or septum

Behavioural Brain Research 231 (2012) 1–10

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Research report

Scopolamine induced memory impairment; possible involvement of NMDA receptor mechanisms of dorsal hippocampus and/or septum Fatemeh Khakpai a , Mohammad Nasehi b , Ali Haeri-Rohani a , Akram Eidi a , Mohammad Reza Zarrindast a,c,d,e,f,g,∗ a

Department of Biology, Faculty of Basic Sciences, Islamic Azad University, Science and Research Branch, Tehran, Iran Department of Biology, Faculty of Basic Sciences, Islamic Azad University, Garmsar Branch, Semnan, Iran c Institute for Cognitive Science Studies (ICSS), Tehran, Iran d Department of Neuroscience, School of Advance Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran e Department of Pharmacology School of Medicine, Tehran University of Medical Sciences, Tehran, Iran f Iranian National Center for Addiction Studies, Tehran University of Medical Sciences, Tehran, Iran g School of Cognitive Sciences, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran b

a r t i c l e

i n f o

Article history: Received 6 December 2011 Received in revised form 23 February 2012 Accepted 27 February 2012 Available online 5 March 2012 Keywords: NMDA D-AP7 Scopolamine Septo-hippocampal Step-through inhibitory avoidance Rat

a b s t r a c t Background and aim: The anatomical connections of septum and hippocampus and the influence of cholinergic and glutamatergic projections in these sites have been reported. In the present study, the effect of pre-training intra-dorsal hippocampal (CA1) and intra-medial septal (MS) administration of scopolamine, a nonselective muscarinic acetylcholine antagonist, and NMDA receptor agents and their interactions, on acquisition of memory have been investigated. Methods: The animals were bilaterally implanted with chronic cannulae in the CA1 regions and in the medial septum. Animals were trained in a step-through type inhibitory avoidance task, and tested 24 h after training to measure step-through latency as memory retrieval. Results: Intra-CA1 or intra-MS injections of scopolamine (0.5, 1 and 2 ␮g/rat) and D-AP7 (a competitive NMDA receptor antagonist; 0.025, 0.05 and 0.1 ␮g/rat) reduced, while NMDA (0.125 and 0.25 ␮g/rat) increased memory. Intra-MS injection of a subthreshold dose of NMDA reduced scopolamine induced amnesia in the MS. However, similar injection of NMDA into CA1 did not alter scopolamine response when injected into CA1. Moreover, intra-MS or -CA1 injection of a subthreshold dose of NMDA did not alter scopolamine response in the CA1 or MS respectively. On the other hand, co-administration subthreshold doses of D-AP7 and scopolamine into CA1 and/or MS induced amnesia. Conclusions: The cholinergic system between septum and CA1 are modulating memory acquisition processes induced by glutamatergic system in the CA1 or septum and co-activation of these systems in these sites can influence learning and memory. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The hippocampus is an important neural structure [1–3], and specially, the CA1 area of the dorsal hippocampus is involved in memory formation [4,5]. The CA1 mediates neural plasticity processes involved in the acquisition, storage and retrieval of memory within the hippocampus [6]. The hippocampus has anatomical connections with various subcortical regions, including the medial septum (MS) which is a part of the forebrain circuitry involved in memory [7,8]. Furthermore, acetylcholine (ACh) is one of the brain

∗ Corresponding author at: Department of Pharmacology School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran. Tel.: +98 21 66402569; fax: +98 21 66402569. E-mail address: [email protected] (M.R. Zarrindast). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2012.02.049

neurotransmitters [9] involved in learning, memory and attention processes [10–12]. The septo-hippocampal cholinergic connection is one major and well-known efferent projection of the MS and the vertical limb of the diagonal band of Broca (MS/VDB) [13–18] which has an important role for learning and memory [19,20]. There is also a suggestion that cholinergic projections from MS to the hippocampus could modulate hippocampal memory process [21]. Specifically, hippocampal learning rates which were assumed to be based on the cholinergic input thus can be reduced by anticholinergic drugs such as scopolamine. The pathway from the hippocampus to MS, self-regulates cholinergic input [22]. The septal cholinergic projections innervate excitatory and inhibitory neurons in the dentate gyrus, CA3 and CA1 of the hippocampus. The septo-hippocampal system has received considerable attention as a pathway with which to evaluate the role of ACh in learning and memory [23].

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Moreover, a glutamatergic septo-hippocampal projection has also been proposed to regulate the activity of septal neurons projecting to the hippocampus [24]. The glutamate as an excitatory neurotransmitter is particularly abundant in the mammalian brain [25–28]. The N-methyl-d-aspartate (NMDA) receptor subtype of glutamate receptor plays a key role in neural physiology, synaptic plasticity and behavioral learning and memory process [29–32]. The receptors are found in high density in the hippocampus and septum [33]. NMDA receptors of the hippocampal formation [34] and MS [24,35] are implicated in cognitive performance, specifically in learning and memory. Considering the cholinergic and glutamatergic afferents from septum to the hippocampal neurons, it was logical to investigate the effects of co-administration of NMDA and acetylcholine receptors agents into the septo-hippocampal pathway on acquisition of inhibitory avoidance memory. For these purposes, at first, we examined the effects of pre-training intra-CA1 and intra-MS administration of scopolamine, NMDA receptor agonist and antagonist (NMDA and D-AP7, respectively) on acquisition of inhibitory avoidance memory. Secondly, the effects of pre-training intra-CA1 and intra-MS co-administration of the drugs on the acquisition process were also investigated. 2. Materials and methods 2.1. Animals Adult male Wistar rats (Institute of Cognitive Science, Tehran, Iran) weighing 220–270 g at the time of surgery, were used. The animals were housed four per cage, in a room under a 12 h light:12 h dark cycle (lights on 07:00 h) and controlled temperature (23 ± 1 ◦ C) with free access to food and water. Animals were allowed to adapt to the laboratory conditions for at least 1 week before surgery. Rats were handled about 3 min each day prior to behavioral testing. All experiments were performed between 8:00 and 12:00 h and each rat was tested only once. All procedures in this study are in accordance with the guide for the Care and Use of Laboratory Animals as adopted by the Ethics Committee of Faculty of Science, Tehran University (357: November 2000). 2.2. Inhibitory avoidance task A learning box consisted of two compartments, one light (white opaque resin, 20 cm × 20 cm × 30 cm) and the other dark (black opaque resin, 20 cm × 20 cm × 30 cm). A 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. Intermittent electric shocks (50 Hz, 3 s, and 1 mA intensity) were delivered to the grid floor of the dark compartment by an isolated stimulator. 2.3. Stereotaxic surgery and microinjections The animals were anaesthetized using a solution containing ketamine hydrochloride (50 mg/kg) plus xylazine (5 mg/kg) and were then positioned in a stereotaxic frame (Stoelting Co, Illinois, USA) with flat-skull position. A midline incision was made in the skin of the skull, and then the underlying periosteum was retracted. A stainless steel guide cannulae (22 gauge) was implanted 2 mm above the MS according to stereotaxic coordinates and two 22-gauge stainless steel guide cannulae were placed (bilaterally) 2 mm above the CA1 region according to stereotaxic coordinate atlas [36]. Stereotaxic coordinates for the MS was +0.3 to +1.1 mm (depending on body weight) anterior of bregma, 0 to ±0.1 mm lateral to the midline and −4.5 to −6.5 mm ventral of the dorsal surface of the skull. Stereotaxic coordinates for the CA1 region of the dorsal hippocampus were −2.6 to −2.9 mm (depending on body weight) posterior to bregma, ±1.6 to −1.8 mm lateral to the midline and −2.5 to −2.8 mm ventral of the dorsal surface of the skull. The cannulae were secured to the bone with dental acrylic cement. A stylet was introduced into the guide cannula to prevent possible obstruction. All animals were allowed about 5–7 days to recover from surgery and from the effect of the anesthetic agents. 2.4. Intra-CA1 and intra-MS injections For drug infusion, the animals were gently restrained by hand; the stylets were removed from the guide cannulae and replaced by dental 27-gauge injection needles (2 mm below the tip of the guide cannula). The needle was connected to a 2.5 ␮l Hamilton microsyringes via polyethylene tubing and the injection were performed manually. The forward movement of a small air bubble inside the polyethylene

tubing interposed between the upper end of the needle and the microsyringe was taken as evidence of drug flow. The MS, left and right CA1 were infused with 0.5 ␮l solution on each site over a 60-s period. Injection needles were left in place for additional 60 s to facilitate the diffusion of the drugs. The drugs were been injected 5 min before training. 2.5. Memory testing Training was based on the protocol used in our previous studies [5,37,38]. All animals were allowed to habituate in the experimental room (with light and sound attenuated) for at least 30 min prior to the experiments. 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 of the animal to enter the dark compartment was recorded. Animals that waited more than 100 s to cross to the dark compartment were eliminated from the experiments (in the present experiments, all of the animals reach the above criterion to enter the dark compartment within 100 s). Once the animal crossed with all four paws to the next compartment, the guillotine door was closed and the animal was immediately withdrawn from the compartment and placed into its home cage. After 25 min the animals received pre-training injection of drugs, and then the trial was repeated for each rat as in the acquisition trial except that as soon as the animal crossed to the dark compartment, the door was closed and a foot shock (50 Hz, 3 s, and 1 mA intensity) was immediately delivered to the grid floor. 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 in the previous trials and if the rat did not enter the dark compartment during 120 s, a successful acquisition of IA was recorded. Otherwise, when the rat entered the dark compartment (before 120 s) a second time, the door was closed and the animal received the shock again. The aim of this experiment with pre-training drug administration was to evaluate effects of drugs on the acquisition process. During the retention session, each animal was gently placed in the light compartment and after 5 s the door was opened, and the step-through latency with which the animal crossed to the dark compartment with all four paws was recorded. The testing process was ended when the animal entered the dark compartment or remained in the light compartment for 300 s. During testing sessions no electric shock was given and the entrance latency into the dark chamber of the inhibitory avoidance apparatus was measured as an index of the inhibitory avoidance memory. After step-through latency test, locomotor activity of each rat was measured. 2.6. Measurement of locomotor activity The locomotion apparatus (Borj Sanat Co, Tehran, Iran) consisted of clear perspex container box (30 cm × 30 cm × 40 cm high). The apparatus has a gray perspex panel (30 cm × 30 cm × 2.2 cm thick) with 16 photocells which divided the box to 16 equalsized squares. Locomotion was recorded as the number of crossings from one square to another during 5 min. 2.7. Drugs The following drugs were used in the experiments: nonselective muscarinic acetylcholine receptor antagonist, scopolamine hydrobromide (Sigma, Poole, Dorset, UK), N-methyl-d-aspartate (NMDA receptor agonist) and dl-2-amino-7phosphonoheptanoate (D-AP7, NMDA receptor antagonist; Tocris, Bristol, UK). All drugs were dissolved in sterile 0.9% saline and were injected into the intra-MS and intra-dorsal hippocampal (intra-CA1) in a volume of 0.5 ␮l/site. 2.8. Statistical analysis Since data displayed normality of distribution and homogeneity of variance, the results were statistically evaluated by analysis of variance one-way (ANOVA) and two-way ANOVA, in which mean ± S.E.M of step-through latencies of experimental groups on the test day were compared. Further analyses for individual betweengroups comparisons were carried out with post hoc Tukey’s test. In all comparisons, P < 0.05 was considered to indicate statistical significance. In experiments 1, 3, 4, and 6 the one-way ANOVA was used for analyzing differences between groups factor of dose. In experiments 2, 5, 7 and 8 two-way ANOVA was used to analyze interaction between scopolamine with NMDA and D-AP7. 2.9. Verification of cannulae placements After the testing sessions each rat was deeply anesthetized and 0.5 ␮l/site of a 4% methylene-blue solution was infused into the CA1 and MS, as described in the drug section, then decapitated and its brain removed and placed in formaldehyde (10%). After several days, the brains were sliced and the sites of injections were verified according to Paxinos and Watson [36]. 2.10. Experimental design Eight animals were used in each experimental group. The animals received more than one injection in the experiments. In all experiments, the first injection was done

F. Khakpai et al. / Behavioural Brain Research 231 (2012) 1–10

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Table 1 All experiments, groups of animals, doses of drugs, and time of drugs injection. Figure

Pre-training treatment Step-through latency

Locomotion activity

First injection in septuma (0.5 ␮l/rat)

Second injection in CA1 (1 ␮l/rat)

Behavioral effect on memory acquisition

Behavioral effect on locomotor activity

A

D

Saline (0.5 ␮l/rat)

Decreased

Not effect

B

E

Saline (0.5 ␮l/rat)

Increased

Not effect

C

F

Saline (0.5 ␮l/rat)

Decreased

Not effect

A

C

Saline (0.5 ␮l/rat)

Decreased

Not effect

B

D

Saline (0.5 ␮l/rat)

Decreased

Not effect

A

B

Saline (0.5 ␮l/rat)

Decreased

Not effect

A

D

Decreased

Not effect

B

E

Saline (1 ␮l/rat)

Increased

Not effect

C

F

Saline (1 ␮l/rat)

Decreased

Not effect

A

C

Saline (1 ␮l/rat)

Decreased

Not effect

B

D

Saline (1 ␮l/rat)

Decreased

Not effect

A

B

Saline (1 ␮l/rat)

Decreased

Not effect

A

D

Scopolamine (0.25, 0.5, 1 and 2 ␮g/rat) NMDA (0.015, 0.03, 0.06, 0.125, 0.25 ␮g/rat) D-AP7 (0.006, 0.012, 0.025, 0.05 and 0.1 ␮g/rat Scopolamine (0.5, 1 and 2 ␮g/rat) with saline (0.5 ␮g/rat) Scopolamine (0.5, 1 and 2 ␮g/rat) with NMDA (0.06 ␮g/rat) Scopolamine (0.25 ␮g/rat) or D-AP7 (0.006, 0.012 ␮g/rat) with saline (1 ␮l/rat) or Scopolamine (0.25 ␮g/rat) Saline (0.5 ␮l/rat)

Scopolamine (0.25, 0.5, 1 and 2 ␮g/rat) NMDA (0.015, 0.03, 0.06, 0.125, 0.25 ␮g/rat) D-AP7 (0.006, 0.012, 0.025, 0.05, 0.1 ␮g/rat) Scopolamine (0.5, 1 and 2 ␮g/rat) with saline (1 ␮l/rat) Scopolamine (0.5, 1, 2 ␮g/rat) with NMDA (0.06 ␮g/rat) Scopolamine (0.25 ␮g/rat) or D-AP7 (0.006, 0.012 ␮g/rat) with saline (1 ␮l/rat) or Scopolamine (0.25 ␮g/rat) Saline (1 ␮l/rat)

Decreased

Not effect

B

E

NMDA (0.03 ␮g/rat)

Decreased

Not effect

C

F

D-AP7 (0.012 ␮g/rat

Decreased

Not effect

A

D

Decreased

Not effect

B

E

NMDA (0.03 ␮g/rat)

Decreased

Not effect

C

F

Scopolamine (0.25, 0.5 and 1 ␮g/rat) Scopolamine (0.25, 0.5 and 1 ␮g/rat) Scopolamine (0.25, 0.5 and 1 ␮g/rat)

Scopolamine (0.25, 0.5 and 1 ␮g/rat) Scopolamine (0.25, 0.5 and 1 ␮g/rat) Scopolamine (0.25, 0.5 and 1 ␮g/rat) Saline (1 ␮l/rat)

D-AP7 (0.012 ␮g/rat

Decreased

Not effect

1

2

3

4

5

6

7

8

9 a

Schematic histology In all experiments, the first injection was done 5 min before training into the medial septum and the second one was administered 5 min after first injection into the CA1.

5 min before training into the medial septum and the second one was administered 5 min after first injection into the CA1. The protocol has been summarized in Table 1.

2.10.1. Experiment 1 The effect of pre-training intra-CA1 administration of scopolamine, NMDA and D-AP7 with intra-MS administration of saline on acquisition of memory formation was examined in this experiment. One group of animals received saline (0.5 ␮l/site) in both CA1 and MS. Other groups of animals received intra-CA1, scopolamine (0.25, 0.5, 1, and 2 ␮g/rat), NMDA (0.015, 0.03, 0.06, 0.125 and 0.25 ␮g/rat) or D-AP7 (0.006, 0.012, 0.025, 0.05 and 0.1 ␮g/rat) with saline (intra-MS) just before training. The locomotor activity behaviors of animals were recorded 5 min after memory testing. Data in this experiment were analyzed separately for each drug by means of one-way ANOVAs involving the between-groups factor of dose.

2.10.2. Experiment 2 In this experiment, the effect of pre-training intra-CA1 injection of NMDA on impairment of memory acquisition by scopolamine in the CA1 was evaluated. Eight groups of animals were used. The animals received intra-CA1, saline (0.5 ␮l/site) or a subthreshold dose of NMDA (0.06 ␮g/rat), 5 min prior to training. These animals had previously received treatment with saline (0.5 ␮l/site) or different doses of scopolamine (0.5, 1 and 2 ␮g/rat) 5 min before the second injection. In addition, all animals received intra-MS saline (0.5 ␮l/rat). The locomotor activity of animals was recorded 5 min after memory testing. Data were analyzed by means of a 2 (NMDA dose) × 4 (scopolamine dose) between-groups factorial ANOVA.

2.10.3. Experiment 3 In this experiment, the effect of pre-training intra-CA1 co-administration of subthreshold doses of scopolamine and D-AP7 on acquisition of memory formation was examined. Six groups of animals were used. The animals received intra-CA1, saline (0.5 ␮l/site) or a subthreshold dose of scopolamine (0.25 ␮g/rat), 5 min prior to training. These animals had previously received saline (0.5 ␮l/site), a subthreshold dose of scopolamine (0.25 ␮g/rat) or D-AP7 (0.006 and 0.012 ␮g/rat), 5 min after the first injection. In addition, all animals received intra-MS saline (0.5 ␮l/rat). The locomotor activity of animals was recorded 5 min after memory testing. In this experiment One-way ANOVA was used for analyzing differences between groups factor of dose. 2.10.4. Experiment 4 The effect of pre-training intra-MS administration of scopolamine, NMDA and D-AP7 with intra-CA1 administration of saline on acquisition of memory formation was examined in this experiment. One group of animals received saline (0.5 ␮l/site) in both MS and CA1. Other groups of animals received intra-MS, scopolamine (0.25, 0.5, 1, and 2 ␮g/rat), NMDA (0.015, 0.03, 0.06, 0.125 and 0.25 ␮g/rat) or D-AP7 (0.006, 0.012, 0.025, 0.05 and 0.1 ␮g/rat) with saline (intra-CA1) just before training. The locomotor activity of animals was recorded 5 min after memory testing. In this experiment, data were analyzed separately for each drug by means of one-way ANOVAs involving the between-groups factor of dose. 2.10.5. Experiment 5 In this experiment, the effect of pre-training intra-MS administration of NMDA on impairment of memory acquisition induced by scopolamine in the MS was evaluated. Eight groups of animals were used. The animals received intra-MS, saline

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(0.5 ␮l/rat) or subthreshold dose of NMDA (0.06 ␮g/rat), 5 min prior to training. These animals had previously received treatment with saline (0.5 ␮l/rat) or different doses of scopolamine (0.5, 1 and 2 ␮g/rat) 5 min before the second injection. In addition, all animals received intra-CA1 saline (0.5 ␮l/site). The locomotor activity of animals was recorded 5 min after memory testing. In this experiment, data were analyzed by means of a 2 (NMDA dose) × 4 (scopolamine dose) between-groups factorial ANOVA. 2.10.6. Experiment 6 In this experiment, the effect of pre-training intra-MS co-administration of subthreshold doses of scopolamine and D-AP7 on acquisition of memory formation was examined. Six groups of animals were used. The animals received intra-MS, saline (0.5 ␮l/rat) or subthreshold dose of scopolamine (0.25 ␮g/rat), 5 min prior to training. These animals had previously received injection of saline (0.5 ␮l/rat), subthreshold doses of scopolamine (0.25 ␮g/rat) or D-AP7 (0.006 and 0.012 ␮g/rat) 5 min before the second injection. In addition, all animals received intra-CA1 saline (0.5 ␮l/site). The locomotor activity of animals was recorded 5 min after memory testing. Data were analyzed by means of one-way ANOVAs involving the betweengroups factor of dose. 2.10.7. Experiment 7 In this experiment, the effect of pre-training intra-CA1 administration of different doses of scopolamine upon memory response induced by NMDA or D-AP7 in the MS was evaluated. Twelve groups of animals were used. The animals received intraCA1 saline (0.5 ␮l/site) or different doses of scopolamine (0.25, 0.5 and 1 ␮g/rat) before training. Moreover, these animals also received intra-MS, saline (0.5 ␮l/rat), a subthreshold dose of NMDA (0.03 ␮g/rat) or a subthreshold dose of D-AP7 (0.012 ␮g/rat) 5 min after the first injection to the MS. The locomotor activity of animals was recorded 5 min after memory testing. In this experiment, separate twoway ANOVAs on NMDA versus D-AP7 were analyzed by means of 2 (NMDA dose) × 4 (scopolamine dose) and 2 (D-AP7 dose) × 4 (scopolamine dose) between-groups factorial ANOVA. 2.10.8. Experiment 8 In this experiment, the effect of pre-training intra-MS administration of different doses of scopolamine upon memory response induced by NMDA and D-AP7 in the CA1 was evaluated. Twelve groups of animals were used. The animals received intra-MS saline (0.5 ␮l/rat) or different doses of scopolamine (0.25, 0.5 and 1 ␮g/rat) before training. Moreover, these animals received intra-CA1, saline (0.5 ␮l/site), subthreshold dose of NMDA (0.03 ␮g/rat) or also a subthreshold dose of D-AP7 (0.012 ␮g/rat) 5 min after the first injection in the MS. The locomotor activity of animals was recorded 5 min after memory testing. In this experiment, separate Two-way ANOVAs on NMDA versus D-AP7 were used to analyzing by means of 2 (NMDA dose) × 4 (scopolamine dose) and 2 (D-AP7 dose) × 4 (scopolamine dose) between-groups factorial ANOVA.

Fig. 1. The effects of pre-training intra-CA1 administration of saline, scopolamine, NMDA or D-AP7 on memory acquisition and locomotor activity. The animals received pre-training intra-CA1 injections of saline (1 ␮l/rat), scopolamine (0.25, 0.5, 1 and 2 ␮g/rat, A), NMDA (0.015, 0.03, 0.06, 0.125 and 0.25 ␮g/rat, B) or DAP7 (0.006, 0.012, 0.025, 0.05 and 0.1 ␮g/rat, C) just before training, while all of above animals received pre-training intra-MS administration of saline (0.5 ␮l/rat). The memory was measured 24 h after injection of drugs. D, E and F showed the effect of drugs on locomotor activity in the test’s day. Data are expressed as mean ± S.E.M of eight animals per group. *P < 0.05, **P < 0.01, ***P < 0.001 different from saline/saline control group.

3. Results 3.1. Effects of pre-training intra-CA1 administration of scopolamine, NMDA and D-AP7 on memory acquisition One way ANOVA analysis revealed that pre-training intraCA1 administration of scopolamine [F (4, 35) = 6.22, P < 0.001, Fig. 1A], NMDA [F (5, 42) = 5.82, P < 0.001, Fig. 1B] and D-AP7 [F (5, 42) = 6.11, P < 0.001, Fig. 1C] altered inhibitory avoidance acquisition. Moreover, post hoc analysis showed that scopolamine at doses of 0.5, 1 and 2 ␮g/rat, and D-AP7 at doses of 0.025, 0.05 and 0.1 ␮g/rat decreased, while NMDA at doses of 0.125 and 0.25 ␮g/rat increased the step-through latency during the retention test. In addition, the results showed that scopolamine [F (4, 35) = 1.63, P > 0.05, Fig. 1D], NMDA [F (5, 42) = 0.8, P > 0.05, Fig. 1E] and D-AP7 [F (5, 42) = 0.69, P > 0.05, Fig. 1F] had no effect on locomotor activity. In conclusion, scopolamine and D-AP7 impaired while NMDA increased acquisition of memory formation in the CA1. 3.2. Effect of pre-training intra-CA1 administration of NMDA on scopolamine-induced memory deficit in the CA1 Two-way ANOVA indicated that there is no interaction between NMDA treatment and scopolamine dose on memory acquisition [within-group comparison: treatment effect: F (1, 56) = 0.49, P > 0.05, dose effect: F (3, 56) = 38.18, P < 0.001, treatment–dose interaction: F (3, 56) = 3.83, P > 0.05] (Fig. 2A and

B) and locomotor activity [within-group comparison: treatment effect: F (1, 56) = 0.31, P > 0.05, dose effect: F (3, 56) = 1.7, P > 0.05, treatment–dose interaction: F (3, 56) = 0.49, P > 0.05] (Fig. 2C and D) in the CA1. In conclusion NMDA did not restore the impairment of memory by scopolamine in the CA1. 3.3. Effects of pre-training co-administration of a subthreshold dose of scopolamine plus also subthreshold doses of D-AP7 on memory acquisition in the CA1 One way ANOVA analysis demonstrated that pre-training intraCA1 co-administration of a subthreshold dose of scopolamine (0.25 ␮g/rat) plus also subthreshold doses of D-AP7 (0.006 ␮g/rat) [F (5, 42) = 11.202, P < 0.001, Fig. 3A] or 0.012 ␮g/rat [F (5, 42) = 15.843, P < 0.001, Fig. 3A] decreased memory acquisition but did not alter locomotor activity [F (5, 42) = 1.458, P > 0.05] and [F (5, 42) = 0.629, P > 0.05] for 0.006 and 0.012 ␮g/rat, respectively (Fig. 3B). In conclusion, there is a synergistic effect between scopolamine and D-AP7 in memory acquisition in the CA1. 3.4. Effects of pre-training intra-MS administration of scopolamine, NMDA and D-AP7 on memory acquisition One way ANOVA analysis revealed that pre-training intra-MS administration of scopolamine [F (4, 35) = 6.83, P < 0.001, Fig. 4A], NMDA [F (5, 42) = 7.19, P < 0.001, Fig. 4B] and D-AP7 [F (5, 42) = 6.07,

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Fig. 2. The effects of pre-training intra-CA1 administration of NMDA on scopolamine-induced memory deficit or locomotor activity in the CA1. The animals received pre-training intra-CA1 saline (1 ␮l/rat, A) or NMDA (0.06 ␮g/rat, B) 5 min after pre-training intra-CA1 injection of saline (1 ␮l/rat) or scopolamine (0.5, 1 and 2 ␮g/rat). In addition, all animals received intra-MS saline (0.5 ␮l/rat). The memory was measured 24 h after injection of drugs. C and D show the effect of drugs on locomotor activity in the test’s day. In addition all animals received pre-training intra-MS administration of saline (0.5 ␮l/rat). Data are expressed as mean ± S.E.M of eight animals per group. *P < 0.05, **P < 0.01, ***P < 0.001 different from saline/saline control group. +P < 0.05, ++P < 0.01, +++P < 0.001 different from saline/NMDA control group.

P < 0.001, Fig. 4C] altered inhibitory avoidance acquisition. Moreover, post hoc analysis showed that scopolamine at doses of 0.5, 1 and 2 ␮g/rat, and D-AP7 at doses of 0.025, 0.05 and 0.1 ␮g/rat decreased, while NMDA at doses of 0.125 and 0.25 ␮g/rat increased the step-through latency during the retention test. In addition, the results showed that scopolamine [F (4, 35) = 0.68, P > 0.05, Fig. 4D], NMDA [F (5, 42) = 1.1, P > 0.05, Fig. 4E] and D-AP7 [F (5, 42) = 2.0, P > 0.05, Fig. 4F] had no effect on locomotor activity. In conclusion, scopolamine and D-AP7 impaired while NMDA increased acquisition of memory formation in the MS. 3.5. Effect of pre-training intra-MS administration of NMDA on scopolamine-induced memory deficit in the MS Two-way ANOVA indicated that there is an interaction between NMDA and scopolamine on memory acquisition [within-group comparison: treatment effect: F (1, 56) = 9.98, P < 0.01, dose effect: F (3, 56) = 29.83, P < 0.001, treatment–dose interaction: F (3, 56) = 3.78, P < 0.05] (Fig. 5A and B) but not locomotor activity [within-group comparison: treatment effect: F (1, 56) = 0.18, P > 0.05, dose effect: F (3, 56) = 2.6, P > 0.05, treatment–dose interaction: F (3, 56) = 1.8, P > 0.05] (Fig. 5A and B) in the CA1. In addition, the post hoc analysis showed that NMDA at a dose of 0.06 ␮g/rat restored impairment of memory induced by scopolamine at a dose of 0.5 ␮g/rat in the MS.

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Fig. 3. The effects of pre-training intra-CA1 co-administration of scopolamine and D-AP7 on memory acquisition or locomotor activity in the CA1. The animals received pre-training intra-CA1 saline (1 ␮l/rat), scopolamine (0.25 ␮g/rat) and D-AP7 (0.006 and 0.012 ␮g/rat) 5 min before pre-training intra-CA1 injection of saline (1 ␮l/rat) or scopolamine (0.25 ␮g/rat). In addition, all animals received intra-MS saline (0.5 ␮l/rat). The memory was measured 24 h after injection of drugs (A). B shows the effect of drugs on locomotor activity in the test’s day. Data are expressed as mean ± S.E.M of eight animals per group. **P < 0.01 and ***P < 0.001 different from saline/saline control group.

3.6. Effects of pre-training co-administration subthreshold dose of scopolamine plus subthreshold doses of D-AP7 on memory acquisition in the MS One way ANOVA analysis demonstrated that pre-training intra-MS co-administration of a subthreshold dose of scopolamine (0.25 ␮g/rat) plus also subthreshold doses of D-AP7 (0.006 ␮g/rat) [F (5, 42) = 10.609, P < 0.001, Fig. 6A] or 0.012 ␮g/rat [F (5, 42) = 14.929, P < 0.001, Fig. 6A] decreased memory acquisition but did not alter locomotor activity [F (5, 42) = 0.791, P > 0.05] and [F (5, 42) = 1.954, P > 0.05] for 0.006 and 0.012 ␮g/rat, respectively (Fig. 6B). Also, the post hoc analysis showed that D-AP7 at doses of 0.006 and 0.012 ␮g/rat expanded impairment of memory induced by scopolamine at a dose of 0.25 ␮g/rat in the septum. In conclusion, there is a synergistic effect between scopolamine and D-AP7 in memory acquisition in the MS.

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Fig. 4. The effects of pre-training intra-septal administration of scopolamine, NMDA or D-AP7 on memory acquisition and locomotor activity. The animals received pretraining intra-septal injections of saline (0.5 ␮l/rat), scopolamine (0.25, 0.5, 1 and 2 ␮g/rat, A), NMDA (0.015, 0.03, 0.06, 0.125 and 0.25 ␮g/rat, B) or D-AP7 (0.006, 0.012, 0.025, 0.05 and 0.1 ␮g/rat, C) just before training. In addition all animals received pre-training intra-CA1 administration of saline (1 ␮l/rat). The memory was measured 24 h after injection of drugs. D, E and F showed the effect of drugs on locomotor activity in the test’s day. Data are expressed as mean ± S.E.M of eight animals per group. *P < 0.05, **P < 0.01, ***P < 0.001 different from saline/saline control group.

3.7. Effect of pre-training intra-MS administration of a subthreshold dose of NMDA or D-AP7 on scopolamine-induced memory deficit in the CA1 Two-way ANOVA indicated that intra-MS administration of a subthreshold dose of NMDA (0.03 ␮g/rat) did not alter impairment of memory acquisition [within-group comparison: treatment effect: F (1, 56) = 1.53, P > 0.05, dose effect: F (3, 56) = 35.5, P < 0.001, treatment–dose interaction: F (3, 56) = 2.7, P > 0.05, Fig. 7A and B] and locomotor activity [within-group comparison: treatment effect: F (1, 56) = 10.53, P < 0.01, dose effect: F (3, 56) = 1.28, P > 0.05, treatment–dose interaction: F (3, 56) = 0.48, P > 0.05, Fig. 7D and E] by pre-training injection of scopolamine in the CA1. In addition, Two-way ANOVA indicated that intra-MS administration of a subthreshold dose of D-AP7 (0.012 ␮g/rat) increased impairment of memory acquisition [within-group comparison: treatment effect: F (1, 56) = 41.53, P < 0.001, dose effect: F (3, 56) = 18.35, P < 0.001, treatment–dose interaction: F (3, 56) = 3.76, P < 0.05, Fig. 7A and C] but not locomotor activity [within-group comparison: treatment effect: F (1, 56) = 7.32, P < 0.01, dose effect: F (3, 56) = 0.5, P > 0.05, treatment–dose interaction: F (3, 56) = 1.34, P > 0.05, Fig. 7D and F] by pre-training injection of scopolamine in the CA1. Moreover, the post hoc analysis showed that D-AP7 at a dose of 0.012 ␮g/rat heighten impairment of memory induced by scopolamine at doses of 0.25, 0.5 and 1 ␮g/rat in the septum and hippocampus.

Fig. 5. The effects of pre-training intra-septal administration of NMDA on scopolamine-induced memory deficit or locomotor activity in the septum. The animals received pre-training intra-septal saline (0.5 ␮l/rat, A) or NMDA (0.06 ␮g/rat, B) 5 min after pre-training intra-septal injection of saline (0.5 ␮l/rat) or scopolamine (0.5, 1 and 2 ␮g/rat). In addition, all animals received intra-CA1 saline (1 ␮l/rat). The memory was measured 24 h after injection of drugs. C and D shows the effect of drugs on locomotor activity in the test’s day. Data are expressed as mean ± S.E.M of eight animals per group. *P < 0.05, **P < 0.01, ***P < 0.001 different from saline/saline control group. +++P < 0.001 different from saline/NMDA control group.  P < 0.05 as compared to respective control group.

In conclusion, while intra-MS injection of NMDA did not alter memory impairment induced by pre-training injection of scopolamine in the CA1, but intra-MS injection of D-AP7 increased this phenomenon. 3.8. Effect of pre-training intra-CA1 administration of a subthreshold dose of NMDA or D-AP7 on scopolamine-induced memory deficit in the MS Two-way ANOVA indicated that intra-CA1 administration of a subthreshold dose of NMDA (0.03 ␮g/rat) did not alter impairment of memory acquisition [within-group comparison: treatment effect: F (1, 56) = 1.35, P > 0.05, dose effect: F (3, 56) = 15.71, P < 0.001, treatment–dose interaction: F (3, 56) = 2.03, P > 0.05, Fig. 8A and B] and locomotor activity [within-group comparison: treatment effect: F (1, 56) = 0.82, P > 0.05, dose effect: F (3, 56) = 0.94, P > 0.05, treatment–dose interaction: F (3, 56) = 0.44, P > 0.05, Fig. 8D and E] by pre-training injection of scopolamine in the MS. In addition, Two-way ANOVA indicated that intra-CA1 administration of subthreshold dose of D-AP7 (0.012 ␮g/rat) increased impairment of memory acquisition [within-group comparison: treatment effect: F (1, 56) = 37.87, P < 0.001, dose effect: F (3, 56) = 17.92, P < 0.001, treatment–dose interaction: F (3, 56) = 3.43, P < 0.05, Fig. 8A and C] but not locomotor activity [within-group comparison: treatment effect: F (1, 56) = 0.16, P > 0.05, dose effect: F (3, 56) = 1.5, P > 0.05, treatment–dose interaction: F (3, 56) = 0.74,

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Fig. 7. The effect of pre-training intra-septal administration of NMDA or D-AP7 on scopolamine-induced memory deficit and locomotor activity in the CA1. The animals received pre-training intra-septal injections of saline (0.5 ␮l/rat, A), NMDA (0.03 ␮g/rat, B) or D-AP7 (0.012 ␮g/rat, C) and pre-training intra-CA1 injection of saline (1 ␮l/rat) or scopolamine (0.25, 0.5 and 1 ␮g/rat). The memory was measured 24 h after injection of drugs. D, E and F showed the effect of drugs on locomotor activity in the test’s day. Data are expressed as mean ± S.E.M of eight animals per group. **P < 0.01 and ***P < 0.001 different from saline/saline control group. ++P < 0.01 and +++P < 0.001 different from saline/NMDA control group. P < 0.001 different from saline/D-AP7 control group. P < 0.05 different from respective saline control group.

Fig. 6. The effects of pre-training intra-septal co-administration of scopolamine and D-AP7 memory acquisition or locomotor activity in the septum. The animals received pre-training intra-septal saline (0.5 ␮l/rat), scopolamine (0.25 ␮g/rat) and D-AP7 (0.006 and 0.012 ␮g/rat) 5 min before pre-training intra-septal injection of saline (0.5 ␮l/rat) or scopolamine (0.25 ␮g/rat). In addition, all animals received intra-CA1 saline (1 ␮l/rat). The memory was measured 24 h after injection of drugs (A). B shows the effect of drugs on locomotor activity in the test’s day. Data are expressed as mean ± S.E.M of eight animals per group. **P < 0.01 and ***P < 0.001 different from saline/saline control group.

P > 0.05, Fig. 8D and F] by pre-training injection of scopolamine in the MS. Furthermore, the post hoc analysis explicit that D-AP7 at a dose of 0.012 ␮g/rat intensified impairment of memory induced by scopolamine at doses of 0.25 and 1 ␮g/rat in the septum and hippocampus. In conclusion, while intra-CA1 injection of NMDA did not alter memory impairment induced by pre-training injection of scopolamine in the MS, but intra-CA1 injection of D-AP7 increased this phenomenon. 3.9. Histology Fig. 9 illustrates the approximate point of drug injections in the CA1 and MS as plotted on the representative sections taken from the rat brain (atlas of Paxinos and Watson [36]). Cannulae were implanted into the CA1 and MS a total of 648 animals, but only the data from 600 animals with correct cannulae implants were included in the statistical analyses.

4. Discussion 4.1. The effect of cholinergic antagonist and glutamatergic receptor system of CA1 on memory acquisition The intra-CA1 scopolamine-induced amnesia showed in the present study is in agreement with other investigation that cholinergic mechanisms modulate learning and memory formation [10,39,40]. Extensive evidence showed that hippocampaldependent learning is associated with an increase in hippocampal acetylcholine (ACh) levels [41] and anticholinergic drugs such as scopolamine impair learning and memory [42,43]. Furthermore, the cholinergic system plays an important modulatory role in memory performance in the hippocampus [44–46]. Our data indicated thatintra-CA1 administration of NMDA at the doses used increased, while D-AP7 reduced memory acquisition indicating involvement of glutamatergic system of CA1 in memory formation. Moreover, subthreshold dose of NMDA which did not alter impairment of memory by scopolamine in the CA1, in combination with subthreshold doses of D-AP7 or scopolamine induced amnesia, indicating the possible interaction between two cholinergic and glutaminergic systems in the memory acquisition in the CA1 site. NMDA receptor mechanism of hippocampal CA1 has been shown to be involved in the regulation of synaptic plasticity and the processes of learning and memory [47,48]. Moreover, Ach potentiates synaptic activity induced by NMDA in the hippocampus, which may show an important role in the learning and memory processes and administration of NMDA receptor antagonists impaired memory

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Fig. 8. The effect of pre-training intra-CA1 administration of NMDA or D-AP7 on scopolamine-induced memory deficit and locomotor activity in the septum. The animals received pre-training intra-CA1 injections of saline (1 ␮l/rat, A), NMDA (0.03 ␮g/rat, B) or D-AP7 (0.012 ␮g/rat, C) and pre-training intra-septal injection of saline (0.5 ␮l/rat) or scopolamine (0.25, 0.5 and 1 ␮g/rat). The memory was measured 24 h after injection of drugs. D, E and F showed the effect of drugs on locomotor activity in the test’s day. Data are expressed as mean ± S.E.M of eight animals per group. **P < 0.01 different from saline/saline control group. +++P < 0.001 different from saline/NMDA control group. P < 0.01 and P < 0.001 different from saline/D-AP7 control group. P < 0.05 and P < 0.01 different from respective saline control group.

performance of rodents in spatial and non-spatial learning tasks that depended on hippocampal function [49–51]. The impairment may be due to decrease in nitric oxide level and subsequent cyclic guanosine monophosphate production in the brain [52]. However, there is a report showing that MK-801, a non-competitive NMDA receptor antagonist, can improve memory [53]. It seems that both cholinergic and NMDA systems not only play a part in the modulation of memory in the dorsal hippocampus of rats, but also have a complex interaction as well. The muscarinic and nicotinic receptors activate glutamatergic pyramidal neurons and in turn, increase glutamate release. NMDA receptors in the CA1 area of dorsal hippocampus interact with cholinergic systems in the modulation of memory [12]. The activation of muscarinic receptors can facilitate NMDA responses in the hippocampus which is thought to underlie the known role of the cholinergic system in learning and memory [49,54]. 4.2. The effect of cholinergic antagonist and glutamatergic receptor system of MS on memory acquisition The present data also demonstrate that intra-MS administration of scopolamine decreased the inhibitory avoidance on the test day, indicating involvement of cholinergic receptor mechanism of MS in memory. Other investigations showed that intra-MS infused muscarinic agonists could decrease memory impairment related to cholinergic blockade [55,56]. Moreover, cholinergic activity of the MS was proposed to be a critical factor for working memory, which may be blocked by scopolamine [57]. ACh could excite

Fig. 9. Approximate location of the injection cannulae tips in the CA1 and MS regions for all rats included in the data analyses was taken from the atlas of Paxinos and Watson [36].

MS neurons [58,59], and that infusion of scopolamine into the MS area dose-dependently decreased ACh release in the hippocampus support this hypothesis [60]. Furthermore, intra-MS infusion of NMDA by itself increased memory acquisition. The data showed that intra-CA1 subthreshold dose of NMDA restored impairment of memory induced by scopolamine in the MS, while a subthreshold dose of D-AP7 increased impairment of memory by scopolamine indicating there is an interaction between scopolamine and D-AP7 in the MS on impairment of memory acquisition. Previously, it has been shown that intra-MS infusions of NMDA agonists or antagonists improve or impair retention of water maze or avoidance tasks respectively [35]. However, there is another report showing that NMDA receptor antagonists also improve memory processes [61]. Thus, it has been argued that the effects of NMDA receptor antagonists on learning and memory are dependent on the type of task [62]. Other investigators also suggested that NMDA receptor antagonists, D-AP7 or MK-801 facilitated retention in a step-down inhibitory avoidance task, but impaired retention in place navigation and step-through dark avoidance tasks [63]. 4.3. Influence of glutamatergic receptor systems of CA1 and MS on impairment of memory induced by scopolamine In the third part of our study, the results indicated that intra-CA1 injection of NMDA could not alter impairment of memory by scopolamine injected into the MS. Intra-MS injection of NMDA could not also change impairment of memory by scopolamine injected into the CA1. However, injections of D-AP7 in combination with a subthreshold dose of scopolamine elicit amnesic response. The data may reveal a glutamatergic mechanism between CA1 and MS may

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regulate the influence of cholinergic system on memory acquisition in either MS or CA1 respectively. The MS and diagonal band of Broca are mutually connected with the hippocampus which may show an important input for spatial learning and hippocampal theta rhythm [57]. While cholinergic septo-hippocampal projections have been well described, but the functional significance of the glutamatergic projection has not been demonstrated [64]. Moreover, about 4–23% of the septo-hippocampal projection is glutamatergic [65,66]. The glutamatergic system in the MS/VDB, possibly through the NMDA receptors is involved in hippocampal-dependent learning and memory [67]. On the other hand, intra-MS injection of glutamate receptor antagonists decreased hippocampal learning and memory [67], hippocampal spatial and emotional learning [24]. 5. Conclusion Firstly, pre-training intra-CA1 or intra-MS administration of NMDA improved, while D-AP7 and scopolamine reduced memory acquisition. Secondly, combination of subthreshold doses of scopolamine with D-AP7 induced amnesia when injected either in CA1 or MS. Thirdly, NMDA injected into the MS, restored impairment of memory induced by scopolamine in this site. Fourthly, NMDA administration in the CA1 did not alter impairment of memory induced by scopolamine in the MS. Moreover, NMDA administration into the MS did not alter scopolamine-induced amnesia when injected into CA1. Finally, there is an interaction between amnesia of scopolamine injected into CA1 with D-AP7 injected into MS and vice versa. It can be concluded that CA1 and MS NMDA receptor mechanisms interact with cholinergic systems of either sites. However, more experiments are required to clarify the interaction between glutamatergic and cholinergic receptor systems in the modulation of memory in these regions. Acknowledgment The authors would like to thank Mohsen Shirazizadeh for his valuable assistance. References [1] Deiana S, Platt B, Riedel G. The cholinergic system and spatial learning. Behav Brain Res 2011;221:389–411. [2] Klinkenberg I, Sambeth A, Blokland A. Acetylcholine and attention. Behav Brain Res 2011;221:430–42. [3] Kirby BP, Rawlins JN. The role of the septo-hippocampal cholinergic projection in T-maze rewarded alternation. Behav Brain Res 2003;143:41–8. [4] Nasehi M, Piri M, Jamali-Raeufy N, Zarrindast MR. Influence of intracerebral administration of NO agents in dorsal hippocampus (CA1) on cannabinoid state-dependent memory in the step-down passive avoidance test. Physiol Behav 2010;100:297–304. [5] Nasehi M, Sahebgharani M, Haeri-Rohani A, Zarrindast MR. Effects of cannabinoids infused into the dorsal hippocampus upon memory formation in 3-days apomorphine-treated rats. Neurobiol Learn Mem 2009;92:391–9. [6] Roesler R, Schroder N, Vianna MR, Quevedo J, Bromberg E, Kapczinski F, et al. Differential involvement of hippocampal and amygdalar NMDA receptors in contextual and aversive aspects of inhibitory avoidance memory in rats. Brain Res 2003;975:207–13. [7] Flood JF, Farr SA, Uezu K, Morley JE. The pharmacology of post-trial memory processing in septum. Eur J Pharmacol 1998;350:31–8. [8] Bunce JG, Sabolek HR, Chrobak JJ. Intraseptal infusion of oxotremorine impairs memory in a delayed-non-match-to-sample radial maze task. Neuroscience 2003;121:259–67. [9] Abreu-Villaca Y, Filgueiras CC, Manhaes AC. Developmental aspects of the cholinergic system. Behav Brain Res 2011;221:367–78. [10] Robinson L, Platt B, Riedel G. Involvement of the cholinergic system in conditioning and perceptual memory. Behav Brain Res 2011;221:443–65. [11] Thiel CM, Huston JP, Schwarting RK. Hippocampal acetylcholine and habituation learning. Neuroscience 1998;85:1253–62. [12] Jafari-Sabet M. NMDA receptor blockers prevents the facilitatory effects of post-training intra-dorsal hippocampal NMDA and physostigmine on memory retention of passive avoidance learning in rats. Behav Brain Res 2006;169:120–7.

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