shock.
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Intermittent electric shocks (50 Hz, 3s, 1mA intensity) were delivered to the grid floor of the dark compartment by an insulated stimulator.
2.5. Behavioral testing 2.5.1. Training The animals were allowed to habituate in the experimental room for 1 h prior to the experiments. Then, each animal was gently placed in the brightly lit compartment of the apparatus; after five seconds, the guillotine door was opened and the animal was allowed to enter the dark compartment. The latency with which the animal crossed into the dark compartment was recorded. Animals that waited more than 100 s to cross to the dark compartment were eliminated from the experiments. Once the animal crossed with all four paws to the next compartment, the guillotine door was closed and the rat was taken into its home cage. The trial was repeated after 30 min as in the acquisition trial where after 5 s the guillotine door was opened and as soon as the animal crossed to the dark (shock) compartment, the door was closed and a foot shock (50 Hz, 1mA and 3 s) was immediately delivered to the grid floor of the dark room. After 20 s, the rat was removed from the apparatus and placed temporarily into its home cage. Two minutes later, the animal was retested in the same way as the prior trials; if the rat did not enter the dark compartment during 120 s, successful acquisition of passive avoidance response was recorded. Otherwise, when the rat entered the dark compartment (before 120 s) for a second time, the door was closed and the animal received the same shock again. After retesting, if the rat acquired the acquisition of passive avoidance, the training was finished successfully.
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2.5.2. Retrieval test Twenty-four hours after training, a retrieval test was performed to determine long-term memory. Each animal was placed in the light compartment for 20 s; the door was opened and the step-through latency was measured for entering into the dark compartment. The test session ended when the animal entered the dark compartment or remained in the light compartment for 300 s (criterion for retrieval). During these sessions, no electric shock was applied. All training and testing sessions were carried out during the light phase between 08.00 h and 14.00 h.
2.6. Experimental design Seven animals were used in each experimental group. The experimental design has been summarized in Table 1. In the experiments where animals received pre-test bilateral intra-BLA of two or three microinjections, the control groups also received two or three saline microinjections. It should be noted that the drug administration intervals were 5 min which was chosen on the basis of our pilot and previous studies (Nazari-Serenjeh and Rezayof, 2013).
2.6.1. Effect of pre-test intra-BLA microinjection of scopolamine on memory retrieval In this experiment, we established a dose-response for amnestic effect of the nonselective muscarinic antagonist, scopolamine. Three groups of the animals were successfully trained in the passive avoidance apparatus. On the test day, they received intra-BLA microinjections of different doses of scopolamine (0.5, 0.75 and 1 µg/rat) and the step-through latency was measured 5 min after the microinjections. One control group received saline (0.6 µl/rat, intra-BLA) 5 min before the test (pre-test).
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2.6.2. Effects of pre-test intra-BLA microinjection of NMDA with or without scopolamine on memory retrieval To evaluate the effect of the activation of BLA NMDA receptors on scopolamineinduced amnesia and create a dose-response curve using data interpolation, eight groups of animals were successfully trained in the passive avoidance apparatus. On the test day, four groups received intra-BLA microinjections of NMDA (0, 0.005, 0.01 and 0.05 µg/rat) plus scopolamine (1 µg/rat) 5 min before testing. The other four groups of animals received intraBLA microinjections of the same doses of NMDA plus saline (0.6 µl/rat) 5 min before testing for examining the effect of the agonist alone. It is important to note that the interval between the two microinjections was 5 min and the step-through latency was measured 5 min after the last microinjection. 2.6.3. Effects of pre-test intra-BLA microinjection of D-AP5 with or without scopolamine on memory retrieval In this experiment, the effects of pre-test intra-BLA microinjection of different doses of D-AP5, a selective NMDA receptor antagonist, alone or with scopolamine on memory retrieval were examined in eight groups of animals. Cubic interpolation was used to create the dose response curve for this effect. All animals were successfully trained in the passive avoidance apparatus. On the test day, four groups of animals received intra-BLA microinjections of different doses of D-AP5 (0, 0.001, 0.003 and 0.005 µg/rat) plus saline (0.6 µl/rat) 5 min before testing. The other four groups of animals received intra-BLA microinjections of the same doses of D-AP5 (0, 0.001, 0.003 and 0.005 µg/rat) plus scopolamine (0.5 µg/rat) 5 min before testing. It is important to note that the interval between the two microinjections was 5 min and the stepthrough latency was measured 5 min after the last microinjection.
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2.6.4. Effects of intra-BLA microinjection of D-AP5 on the NMDA-induced reversal effect of scopolamine-induced amnesia This experiment examined the effect of intra-BLA microinjection of D-AP5 on the induced changes by scopolamine (1 µg/rat) in combination with NMDA in the passive avoidance learning task. On the training day, all animals were successfully trained. In three groups, the animals received pre-test intra-BLA microinjection of D-AP5 (0.001, 0.002 and 0.003 µg/rat). After 5 min, they were injected with NMDA (0.05 µg/rat; intra-BLA) and 5 min later, they received scopolamine (1 µg/rat; intra-BLA). In this experiment, we used three control groups. The first group received intra-BLA microinjections of saline (0.6 µl/rat) for three times with 5 min intervals. The second one received intra-BLA microinjections of saline (0.6 µl/rat) plus saline (0.6 µl/rat) with 5 min interval, and 5 min later they were injected with scopolamine (1 µg/rat; intra-BLA). The third group received saline (0.6 µl/rat; intra-BLA) plus NMDA (0.05 µg/rat intra-BLA) and scopolamine (1 µg/rat; intra-BLA) with 5 min intervals. Step-through latency was measured 5 min after the last microinjection.
2.7. Verification of cannula placements After the testing sessions, each rat was deeply anesthetized and 0.6 μl of a 1% methyleneblue solution was bilaterally injected into the BLA (0.6 μl/side) as described in the drug section. The animal was then decapitated and its brain was removed and placed in a 10% formalin solution. After 10 days, the brains were sliced and the sites of microinjections were verified according to the atlas of Paxinos and Watson (2007). Data from the animals with microinjection sites located outside the BLA regions were not used in the analysis. 2.8. Statistics
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The data are expressed as means ± S.E.M. The statistical analyses were performed using one- and two-way analysis of variance (ANOVA). Post-hoc comparison of means was carried out with the Tukey test for multiple comparisons, when appropriate. The level of statistical significance was set at P < 0.05. Calculations were performed using the SPSS statistical package. In order to get a better understanding of the mean response at intermediate values, cubic interpolation was performed on the experimental data. Considering that this kind of analysis is a convenient tool for interpolation of dose response data (Yankov, 2010) and also that the obtained data have great precision, one may suggest that this approach can reduce the number of animal use in behavioral experiments.
3. Results 3.1. Histology Fig. 1 shows the placements of the injection cannulas in the BLA. The representative photomicrograph of the BLA is shown in the left plate of Fig. 1. The right plate of Fig. 1 also shows the representative section taken from the rat brain atlas of Paxinos and Watson (2007). Shaded and dark regions represent the approximate points in which the cannula was positioned for each animal.
3.2. Effect of pre-test intra-BLA microinjection of scopolamine on memory retrieval Fig. 2 presents the effects of pre-test intra-BLA microinjection of scopolamine on stepthrough latency. One-way ANOVA revealed that intra-BLA microinjection of different doses of scopolamine dose-dependently decreased the step-through latency on the test day, indicating an amnesic effect of the drug [F(3, 24) = 22.8, P<0.001]
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3.3. Effects of pre-test intra-BLA microinjection of NMDA with or without scopolamine on memory retrieval Fig. 3A and 3B show the effects of pre-test intra-BLA microinjection of NMDA with or without scopolamine on memory retrieval. Two-way ANOVA revealed an interaction between the effects of pre-test intra-BLA microinjection of NMDA (0.005, 0.01 and 0.05 μg/rat) alone and NMDA plus scopolamine (1 µg/rat, intra-BLA) on memory retrieval [within-group comparison: treatment effect: F(1,48) = 33.2, P<0.001; dose effect: F(3,48) = 4.6, P<0.01; treatment × dose interaction: F(3,48) = 5.5, P<0.01]. The analysis also indicated that there was no significant change in the retrieval latencies in the animals that were injected with NMDA (0, 0.005, 0.01 and 0.05 μg/rat, intra-BLA) plus saline (0.6 µl/rat, intra-BLA) compared to the control group [F(3,24) = 1.0, P>0.05]. One-way ANOVA [F(3,24) = 8.3, P<0.01] revealed that pre-test intra-BLA microinjection of NMDA reversed the impairment of memory formation induced by a higher dose of scopolamine (1 μg/rat, right panel of Fig. 3A). The maximum response was obtained with 0.05 µg/rat of NMDA (P<0.01). Moreover, cubic interpolation of dose response data in Fig. 3B shows the kinetics of intermediate NMDA concentrations on the memory formation. Increasing concentrations of NMDA without scopolamine made no significant change in the retrieval latency (Fig. 3B, Back of the graph), while this increasing with scopolamine presented strongest effect on the latency in concentrations between 0.005 and 0.01 μg/rat as indicated by the slope of the interpolated surface (Fig. 3B, Front of the graph).
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3.4. Effects of pre-test intra-BLA microinjection of D-AP5 with or without scopolamine on memory retrieval Fig. 4A and 4B show the effects of pre-test intra-BLA microinjection of D-AP5 with or without on memory retrieval. Two-way ANOVA revealed an interaction between the effects of pre-test intra-BLA microinjection of D-AP5 (0, 0.001, 0.003 and 0.005 μg/rat) alone and D-AP5 plus scopolamine (0.5 µg/rat, intra-BLA) on memory retrieval [within-group comparison: treatment effect: F(1,48) = 34.2, P<0.001; dose effect: F(3,48) = 13.6, P<0.001; treatment × dose interaction: F(3,48) = 8.5, P<0.001]. Furthermore, one-way ANOVA showed no significant change in the retrieval latencies of the animals that were injected with D-AP5 plus saline (0.6 µl/rat, intra-BLA) compared to the control group [F(3,24) = 0.7, P>0.05]. In addition, as shown in Fig. 4A and 4B (interpolation of the experimental data), pre-test co-administration of D-AP5 (0.001, 0.003 and 0.005 μg/rat, intra-BLA) and 0.5 µg/rat of scopolamine, which alone did not induce amnesia, inhibited memory retrieval and induced amnesia [One-way ANOVA, F(3,24) = 13.3, P<0.001]. Cubic interpolation of dose response data revealed the kinetics of intermediate D-AP5 concentrations on the memory formation (Fig. 4B). This analysis revealed that increasing concentrations of D-AP5 had no significant change in the retrieval latency in the absence of scopolamine (Fig. 4B, Right of the graph), but once combined with scopolamine microinjection, the slope of the interpolated D-AP5 concentration surface indicated that this effect is almost constant for the whole range used in this trial (Fig. 4B, Left of the graph). This is in contrast to the kinetics detected for NMDA (Fig. 3B).
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3.5. Effects of intra-BLA microinjection of D-AP5 on the NMDA-induced reversal effect of scopolamine-induced amnesia Fig. 5 shows the effect of pre-test intra-BLA microinjections of different doses of D-AP5 on the NMDA-induced reversal effect of scopolamine (1 µg/rat, intra-BLA) response. One-way ANOVA indicated that intra-BLA microinjection of different doses of D-AP5 (0.001, 0.002 and 0.003 μg/rat) inhibited the reversal effect of NMDA (0.05 µg/rat, intra-BLA) on scopolamine response [F(5, 36) = 21.5, P<0.001]. The maximum response was obtained with 0.003 µg/rat of D-AP5 (P<0.001).
4. Discussion The results of the first experiment showed that pre-test intra-basolateral amygdala (BLA) microinjection of scopolamine impaired memory retrieval in inhibitory avoidance task in a dosedependent manner. In agreement with our results, it has been shown that intra-BLA or intrahippocampal microinjection of scopolamine attenuates working (Ohno et al., 1993) or spatial memory (Carli et al., 2009) in rats respectively. On the basis of these findings, it seems that the BLA muscarinic receptors play a critical role in memory formation. Immunohistochemistry studies have also shown that there are highly cholinergic projections to the BLA (Carlsen et al., 1985) which widely express muscarinic cholinergic receptors (McDonald and Mascagni, 2010). Boccia and coworkers performed a similar series of experiments in 2009 and demonstrated that the activation of the basolateral amygdala muscarinic cholinergic receptors potentiated the memory consolidation of contextual fear conditioning. Additionally, it has been proposed in a study conducted by Power and coworkers (2003) that the activation of the BLA M1 muscarinic receptors increases the excitability of the pyramidal glutamatergic inputs, while the BLA M2
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muscarinic receptors activation inhibits GABAergic interneurons function in this site. Therefore, it seems that the BLA muscarinic receptors play a key role in memory formation. Pharmacological data have also hypothesized that muscarinic cholinergic receptors and NMDA receptors jointly modulate synaptic plasticity and memory formation (Calabresi et al., 1999). In accordance with this fact that there is a high density of muscarinic cholinergic and NMDA receptors binding sites in the BLA (see introduction) which are involved in amygdalarelated memory (Power et al., 2003), the second experiment was designed to clarify whether the activation of the BLA NMDA receptors affects scopolamine-induced memory retrieval impairment. A significant finding of the present study was that pre-test intra-BLA microinjection of NMDA reversed scopolamine-induced amnesia. It should be noted that pre-test intra-BLA microinjection of NMDA alone had no effect on memory retrieval. Also, the cubic interpolation analysis supported the interaction between NMDA and scopolamine in memory retrieval. In agreement with our results, intra-hippocampal or intra-ventral tegmental area microinjection of NMDA receptors agonists have been reported to reverse the impairment of working (Kishi et al., 1996) or passive avoidance (Mahmoodi et al., 2010) memory induced by scopolamine respectively. Electrophysiological studies suggest that there is a synergetic interaction between NMDA and muscarinic receptors in the hippocampal LTP induction (Marino et al., 1998). On the other hand, the activation of the BLA NMDA receptors potentiated the improving effect of physostigmine as an anticholinesterase drug on memory consolidation (Jafari-Sabet, 2006). The most obvious finding to emerge from these studies is that there is an interaction between the BLA NMDA and muscarinic receptors in learning and memory processes. Considering that the activation of the neocortical muscarinic receptors increased the glutamate release (Chessell and Humphrey, 1995) and that NMDA reversed scopolamine-induced amnesia, it seems that the
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increase of glutamatergic neurotransmission may be a useful therapeutic option in Alzheimer's disease. Our findings also showed that pre-test intra-BLA co-administration of ineffective doses of D-AP5 and scopolamine (0.5 µg/rat), which alone did not induce amnesia, inhibited memory retrieval. It is important to note that the interaction between D-AP5 and scopolamine was also confirmed by cubic interpolation analysis in different doses of drugs. In accordance with the present data, pre-training systemic administration of the D-AP5 and scopolamine impaired the spatial memory in Y maze task (Riedel et al., 1994). Moreover, systemic co-administration of scopolamine and MK-801 (a noncompetitive NMDA receptor antagonist) or intra-hippocampal microinjection of scopolamine and DAP7 (a selective NMDA receptor antagonist) impaired memory formation (Khakpai et al., 2012). Our results also showed that pre-test microinjection of D-AP5 inhibited the reversal effect of NMDA on scopolamine response in the passive avoidance task. It has previously been shown that intracerebroventricular administration of the D-AP5 reversed the NMDA-induced spatial memory formation in rats and led to memory impairments (Morris et al., 2013). Intra-hippocampal administration of the D-AP5 blocked NMDA-induced long-term potentiation (LTP; Davis et al., 1992). Interestingly, Li and coworkers (1997) have shown that an ineffective dose of MK-801 augmented the scopolamine-induced spatial memory impairments in rats. Thus, it can be concluded that the BLA muscarinic receptors play a critical role in passive avoidance learning. Additionally, there may be, either directly or indirectly, a functional interaction between the BLA muscarinic and NMDA receptors in memory retrieval. Although this hypothesis of interaction in the BLA should be investigated using molecular and cellular studies in the future, some previous studies have shown the existence of an interactive mechanism between these receptors in other brain sites. For example, the activation of
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muscarinic receptors in the hippocampus induces long-lasting facilitation of excitatory postsynaptic potentials which is mediated by NMDA receptors (Markram and Segal, 1990; Dennis et al., 2014). Calabresi et al. (1998) also suggested that there is a positive modulatory action of muscarinic receptors on NMDA receptor mechanism in striatal spiny neurons which is associated with the activation of protein kinase C.
5. Conclusion Thus, it can be concluded that the BLA muscarinic receptors play a critical role in passive avoidance learning and blockade of these receptors induced amnesia. The results also revealed that the inactivation or activation of the BLA NMDA receptors affect the scopolamine, as a nonselective potent antagonist of muscarinic receptors induced amnesia suggesting that there is a functional interaction between the BLA muscarinic and NMDA receptors in memory retrieval formation.
Acknowledgments None.
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Pidoplichko, V.I., Prager, E.M., Aroniadou-Anderjaska, V., Braga, M.F., 2013. α7-Containing nicotinic acetylcholine receptors on interneurons of the basolateral amygdala and their role in the regulation of the network excitability. J Neurophysiol. 110 (10), 2358-69. Polepalli, J., Sah, P., 2011. Interneurons in the basolateral amygdala. Neuropharmacology. 60 (5), 765-73. Poulin, A.N., 2006. Vesicular glutamate transporter 3 immunoreactivity is present in cholinergic basal forebrain neurons projecting to the basolateral amygdala in rat. J Comp Neurol. 498 (5), 690-711. Power, A.E., McIntyre, C.K., Litmanovich, A., McGaugh, J.L., 2003. Cholinergic modulation of memory in the basolateral amygdala involves activation of both m1 and m2 receptors. Behav Pharmacol. 14(3):207-13. Riedel, G., Wetzel, W., Reymann, K.G., 1994. Computer-assisted shock-reinforced Y-maze training: a method for studying spatial alternation behaviour. Neuroreport. 5 (16), 2061-4. Roesler, R., Vianna, M.R., De-Paris, F., Quevedo, J., Walz, R., Bianchin, M., 2000. Infusions of AP5 intothe basolateral amygdala impair the formation, but not the expression, of step-downinhibitory avoidance. Braz J Med Biol Res. 33(7), 829-34. Roozendaal, B., McReynolds, J.R., McGaugh, J.L., 2004. The basolateral amygdala interacts with the medial prefrontal cortex in regulating glucocorticoid effects on working memory impairment. J Neurosci. 24 (6), 1385-92. Sardari, M., Rezayof, A., Zarrindast, M.R., 2015. 5-HT1A receptor blockade targeting the basolateral amygdala improved stress-induced impairment of memory consolidation and retrieval in rats. Neuroscience. 300, 609-18. Shinoe, T., Matsui, M., Taketo, M.M., Manabe, T., 2005. Modulation of synaptic plasticity by physiological activation of M1 muscarinic acetylcholine receptors in the mouse hippocampus. J Neurosci. 25(48), 11194-200. Spampanato, J., Polepalli,
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Tomaz, C., Dickinson-Anson, H., McGaugh, J.L., 1992. Basolateral amygdala lesions block diazepam-induced anterograde amnesia in an inhibitory avoidance task. Proc Natl Acad Sci U S A. 89(8):3615-9. Unal, C.T., Pare, D., Zaborszky, L., 2015. Impact of basal forebrain cholinergic inputs on basolateral amygdala neurons. J Neurosci. 35(2), 853-63. van der Zee, EA., Luiten, P.G., 1999. Muscarinic acetylcholine receptors in the hippocampus, neocortex and amygdala: a review of immunocytochemical localization in relation to learning and memory. Prog Neurobiol. 58(5), 409-71. Watanabe, Y., Ikegaya, Y., Saito, H., Abe, K., 1995. Roles of GABAA, NMDA and muscarinic receptors in induction of long-term potentiation in the medial and lateral amygdala in vitro. Neurosci Res. 21 (4), 317-22. Yankov, K., 2010. Dose-effect modeling of experimental data. Journal of Information, Control and Management Systems. 8, No. 3 257.
Legends Fig. 1. Representative photomicrograph of microinjection into the BLA. Left plate (A) shows the location of the microinjection cannula tip in the BLA for all rats included in the data analyses. The right plate (B) shows the representative section taken from the rat brain atlas of Paxinos and Watson, 2007. Shaded and dark areas show the site of microinjections into the BLA. The number indicates A-P coordinate relative to the bregma.
Fig. 2. Effect of pre-test intra-BLA microinjection of scopolamine on the step-through latencies. The animals were successfully trained in the passive avoidance apparatus. On the test day, they received intra-BLA microinjections of saline (0.6 µl/rat) or different doses of scopolamine (0.5, 0.75 and 1 µg/rat) and the step-through latency was measured 5 min after the microinjection. 22
Each value represents the mean ± S.E.M. of seven animals per group. **P < 0.0, ***P < 0.001 compared with the saline control group.
Fig. 3. Effects of pre-test intra-BLA microinjection of NMDA with or without scopolamine on memory retrieval. The animals were successfully trained in the passive avoidance apparatus. On the test day, four groups of animals received intra-BLA microinjections of NMDA (0, 0.005, 0.01 and 0.05 µg/rat) plus saline (0.6 µl/rat) 5 min before testing (Fig 3A, left panel). The other four groups of animals received intra-BLA microinjections of the same doses of NMDA plus scopolamine (1 µg/rat) 5 min before testing (Fig. 3A, right panel). Cubic interpolation of the latency was detected upon intra-BLA microinjection of NMDA/saline and NMDA/scopolamine. Solid symbols represent the experimental values. Color shading reflects the magnitude of step through latency (Fig. 3B). Each value represents the mean ± S.E.M. of seven animals per group. ***P < 0.01 compared with the saline/saline control group. +P < 0.05 and
++
P < 0.01 compared
with the saline/scopolamine control group.
Fig. 4. Effects of pre-test intra-BLA microinjection of D-AP5 with or without scopolamine on memory retrieval. The animals were successfully trained in the passive avoidance apparatus. On the test day, four groups of animals received intra-BLA microinjections of D-AP5 (0, 0.001, 0.003 and 0.005 µg/rat) plus saline (0.6 µl/rat) 5 min before testing (Fig 4A, left panel). The other four groups of animals received intra-BLA microinjections of the same doses of D-AP5 plus scopolamine (0.5 µg/rat) 5 min before testing (Fig 4A, right panel). Cubic interpolation of latency was detected upon intra-BLA microinjections of D-AP5/saline and D-AP5/scopolamine. Solid symbols represent the experimental values. Color shading reflects the magnitude of step
23
through latency (Fig. 4B). Each value represents the mean ± S.E.M. of seven animals per group. *P < 0.05, ***P < 0.001 compared with the saline/scopolamine control group.
Fig.5. Effect of intra-BLA microinjection of D-AP5 on the NMDA-induced reversal effect of scopolamine-induced amnesia. The animals were successfully trained in the passive avoidance apparatus. On the test day, two groups received intra-BLA microinjections of saline (0.6 µl/rat) and saline (0.6 µl/rat) with 5 min interval, and after 5 min they were injected with saline (0.6 µl/rat) or scopolamine (1 µg/rat). Four groups received intra-BLA microinjections of saline (0.6 µl/rat) or D-AP5 (0.001, 0.002 and 0.003 µg/rat, intra-BLA), NMDA (0.05 µg/rat) and finally, scopolamine (1 µg/rat) with 5 min intervals. Data are expressed as mean ± S.E.M. of seven animals per group. ***P <0.001 different from saline/saline/saline group. +++P<0.001 different from saline/saline/ scopolamine group.
###
P <0.001 different from saline/NMDA/scopolamine
group.
24
Figure 2
350
Step-through latency (S)
300
250
200
**
150
100
***
50
0 0
0.5
0.75
scopolamine (g/rat)
Fig. 2
Fig. 2
1
Figure 3A
350
Scopolamine (1 g/rat)
Saline (0.6 l/rat)
Step-through latency (S)
300
++
250
+
200
150
100
***
50
0 0
0.005
0.01
0
0.05
NMDA (g/rat)
Fig. 3A
Fig. 3A
0.005
0.01
0.05
Figure 4A
Scopolamine (0.5 g/rat)
Saline (0.6 l/rat)
350
Step-through latency (S)
300
250
*
200
150
100
*** 50
0 0
0.001 0.003
0.005
0
DAP-5 (g/rat)
Fig . 4A
Fig. 4A
0.001
0.003
0.005
Figure 5
350
+++
Step-through latency (S)
300
250
200
150
100
### ***
50
0 saline + saline + saline
saline + saline
0.001
0.002
0.003
DAP-5 (g/rat) NMDA (0.05 g/rat) Scopolamine (1 g/rat)
Fig. 5
Fig. 5
Table 1. Summary of experimental designs.
Pre-test treatment Intra-BLA microinjection (μg/rat) Figure
2 Left Panel 3
Right Panel Left Panel
4 Right Panel 5
Saline ( μl/rat)
Scopolamine (μg/rat)
NMDA (μg/rat)
0.6
0.5-1
-
-
-
0.005-0.05
-
0.6 0.6
1 -
0.005-0.05
-
0.6
0.5
0.6
0.6
1
25
DAP-5 (μg/rat)
-
0.001-0.005 0.001-0.005
0.05
0.001-0.003
Highlights (for review)
Highlights ►Intra-BLA injection of scopolamine induced memory retrieval impairment. ►Pre-test intra-BLA injection of NMDA reversed scopolamine induced amnesia. ►Intra-BLA injection of D-AP5 potentiated scopolamine induced memory impairment. ►Blockade of the NMDA receptors inhibited the effects of NMDA on scopolamine response. ► Intra-BLA injection of NMDA or D-AP5 alone had no effects on memory formation.
S1074-7427(16)30389-6 http://dx.doi.org/10.1016/j.nlm.2016.12.008 YNLME 6596
To appear in:
Neurobiology of Learning and Memory
Received Date: Revised Date: Accepted Date:
1 June 2016 6 December 2016 13 December 2016
Please cite this article as: Nazarinia, E., Rezayof, A., Sardari, M., Yazdanbakhsh, N., Contribution of the basolateral amygdala NMDA and muscarinic receptors in rat's memory retrieval, Neurobiology of Learning and Memory (2016), doi: http://dx.doi.org/10.1016/j.nlm.2016.12.008
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Contribution of the basolateral amygdala NMDA and muscarinic receptors in rat's memory retrieval Efat Nazarinia a, Ameneh Rezayofa,b, Maryam Sardaria, Nima Yazdanbakhshc a
Department of Animal Biology, School of Biology and Center of Excellence in Phylogeny of
Living Organisms, College of Science, University of Tehran, Tehran, Iran. b
School of Cognitive Sciences, Institute for Research in Fundamental Sciences (IPM), Tehran,
Iran c
School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of
Science, University of Tehran, Tehran, Iran
Correspondence to: A. Rezayof, PhD. Professor, Department of Animal Biology, School of Biology, College of Science, University of Tehran, P. O. Box 4155-6455, Tehran, Iran Fax: (+9821)-66405141 Tel: (+9821)-61112483 e-mail: [email protected]
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Abstract The present study was designed to investigate the involvement of the muscarinic cholinergic receptors in the basolateral amygdala (BLA) in memory retrieval. Also, the possible relationship between the BLA muscarinic cholinergic and the NMDA receptor systems was evaluated in the inhibitory avoidance learning. Male Wistar rats were bilaterally cannulated into the BLAs and memory retrieval was measured in a step-through type inhibitory avoidance apparatus. IntraBLA microinjection of different doses of a non-selective muscarinic receptor antagonist, scopolamine (0.5-1 μg/rat, intra-BLA), 5 min before the testing phase dose-dependently induced amnesia. Pre-test intra-BLA microinjection of different doses of NMDA (0.005-0.05 μg/rat) reversed scopolamine-induced amnesia and improved memory retrieval. In addition, different doses of a selective NMDA receptor antagonist, D-AP5 (0.001-0.005 μg/rat, intra-BLA) potentiated the response of an ineffective dose of scopolamine (0.5 µg/rat) to inhibit memory retrieval. It should be considered that pre-test intra-BLA microinjection of the same doses of NMDA or D-AP5 by themselves had no effect on memory retrieval. Similar to ANOVA analysis, our cubic interpolation analysis also predicted that the activation or inactivation of the NMDA receptors by different doses of drugs can affect the scopolamine response. On the other hand, pre-test intra-BLA microinjection of D-AP5 inhibited the reversal effect of NMDA on scopolamine-induced amnesia. It can be concluded that the BLA cholinergic system, via muscarinic receptors, has a critical role in memory retrieval. Our results also suggest that a cooperative interaction between the BLA NMDA and muscarinic acetylcholine receptors modulates memory formation of inhibitory avoidance task in rats. Keywords: Basolateral amygdala; Muscarinic receptors; NMDA receptors; Passive avoidance learning; Rat(s)
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1. Introduction Cholinergic system, via nicotinic and muscarinic receptors, seems to be a major neuropharmacological target for the treatment of Alzheimer diseases (AD)-related memory deficits because these receptors’ expressions may be decreased in AD (Kihara and Shimohama, 2004). Behavioral studies confirm that scopolamine as a non-selective muscarinic receptor antagonist induces amnesic effects in different animal models which can be used as valuable tools for human dementia and AD researches (Biradar et al., 2012). From the molecular point of view, five different muscarinic receptors (M1–M5) which belong to the large family of receptors coupled to G proteins (Haga, 2013) have been identified to mediate learning and memory processes in different brain sites including the hippocampus and the amygdala (Shinoe et al., 2005; van der Zee and Luiten, 1995). Ingles and coworkers (1993) have shown that intraamygdala microinjection of scopolamine impairs working and reference memory in the double Y-maze. Extensive evidence suggests that there is a high density of cholinergic inputs to the amygdala (Emre et al., 1993) which plays critical roles in long-term potentiation (LTP; Mansvelder et al., 2009) and memory formation (Boccia et al., 2009). Using the combined in vivo/in vitro electrophysiological and behavioural studies, it has been shown that long-term fear memory formation may be associated with increased activity of the amygdala cholinergic system (Pape et al, 2005). Furthermore, the blockade of the amygdala muscarinic receptors inhibited the induction of LTP in the medial and lateral amygdala (Watanabe, et al., 1995). A growing body of evidence suggests the involvement of the basolateral amygdala (BLA) in different animal models for learning and memory (Ferry and McGough, 2008; Roesler et al., 2000, Roozendaal et al., 2004). Interestingly, lesion of the BLA impaired memory consolidation or retrieval of the passive avoidance learning in rats (Tomaz et al., 1992). Recently, using
3
optogenetic methods and in vitro patch-clamp recordings, Unal et al. (2015) reported that basal forebrain cholinergic inputs on principal BLA neurons may, via the muscarinic receptors, facilitate memory formation in BLA-dependent tasks. Moreover, it is important to note that pyramidal glutamatergic neurons constitute the majority of the BLA neuronal population (for review see Spampanato et al., 2011) which express the metabotropic and ionotropic glutamate receptors to mediate cognitive functions (Ji et al., 2010; Davis, 2011). A study conducted by LaLumiere and coworkers (2004) has shown that blockade of the BLA N-methyl-D-aspartate (NMDA; heteromeric ion channels) subtype of glutamate receptors inhibits the passive avoidance memory formation. Additionally, the BLA NMDA receptors have critical role in the acquisition and storage of fear-motivated learning in rats (Delaney et al., 2013). It seems that the basolateral amygdala activation may, via NMDA receptor mechanism, be able to modulate hippocampal long-term potentiation (LTP) which is a cellular model of learning and memory (Maren and Fanselow, 1995; Li and Richter-Levin, 2013). On the other hand, a functional interaction between glutamatergic and cholinergic systems in the BLA is suggested to modulate memory formation (Sugita et al., 1991; Pidoplichko et al., 2013). The activation of the BLA NMDA receptors potentiated the improving effect of physostigmine, as an anticholinesterase agent, on memory retrieval in an inhibitory avoidance learning task (Jafari-sabet, 2006). Moreover, using in situ hybridization studies, it has been reported that the cholinergic projections to the BLA contain vesicular glutamate transporters which may increase the capacity for the corelease of glutamate and acetylcholine to mediate emotionally motivated learning (Poulin et al., 2006). Considering that the BLA has an important role in passive avoidance learning task (Khajehpour et al., 2011) and that there is a high distribution of NMDA and muscarinic receptors in the BLA (Monaghan and Cotman, 1985 ; McDonald and Mascagni, 2010), the aim of the
4
present study was to investigate (i) whether the blockade of the BLA muscarinic receptors by scopolamine affects memory retrieval in a passive avoidance learning task, (ii) and the role of the activation or inactivation of the BLA NMDA receptors in scopolamine effects on passive avoidance memory retrieval.
2. Materials and methods 2.1. Animals Male Wistar rats bred in an animal house, weighing 200-250 g at the time of surgery, were used. Animals were housed four per cage in a room with a 12:12 h light / dark cycle (lights on 07:00 hour and controlled temperature (23 ± 1°C). Animals had access to food and water ad libitum and were allowed to adapt to the laboratory conditions for at least 1 week before surgery. Rats were handled for about 5 min each day prior to behavioral testing. All experiments were performed between 9:00 and 14:00 and each rat was tested only once. All procedures for the treatment of animals were approved by the Research and Ethics Committee of the School of Biology, University of Tehran and were done in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH publications no. 80-23).
2.2. Surgery
Animals were anesthetized with intraperitoneal microinjection of ketamine hydrochloride (50 mg/kg) plus xylazine (5 mg/kg). Stainless steel, 22-gauge guide cannulas were implanted (bilaterally) 1 mm above the intended site of the microinjection according to the atlas of Paxinos and Watson (2007). Stereotaxic coordinates for the BLA were incisor bar (-3.3 mm), -2.4 mm posterior to the bregma, 5 mm lateral to the sagital suture and −8.5 mm from the top of the skull. 5
Cannulas were secured to anchor jewelers' screws with dental acrylic. To prevent clogging, stainless steel stylets (27 gauge) were placed in the guide cannulas until the animals were given the BLA microinjection. All animals were allowed 1 week to recover from surgery and clear from anesthetic drugs. For drug infusion, the animals were gently restrained by hand; the stylets were removed from the guide cannulas and replaced with 27-gauge microinjection needles (1 mm below the tip of the guide cannulas). Each microinjection unit was connected by polyethylene tubing to 2 µl Hamilton syringe. The left and right BLA were injected with a 0.3 µl solution on each side (0.6 µl/rat) over a 60-s period. The microinjection needles were left in place for an additional 60 s to allow diffusion and the stylets were then reinserted into the guide cannulas.
2.3. Drugs The drugs included scopolamine hydrochloride, NMDA (N-methyl-D-aspartic acid) and D-AP5 [D-(-)-2-amino-5-phosphonopentanoic acid] (Tocris, Bristol, UK). All drugs were dissolved in sterile 0.9% saline just before the experiments. Control animals received saline. All drugs were injected into the BLA.
2.4. Passive avoidance apparatus The learning box consisted of two compartments, one light (white compartment, 20 cm × 20 cm × 30 cm) and the other dark (black compartment, 20 cm × 20 cm × 30 cm). The guillotine door opening (7 cm × 9 cm) was made on the floor in the center of the partition between the two compartments. Stainless steel grids (2.5 mm in diameter) were placed at 1-cm intervals (distance between the centers of grids) on the floor of the dark compartment to produce foot shock.
6
Intermittent electric shocks (50 Hz, 3s, 1mA intensity) were delivered to the grid floor of the dark compartment by an insulated stimulator.
2.5. Behavioral testing 2.5.1. Training The animals were allowed to habituate in the experimental room for 1 h prior to the experiments. Then, each animal was gently placed in the brightly lit compartment of the apparatus; after five seconds, the guillotine door was opened and the animal was allowed to enter the dark compartment. The latency with which the animal crossed into the dark compartment was recorded. Animals that waited more than 100 s to cross to the dark compartment were eliminated from the experiments. Once the animal crossed with all four paws to the next compartment, the guillotine door was closed and the rat was taken into its home cage. The trial was repeated after 30 min as in the acquisition trial where after 5 s the guillotine door was opened and as soon as the animal crossed to the dark (shock) compartment, the door was closed and a foot shock (50 Hz, 1mA and 3 s) was immediately delivered to the grid floor of the dark room. After 20 s, the rat was removed from the apparatus and placed temporarily into its home cage. Two minutes later, the animal was retested in the same way as the prior trials; if the rat did not enter the dark compartment during 120 s, successful acquisition of passive avoidance response was recorded. Otherwise, when the rat entered the dark compartment (before 120 s) for a second time, the door was closed and the animal received the same shock again. After retesting, if the rat acquired the acquisition of passive avoidance, the training was finished successfully.
7
2.5.2. Retrieval test Twenty-four hours after training, a retrieval test was performed to determine long-term memory. Each animal was placed in the light compartment for 20 s; the door was opened and the step-through latency was measured for entering into the dark compartment. The test session ended when the animal entered the dark compartment or remained in the light compartment for 300 s (criterion for retrieval). During these sessions, no electric shock was applied. All training and testing sessions were carried out during the light phase between 08.00 h and 14.00 h.
2.6. Experimental design Seven animals were used in each experimental group. The experimental design has been summarized in Table 1. In the experiments where animals received pre-test bilateral intra-BLA of two or three microinjections, the control groups also received two or three saline microinjections. It should be noted that the drug administration intervals were 5 min which was chosen on the basis of our pilot and previous studies (Nazari-Serenjeh and Rezayof, 2013).
2.6.1. Effect of pre-test intra-BLA microinjection of scopolamine on memory retrieval In this experiment, we established a dose-response for amnestic effect of the nonselective muscarinic antagonist, scopolamine. Three groups of the animals were successfully trained in the passive avoidance apparatus. On the test day, they received intra-BLA microinjections of different doses of scopolamine (0.5, 0.75 and 1 µg/rat) and the step-through latency was measured 5 min after the microinjections. One control group received saline (0.6 µl/rat, intra-BLA) 5 min before the test (pre-test).
8
2.6.2. Effects of pre-test intra-BLA microinjection of NMDA with or without scopolamine on memory retrieval To evaluate the effect of the activation of BLA NMDA receptors on scopolamineinduced amnesia and create a dose-response curve using data interpolation, eight groups of animals were successfully trained in the passive avoidance apparatus. On the test day, four groups received intra-BLA microinjections of NMDA (0, 0.005, 0.01 and 0.05 µg/rat) plus scopolamine (1 µg/rat) 5 min before testing. The other four groups of animals received intraBLA microinjections of the same doses of NMDA plus saline (0.6 µl/rat) 5 min before testing for examining the effect of the agonist alone. It is important to note that the interval between the two microinjections was 5 min and the step-through latency was measured 5 min after the last microinjection. 2.6.3. Effects of pre-test intra-BLA microinjection of D-AP5 with or without scopolamine on memory retrieval In this experiment, the effects of pre-test intra-BLA microinjection of different doses of D-AP5, a selective NMDA receptor antagonist, alone or with scopolamine on memory retrieval were examined in eight groups of animals. Cubic interpolation was used to create the dose response curve for this effect. All animals were successfully trained in the passive avoidance apparatus. On the test day, four groups of animals received intra-BLA microinjections of different doses of D-AP5 (0, 0.001, 0.003 and 0.005 µg/rat) plus saline (0.6 µl/rat) 5 min before testing. The other four groups of animals received intra-BLA microinjections of the same doses of D-AP5 (0, 0.001, 0.003 and 0.005 µg/rat) plus scopolamine (0.5 µg/rat) 5 min before testing. It is important to note that the interval between the two microinjections was 5 min and the stepthrough latency was measured 5 min after the last microinjection.
9
2.6.4. Effects of intra-BLA microinjection of D-AP5 on the NMDA-induced reversal effect of scopolamine-induced amnesia This experiment examined the effect of intra-BLA microinjection of D-AP5 on the induced changes by scopolamine (1 µg/rat) in combination with NMDA in the passive avoidance learning task. On the training day, all animals were successfully trained. In three groups, the animals received pre-test intra-BLA microinjection of D-AP5 (0.001, 0.002 and 0.003 µg/rat). After 5 min, they were injected with NMDA (0.05 µg/rat; intra-BLA) and 5 min later, they received scopolamine (1 µg/rat; intra-BLA). In this experiment, we used three control groups. The first group received intra-BLA microinjections of saline (0.6 µl/rat) for three times with 5 min intervals. The second one received intra-BLA microinjections of saline (0.6 µl/rat) plus saline (0.6 µl/rat) with 5 min interval, and 5 min later they were injected with scopolamine (1 µg/rat; intra-BLA). The third group received saline (0.6 µl/rat; intra-BLA) plus NMDA (0.05 µg/rat intra-BLA) and scopolamine (1 µg/rat; intra-BLA) with 5 min intervals. Step-through latency was measured 5 min after the last microinjection.
2.7. Verification of cannula placements After the testing sessions, each rat was deeply anesthetized and 0.6 μl of a 1% methyleneblue solution was bilaterally injected into the BLA (0.6 μl/side) as described in the drug section. The animal was then decapitated and its brain was removed and placed in a 10% formalin solution. After 10 days, the brains were sliced and the sites of microinjections were verified according to the atlas of Paxinos and Watson (2007). Data from the animals with microinjection sites located outside the BLA regions were not used in the analysis. 2.8. Statistics
10
The data are expressed as means ± S.E.M. The statistical analyses were performed using one- and two-way analysis of variance (ANOVA). Post-hoc comparison of means was carried out with the Tukey test for multiple comparisons, when appropriate. The level of statistical significance was set at P < 0.05. Calculations were performed using the SPSS statistical package. In order to get a better understanding of the mean response at intermediate values, cubic interpolation was performed on the experimental data. Considering that this kind of analysis is a convenient tool for interpolation of dose response data (Yankov, 2010) and also that the obtained data have great precision, one may suggest that this approach can reduce the number of animal use in behavioral experiments.
3. Results 3.1. Histology Fig. 1 shows the placements of the injection cannulas in the BLA. The representative photomicrograph of the BLA is shown in the left plate of Fig. 1. The right plate of Fig. 1 also shows the representative section taken from the rat brain atlas of Paxinos and Watson (2007). Shaded and dark regions represent the approximate points in which the cannula was positioned for each animal.
3.2. Effect of pre-test intra-BLA microinjection of scopolamine on memory retrieval Fig. 2 presents the effects of pre-test intra-BLA microinjection of scopolamine on stepthrough latency. One-way ANOVA revealed that intra-BLA microinjection of different doses of scopolamine dose-dependently decreased the step-through latency on the test day, indicating an amnesic effect of the drug [F(3, 24) = 22.8, P<0.001]
11
3.3. Effects of pre-test intra-BLA microinjection of NMDA with or without scopolamine on memory retrieval Fig. 3A and 3B show the effects of pre-test intra-BLA microinjection of NMDA with or without scopolamine on memory retrieval. Two-way ANOVA revealed an interaction between the effects of pre-test intra-BLA microinjection of NMDA (0.005, 0.01 and 0.05 μg/rat) alone and NMDA plus scopolamine (1 µg/rat, intra-BLA) on memory retrieval [within-group comparison: treatment effect: F(1,48) = 33.2, P<0.001; dose effect: F(3,48) = 4.6, P<0.01; treatment × dose interaction: F(3,48) = 5.5, P<0.01]. The analysis also indicated that there was no significant change in the retrieval latencies in the animals that were injected with NMDA (0, 0.005, 0.01 and 0.05 μg/rat, intra-BLA) plus saline (0.6 µl/rat, intra-BLA) compared to the control group [F(3,24) = 1.0, P>0.05]. One-way ANOVA [F(3,24) = 8.3, P<0.01] revealed that pre-test intra-BLA microinjection of NMDA reversed the impairment of memory formation induced by a higher dose of scopolamine (1 μg/rat, right panel of Fig. 3A). The maximum response was obtained with 0.05 µg/rat of NMDA (P<0.01). Moreover, cubic interpolation of dose response data in Fig. 3B shows the kinetics of intermediate NMDA concentrations on the memory formation. Increasing concentrations of NMDA without scopolamine made no significant change in the retrieval latency (Fig. 3B, Back of the graph), while this increasing with scopolamine presented strongest effect on the latency in concentrations between 0.005 and 0.01 μg/rat as indicated by the slope of the interpolated surface (Fig. 3B, Front of the graph).
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3.4. Effects of pre-test intra-BLA microinjection of D-AP5 with or without scopolamine on memory retrieval Fig. 4A and 4B show the effects of pre-test intra-BLA microinjection of D-AP5 with or without on memory retrieval. Two-way ANOVA revealed an interaction between the effects of pre-test intra-BLA microinjection of D-AP5 (0, 0.001, 0.003 and 0.005 μg/rat) alone and D-AP5 plus scopolamine (0.5 µg/rat, intra-BLA) on memory retrieval [within-group comparison: treatment effect: F(1,48) = 34.2, P<0.001; dose effect: F(3,48) = 13.6, P<0.001; treatment × dose interaction: F(3,48) = 8.5, P<0.001]. Furthermore, one-way ANOVA showed no significant change in the retrieval latencies of the animals that were injected with D-AP5 plus saline (0.6 µl/rat, intra-BLA) compared to the control group [F(3,24) = 0.7, P>0.05]. In addition, as shown in Fig. 4A and 4B (interpolation of the experimental data), pre-test co-administration of D-AP5 (0.001, 0.003 and 0.005 μg/rat, intra-BLA) and 0.5 µg/rat of scopolamine, which alone did not induce amnesia, inhibited memory retrieval and induced amnesia [One-way ANOVA, F(3,24) = 13.3, P<0.001]. Cubic interpolation of dose response data revealed the kinetics of intermediate D-AP5 concentrations on the memory formation (Fig. 4B). This analysis revealed that increasing concentrations of D-AP5 had no significant change in the retrieval latency in the absence of scopolamine (Fig. 4B, Right of the graph), but once combined with scopolamine microinjection, the slope of the interpolated D-AP5 concentration surface indicated that this effect is almost constant for the whole range used in this trial (Fig. 4B, Left of the graph). This is in contrast to the kinetics detected for NMDA (Fig. 3B).
13
3.5. Effects of intra-BLA microinjection of D-AP5 on the NMDA-induced reversal effect of scopolamine-induced amnesia Fig. 5 shows the effect of pre-test intra-BLA microinjections of different doses of D-AP5 on the NMDA-induced reversal effect of scopolamine (1 µg/rat, intra-BLA) response. One-way ANOVA indicated that intra-BLA microinjection of different doses of D-AP5 (0.001, 0.002 and 0.003 μg/rat) inhibited the reversal effect of NMDA (0.05 µg/rat, intra-BLA) on scopolamine response [F(5, 36) = 21.5, P<0.001]. The maximum response was obtained with 0.003 µg/rat of D-AP5 (P<0.001).
4. Discussion The results of the first experiment showed that pre-test intra-basolateral amygdala (BLA) microinjection of scopolamine impaired memory retrieval in inhibitory avoidance task in a dosedependent manner. In agreement with our results, it has been shown that intra-BLA or intrahippocampal microinjection of scopolamine attenuates working (Ohno et al., 1993) or spatial memory (Carli et al., 2009) in rats respectively. On the basis of these findings, it seems that the BLA muscarinic receptors play a critical role in memory formation. Immunohistochemistry studies have also shown that there are highly cholinergic projections to the BLA (Carlsen et al., 1985) which widely express muscarinic cholinergic receptors (McDonald and Mascagni, 2010). Boccia and coworkers performed a similar series of experiments in 2009 and demonstrated that the activation of the basolateral amygdala muscarinic cholinergic receptors potentiated the memory consolidation of contextual fear conditioning. Additionally, it has been proposed in a study conducted by Power and coworkers (2003) that the activation of the BLA M1 muscarinic receptors increases the excitability of the pyramidal glutamatergic inputs, while the BLA M2
14
muscarinic receptors activation inhibits GABAergic interneurons function in this site. Therefore, it seems that the BLA muscarinic receptors play a key role in memory formation. Pharmacological data have also hypothesized that muscarinic cholinergic receptors and NMDA receptors jointly modulate synaptic plasticity and memory formation (Calabresi et al., 1999). In accordance with this fact that there is a high density of muscarinic cholinergic and NMDA receptors binding sites in the BLA (see introduction) which are involved in amygdalarelated memory (Power et al., 2003), the second experiment was designed to clarify whether the activation of the BLA NMDA receptors affects scopolamine-induced memory retrieval impairment. A significant finding of the present study was that pre-test intra-BLA microinjection of NMDA reversed scopolamine-induced amnesia. It should be noted that pre-test intra-BLA microinjection of NMDA alone had no effect on memory retrieval. Also, the cubic interpolation analysis supported the interaction between NMDA and scopolamine in memory retrieval. In agreement with our results, intra-hippocampal or intra-ventral tegmental area microinjection of NMDA receptors agonists have been reported to reverse the impairment of working (Kishi et al., 1996) or passive avoidance (Mahmoodi et al., 2010) memory induced by scopolamine respectively. Electrophysiological studies suggest that there is a synergetic interaction between NMDA and muscarinic receptors in the hippocampal LTP induction (Marino et al., 1998). On the other hand, the activation of the BLA NMDA receptors potentiated the improving effect of physostigmine as an anticholinesterase drug on memory consolidation (Jafari-Sabet, 2006). The most obvious finding to emerge from these studies is that there is an interaction between the BLA NMDA and muscarinic receptors in learning and memory processes. Considering that the activation of the neocortical muscarinic receptors increased the glutamate release (Chessell and Humphrey, 1995) and that NMDA reversed scopolamine-induced amnesia, it seems that the
15
increase of glutamatergic neurotransmission may be a useful therapeutic option in Alzheimer's disease. Our findings also showed that pre-test intra-BLA co-administration of ineffective doses of D-AP5 and scopolamine (0.5 µg/rat), which alone did not induce amnesia, inhibited memory retrieval. It is important to note that the interaction between D-AP5 and scopolamine was also confirmed by cubic interpolation analysis in different doses of drugs. In accordance with the present data, pre-training systemic administration of the D-AP5 and scopolamine impaired the spatial memory in Y maze task (Riedel et al., 1994). Moreover, systemic co-administration of scopolamine and MK-801 (a noncompetitive NMDA receptor antagonist) or intra-hippocampal microinjection of scopolamine and DAP7 (a selective NMDA receptor antagonist) impaired memory formation (Khakpai et al., 2012). Our results also showed that pre-test microinjection of D-AP5 inhibited the reversal effect of NMDA on scopolamine response in the passive avoidance task. It has previously been shown that intracerebroventricular administration of the D-AP5 reversed the NMDA-induced spatial memory formation in rats and led to memory impairments (Morris et al., 2013). Intra-hippocampal administration of the D-AP5 blocked NMDA-induced long-term potentiation (LTP; Davis et al., 1992). Interestingly, Li and coworkers (1997) have shown that an ineffective dose of MK-801 augmented the scopolamine-induced spatial memory impairments in rats. Thus, it can be concluded that the BLA muscarinic receptors play a critical role in passive avoidance learning. Additionally, there may be, either directly or indirectly, a functional interaction between the BLA muscarinic and NMDA receptors in memory retrieval. Although this hypothesis of interaction in the BLA should be investigated using molecular and cellular studies in the future, some previous studies have shown the existence of an interactive mechanism between these receptors in other brain sites. For example, the activation of
16
muscarinic receptors in the hippocampus induces long-lasting facilitation of excitatory postsynaptic potentials which is mediated by NMDA receptors (Markram and Segal, 1990; Dennis et al., 2014). Calabresi et al. (1998) also suggested that there is a positive modulatory action of muscarinic receptors on NMDA receptor mechanism in striatal spiny neurons which is associated with the activation of protein kinase C.
5. Conclusion Thus, it can be concluded that the BLA muscarinic receptors play a critical role in passive avoidance learning and blockade of these receptors induced amnesia. The results also revealed that the inactivation or activation of the BLA NMDA receptors affect the scopolamine, as a nonselective potent antagonist of muscarinic receptors induced amnesia suggesting that there is a functional interaction between the BLA muscarinic and NMDA receptors in memory retrieval formation.
Acknowledgments None.
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Legends Fig. 1. Representative photomicrograph of microinjection into the BLA. Left plate (A) shows the location of the microinjection cannula tip in the BLA for all rats included in the data analyses. The right plate (B) shows the representative section taken from the rat brain atlas of Paxinos and Watson, 2007. Shaded and dark areas show the site of microinjections into the BLA. The number indicates A-P coordinate relative to the bregma.
Fig. 2. Effect of pre-test intra-BLA microinjection of scopolamine on the step-through latencies. The animals were successfully trained in the passive avoidance apparatus. On the test day, they received intra-BLA microinjections of saline (0.6 µl/rat) or different doses of scopolamine (0.5, 0.75 and 1 µg/rat) and the step-through latency was measured 5 min after the microinjection. 22
Each value represents the mean ± S.E.M. of seven animals per group. **P < 0.0, ***P < 0.001 compared with the saline control group.
Fig. 3. Effects of pre-test intra-BLA microinjection of NMDA with or without scopolamine on memory retrieval. The animals were successfully trained in the passive avoidance apparatus. On the test day, four groups of animals received intra-BLA microinjections of NMDA (0, 0.005, 0.01 and 0.05 µg/rat) plus saline (0.6 µl/rat) 5 min before testing (Fig 3A, left panel). The other four groups of animals received intra-BLA microinjections of the same doses of NMDA plus scopolamine (1 µg/rat) 5 min before testing (Fig. 3A, right panel). Cubic interpolation of the latency was detected upon intra-BLA microinjection of NMDA/saline and NMDA/scopolamine. Solid symbols represent the experimental values. Color shading reflects the magnitude of step through latency (Fig. 3B). Each value represents the mean ± S.E.M. of seven animals per group. ***P < 0.01 compared with the saline/saline control group. +P < 0.05 and
++
P < 0.01 compared
with the saline/scopolamine control group.
Fig. 4. Effects of pre-test intra-BLA microinjection of D-AP5 with or without scopolamine on memory retrieval. The animals were successfully trained in the passive avoidance apparatus. On the test day, four groups of animals received intra-BLA microinjections of D-AP5 (0, 0.001, 0.003 and 0.005 µg/rat) plus saline (0.6 µl/rat) 5 min before testing (Fig 4A, left panel). The other four groups of animals received intra-BLA microinjections of the same doses of D-AP5 plus scopolamine (0.5 µg/rat) 5 min before testing (Fig 4A, right panel). Cubic interpolation of latency was detected upon intra-BLA microinjections of D-AP5/saline and D-AP5/scopolamine. Solid symbols represent the experimental values. Color shading reflects the magnitude of step
23
through latency (Fig. 4B). Each value represents the mean ± S.E.M. of seven animals per group. *P < 0.05, ***P < 0.001 compared with the saline/scopolamine control group.
Fig.5. Effect of intra-BLA microinjection of D-AP5 on the NMDA-induced reversal effect of scopolamine-induced amnesia. The animals were successfully trained in the passive avoidance apparatus. On the test day, two groups received intra-BLA microinjections of saline (0.6 µl/rat) and saline (0.6 µl/rat) with 5 min interval, and after 5 min they were injected with saline (0.6 µl/rat) or scopolamine (1 µg/rat). Four groups received intra-BLA microinjections of saline (0.6 µl/rat) or D-AP5 (0.001, 0.002 and 0.003 µg/rat, intra-BLA), NMDA (0.05 µg/rat) and finally, scopolamine (1 µg/rat) with 5 min intervals. Data are expressed as mean ± S.E.M. of seven animals per group. ***P <0.001 different from saline/saline/saline group. +++P<0.001 different from saline/saline/ scopolamine group.
###
P <0.001 different from saline/NMDA/scopolamine
group.
24
Figure 2
350
Step-through latency (S)
300
250
200
**
150
100
***
50
0 0
0.5
0.75
scopolamine (g/rat)
Fig. 2
Fig. 2
1
Figure 3A
350
Scopolamine (1 g/rat)
Saline (0.6 l/rat)
Step-through latency (S)
300
++
250
+
200
150
100
***
50
0 0
0.005
0.01
0
0.05
NMDA (g/rat)
Fig. 3A
Fig. 3A
0.005
0.01
0.05
Figure 4A
Scopolamine (0.5 g/rat)
Saline (0.6 l/rat)
350
Step-through latency (S)
300
250
*
200
150
100
*** 50
0 0
0.001 0.003
0.005
0
DAP-5 (g/rat)
Fig . 4A
Fig. 4A
0.001
0.003
0.005
Figure 5
350
+++
Step-through latency (S)
300
250
200
150
100
### ***
50
0 saline + saline + saline
saline + saline
0.001
0.002
0.003
DAP-5 (g/rat) NMDA (0.05 g/rat) Scopolamine (1 g/rat)
Fig. 5
Fig. 5
Table 1. Summary of experimental designs.
Pre-test treatment Intra-BLA microinjection (μg/rat) Figure
2 Left Panel 3
Right Panel Left Panel
4 Right Panel 5
Saline ( μl/rat)
Scopolamine (μg/rat)
NMDA (μg/rat)
0.6
0.5-1
-
-
-
0.005-0.05
-
0.6 0.6
1 -
0.005-0.05
-
0.6
0.5
0.6
0.6
1
25
DAP-5 (μg/rat)
-
0.001-0.005 0.001-0.005
0.05
0.001-0.003
Highlights (for review)
Highlights ►Intra-BLA injection of scopolamine induced memory retrieval impairment. ►Pre-test intra-BLA injection of NMDA reversed scopolamine induced amnesia. ►Intra-BLA injection of D-AP5 potentiated scopolamine induced memory impairment. ►Blockade of the NMDA receptors inhibited the effects of NMDA on scopolamine response. ► Intra-BLA injection of NMDA or D-AP5 alone had no effects on memory formation.