Involvement of nitrergic system of CA1in harmane induced learning and memory deficits

Physiology & Behavior 109 (2013) 23–32

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Involvement of nitrergic system of CA1in harmane induced learning and memory deficits Mohammad Nasehi a, Morteza Piri b, Mojgan Abdollahian c, Mohammad Reza Zarrindast c, d, e, f, g,⁎ a

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

H I G H L I G H T S ► ► ► ► ►

Intraperitoneal pre-training injection of harmane (HA) impairs memory encoding. Pre-training/testing intra-CA1 injection of L-NAME did not alter memory formation. Pre-training/testing intra-CA1 injection of L-NAME reversed HA-induced amnesia. Pre-training/testing intra-CA1 injection of L-arginine did not alter memory formation. Pre-testing but not pre-training injection of L-arginine increased HA-induced amnesia.

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Article history: Received 30 May 2012 Received in revised form 18 September 2012 Accepted 19 October 2012 Available online 25 October 2012 Keywords: Harmane Nitrergic system Step-down passive avoidance Hole-board Memory Anxiety-like behavior Mice

a b s t r a c t Harmane (HA) is a β-carboline alkaloid derived from the Peganum harmala plant which induces memory impairment. On the other hand some of the investigations showed that β-carboline alkaloids inhibit NO production. Thus, the aim of the present study was to investigate the role of nitrergic system of the dorsal hippocampus (CA1) in HA-induced amnesia in male adult mice. One-trial step-down passive avoidance and hole-board apparatuses were used for the assessment of memory retrieval and exploratory behaviors respectively. The data indicated that pre-training intraperitoneal (i.p.) administration of HA (12 and 16 mg/kg) decreased memory acquisition. Sole pre-training or pre-testing administration of L-NAME, a nitric oxide synthesis inhibitor (5, 10 and 15 μg/mice, intra-CA1) did not alter memory retrieval. On the other hand, pre-training (10 and 15 μg/mice, intra-CA1) and pre-testing (5, 10 μg/mice, intra-CA1) injections of L-NAME restored HA-induced amnesia (16 mg/kg, i.p.). Furthermore, neither sole pre-training nor pre-testing administration of L-arginine, a NO precursor (3, 6 and 9 μg/mice, intra-CA1), altered memory retrieval. In addition, pre-testing (6 and 9 μg/mice, intra-CA1), but not pre-training, injection of L-arginine increased HA-induced amnesia (16 mg/kg, i.p.). These results suggest that the nitrergic system of CA1 is involved in HA-induced amnesia. © 2012 Elsevier Inc. All rights reserved.

1. Introduction A number of β-carbolines which are known as harmala alkaloids, have been identified in common plant-derived foodstuffs, beverages, and inhaled substances [1]. The first endogenous β-carboline, pinoline (6-methoxytetrahydro-β-carboline), with an indole nucleus and a pyridine ring, was found in an extract of pinal gland tissue [2]. The alkaloids have also been found in hallucinogenic plants used ⁎ Corresponding author at: Department of Neuroscience, School of Advanced Medical Technologies, P.O. Box 13145-784, Tehran, Iran. Tel./fax: +98 21 66402569. E-mail address: [email protected] (M.R. Zarrindast). 0031-9384/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.physbeh.2012.10.006

in South American Indian cultures [3]. Depending upon their degree of ring saturation, the β-carbolines (called also harmala) can be divided into three structural groups including, fully unsaturated pyridine ring (harmane; HA), the dihydro derivative (harmaline) and the tetrahydro derivatives [1,4–6]. Endogenous harmala alkaloids may also exist in normal body constituents, such as blood plasma, heart, kidney, liver and brain tissue [7–9]. Moreover, our previous study reported that harmane impaired memory consolidation [1,10]. On the other hand, the alkaloid extract of Peganum harmala and harman induce antinociceptive activities [11] and anti-allodynia in a dose-dependent manner [12], respectively, which may influence on the drug memory response.

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β-Carbolines can also act on a wide range of central nervous system binding sites [1,6,13], such as 5HT2 and 5HT1A receptors [6], imidazoline receptors [13] and interact with NMDA receptors [14]. The HA inhibits enzymes such as monoamine oxidase A and B (main effect of HA in the body) [1,15–17] and inducible nitric oxide synthase (iNOS) [18]. β-Carboline alkaloid is also reported to decrease the level of iNOS protein and NOS promoter activities in a concentrationdependent manner [19]. Nitric oxide (NO) has a critical role, as a prominent second messenger, in the central and peripheral nervous systems [20]. It participates in certain forms of long-term potentiation and expression [20–22]. NO is involved in many central effects of drugs such as nicotine [23], morphine [24], cannabinoid [20], ethanol [25], lithium [26] and histamine [27]. Moreover, the hippocampus, which has a principal role in learning and memory [28], is necessary to mediate the expression of passive avoidance learning [1,20,29]. A number of studies have also indicated that NO [20,25,30] and harmane [10] are involved in hippocampusdependent learning and memory. On the basis of the previous studies including; 1 — memory impairment induced by HA, 2 — inhibition of monoamine oxidase and NOS enzymes by β-carboline alkaloids, 3 — close interaction between NO and dopamine in modulation of learning and memory [31], and 4 — the pivotal role of CA1 on HA-induced amnesia [10], the aim of the present study is to investigate the effect of CA1 nitrergic system on HA (fully aromatic) alkaloid-induced amnesia in male mice in the step-down task. Since the drug can produce non-mnemonic effects (fear, locomotor activity, etc.) which can confound the results on the passive-avoidance task, we used hole-board task immediately after memory measurement by passive avoidance task on the test day. 2. Materials and methods 2.1. Animals Male albino NMRI mice weighing 25–30 g were used. Animals were kept in an animal house with a 12/12-h light–dark cycle and controlled temperature (22 ± 2 °C). They were housed in groups of 10 in Plexiglas cages and had free access to food and water except during the limited periods of experiments. Ten animals were used in each group; each animal was used once only. Behavioral experiments were done during the light phase of the light/dark cycle. All procedures were carried out in accordance with institutional guidelines for animal care and use. 2.2. Cannula guide implantation Mice were anesthetized with intra-peritoneal injection of ketamine hydrochloride (50 mg/kg) plus xylazine (5 mg/kg) and were placed in a stereotaxic apparatus. The skin was then incised and the skull was cleaned. 22-gauge guide cannulae were placed (bilaterally) 1 mm above the intended site of injection according to the atlas of Paxinos [32]. Stereotaxic coordinates for the CA1 regions of the dorsal hippocampus were AP: − 2 mm from bregma, L: ±1.6 from the sagittal suture and V: − 1.5 mm from the skull surface. The cannulae were secured with dental acrylic. Stainless steel stylets (27-gauge) were inserted into the guide cannulae to keep them free of debris. All animals were allowed 1 week to recover from the surgery and from the effect of the anesthetic agents. 2.3. Intra-CA1 injections For drug infusion, the animals were gently restrained by hand. The stylets were removed from the guide cannulae and replaced by 27-gauge injection needles (1 mm below the tip of the guide cannulae). The injection solutions were manually administered in a total volume of

1 μl/mice (0.5 μl in each side) over a 60 s period. Injection needles were left in place for an additional 60 s to facilitate the diffusion of the drugs. 2.4. Memory testing and apparatus The step-down latency in passive avoidance is suitable for testing learning and memory in mice [33–35]. An inhibitory avoidance apparatus consisted of a wooden box (30×30×40 cm3) with a floor that consisted of parallel stainless steel rods (0.3 cm in diameter, spaced 1 cm apart). A wooden platform (4×4×4 cm3) was set in the center of the grid floor. Electric shocks (1 Hz, 0.5 s and 45 V DC) were delivered to the grid floor by an isolated stimulator (Borj Sanat Co, Tehran, Iran). For testing, each mouse was gently placed on the wooden platform. When the mouse stepped down from the platform and placed all its paws on the grid floor, intermittent electric shocks were delivered continuously for 15 s [36,37]. This training procedure was carried out between 9:00 a.m. and 2:00 p.m. Twenty-four hours after training, each mouse was placed on the platform again, and the step-down latency was measured with a stop-watch as passive avoidance behavior. An upper cut-off time of 300 s was set. The retrieval test was also carried out between 9:00 a.m. and 2:00 p.m. 2.5. Exploratory behavior testing and apparatus The hole-board test as a simple method for examining the response of an animal to an unfamiliar environment was first introduced by Boissier and Simon [38]. The test has since been used to evaluate emotionality, anxiety and/or responses to stress in animals [39]. Different behaviors which can be observed and measured in this test, makes possible the description of animal's behavior. The hole-board apparatus (Borj Sanat Co, Tehran, Iran) consisted of gray Perspex panels (40 cm × 40 cm, 2.2 cm thick) with 16 equidistant holes 3 cm in diameter in the floor made on the basis of methods used previously [40]. The board was positioned 15 cm above a table. For anxiety testing, 5 min after memory testing, animals were placed singly in the center of the board facing away from the observer and head-dip numbers were recorded by photocells arranged below the holes over 5 min. Increase or decrease in head-dips indicated anxiolytic-like or anxiogenic-like behaviors respectively. Locomotor activity was also measured by an observer unaware of the treatments measured during the testing phase. For this purpose, the ground area of the hole-board was divided into four equal sized squares. Locomotion was measured as the number of locomotor activity crossings from one square to another. Other behavioral performances such as latency to the first head-dipping, rearing, grooming and defecation were manually recorded by the experimenter during the test. 2.6. Drugs The drugs used in the present study were ketamine and xylazine (Alfasan Chemical Co, Woerden, and Holland), harmane (HA; 1-methyl-9H-pyrido [3,4-b] indole, C12H10N2) from Sigma (St. Louis, MO), L-arginine and L-NAME (Tocris Cookson, Bristol, UK). The time of injections and doses of compounds used in the experiments were chosen according to published works in scientific literature [41–44]. All compounds were tested at three doses: harmane (HA) 8, 12 and 16 mg/kg; L-NAME, 5, 10 and 15 μg/mice; and L-arginine, 3, 6 and 9 μg/mice. HA was dissolved in sterile 0.9% NaCl solution and the compound was stirred for 1 h before obtaining the final solution; L-NAME and L-arginine were dissolved in 0.9% physiological saline just before the experiments. 2.7. Histology After testing sessions, each mouse was deeply anesthetized and 1 μl of a 4% methylene-blue solution was bilaterally infused into the

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CA1 (0.5 μl/side) as described in the drug section; the mouse was then decapitated and its brain was removed and placed in formaldehyde (10%). After several days, the brains were sliced and the sites of injections were verified according to atlas of Paxinos [32]. 2.8. Drug treatment Ten animals were used in each experimental group. In experiments where animals received two injections, control groups received saline injections. HA was injected intraperitoneally (pre-training) in a volume of 10 ml/kg. The timing of the pre-training or pre-testing drug (L-NAME and L-arginine) injection was selected based on pilot and previous studies [1,20,25]. 2.8.1. Experiment 1: effects of HA on memory acquisition In this experiment, four groups of animals were used. The animals received saline (10 ml/kg, i.p.) or different doses of HA (8, 12 and 16 mg/kg, i.p.) 15 min before training; on the test day all groups received saline (10 ml/kg, i.p.) 15 min before testing. The exploratory behaviors of animals were recorded by hole-board task 5 min after memory testing. 2.8.2. Experiment 2: effects of pre-training administration of L-NAME (intra-CA1) on memory acquisition and exploratory behavior under the influence of HA treatment In this experiment, eight groups of animals were used. The animals received saline (1 μl/mice, intra-CA1) or L-NAME (5, 10 and 15 μg/mice, intra-CA1) 5 min before training. These animals had previously received treatment with saline (10 ml/kg, i.p.) or HA (16 mg/kg, i.p.) 15 min before training. The exploratory behaviors of the animals were recorded by hole-board task 5 min after memory testing. 2.8.3. Experiment 3: effects of pre-testing administration of L-NAME (intraCA1) on memory retrieval and exploratory behavior under the influence of HA treatment In this experiment, eight groups of animals were used. Four groups received pre-training saline (10 ml/kg, i.p.) and 24 h after training, they received saline (1 μl/mice, intra-CA1) or L-NAME (5, 10 and 15 μg/mice, intra-CA1) 5 min before testing. The other four groups received pre-training HA (16 mg/kg, i.p.), and 24 h after training, they received saline (1 μl/mice, intra-CA1) or L-NAME (5, 10 and 15 μg/mice, intra-CA1) 5 min before testing. The exploratory behaviors of animals were recorded by hole-board task 5 min after memory testing. 2.8.4. Experiment 4: effects of pre-training administration of L-arginine (intra-CA1) on memory acquisition and exploratory behavior under the influence of HA treatment In this experiment, eight groups of animals were used. The animals received saline (1 μl/mice, intra-CA1) or L-arginine (3, 6 and 9 μg/mice, intra-CA1) 5 min before training. These animals had previously received treatment with saline (10 ml/kg, i.p.) or HA (16 mg/kg, i.p.) 15 min before training. The exploratory behaviors of animals were recorded by hole-board task 5 min after memory testing. 2.8.5. Experiment 5: effects of pre-testing administration of L-arginine (intra-CA1) on memory retrieval and exploratory behavior under the influence of HA treatment In this experiment, eight groups of animals were used. Four groups received pre-training saline (10 ml/kg, i.p.) and 24 h after training, they received saline (1 μl/mice, intra-CA1) or L-arginine (3, 6 and 9 μg/mice, intra-CA1) 5 min before testing. The other four groups received pre-training HA (16 mg/kg, i.p.), and 24 h after training, they received saline (1 μl/mice, intra-CA1) or L-arginine (3, 6 and 9 μg/mice, intra-CA1) 5 min before testing. The exploratory behaviors of animals were recorded by hole-board task 5 min after memory testing.

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2.9. Statistical analysis Because of individual variations, the data obtained from the step-down apparatus were analyzed using the Kruskal–Wallis nonparametric one-way analysis of variance (ANOVA) followed by a two-tailed Mann–Whitney's U-test. Holmes Sequential Bonferroni Correction Test was used for the paired comparisons when appropriate. The step-down latencies for the animals in each experimental group were expressed as median and inter-quartile ranges. The data obtained from the hole-board apparatus are, however, presented as the mean ± S.E.M. One-way repeated measures analysis of variance (ANOVA) followed by post hoc test was used for statistical evaluations. In all statistical evaluations, P b 0.05 was used as the criterion for statistical significance. 3. Results 3.1. Histology Cannulae were implanted into the CA1 regions of the dorsal hippocampus of a total of 408 animals, but only the data from 360 animals with correct cannula implants were included in the statistical analyses. 3.2. Effects of HA on memory acquisition and exploratory behaviors Kruskal–Wallis [H (3) = 28.59, P b 0.001] and Mann–Whitney's U-test ANOVA show that the administration of different doses of HA (12 and 16 mg/kg, i.p.), 15 min before training, decreased memory acquisition (Fig. 1A). Fig. 1B, C and D also shows the effects of HA on exploratory behaviors. One-way ANOVA analyses revealed that HA in the doses used had no effect on head-dip counts [F (3, 36) = 1.18, P > 0.05] (panel B), latency to head-dipping [F (3, 36) = 1.13, P > 0.05] (panel C), locomotor activity [F (3, 36) = 0.72, P > 0.05] (panel D), number of rearings [F (3, 36) = 1.3, P > 0.05], number of groomings [F (3, 36) = 1.07, P > 0.05] and number of defecations [F (3, 36) = 1.8, P > 0.05]. In conclusion, the data showed that the administration of HA (12 and 16 mg/kg, i.p.), 15 min before training, decreased memory acquisition and had no effect on anxiety-like behavior. 3.3. Effects of pre-training administration of L-NAME on memory acquisition and exploratory behaviors under the influence of HA treatment Fig. 2A (left panel) shows that the administration of different doses of L-NAME (5, 10 and 15 μg/mice, intra-CA1), 15 min before training, did not alter memory acquisition [Kruskal–Wallis ANOVA, H (3) = 0.49, P > 0.05]. In addition, Fig. 2B, C and D (left panels) shows the effects of L-NAME on exploratory behaviors. One-way ANOVA and post hoc analyses revealed that L-NAME in the doses used had no effect on head-dip counts[F (3, 36) = 0.66, P > 0.05] (panel B), latency to head-dipping [F (3, 36) = 0.1, P > 0.05] (panel C), locomotor activity [F (3, 36) = 1.12, P > 0.05] (panel D), number of rearings [F (3, 36) = 1.06, P > 0.05], number of groomings [F (3, 36) = 0.05, P > 0.05] and number of defecations [F (3, 36) = 1.52, P > 0.05]. In conclusion, the data showed that sole injection of L-NAME (intra-CA1) had no effect on memory acquisition anxietylike behaviors Moreover, Fig. 2A (right panel) shows the effect of pre-training injection of L-NAME on HA induced amnesia. Mann–Whitney's U-test analysis revealed that different doses of L-NAME (10 and 15 μg/mice) partially reversed the memory impairment caused by pre-training administration of HA (16 mg/kg, i.p.) [Kruskal–Wallis ANOVA, H (3) = 25.29, Pb 0.001]. Fig. 2B, C and D (right panel) also shows the effects of L-NAME on exploratory behaviors induced by pre-training administration of HA. One-way ANOVA and post hoc analyses revealed that

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Fig. 1. Effects of pre-training administration of HA on memory acquisition and exploratory behaviors. The animals received pre-training administration of saline (10 ml/kg, i.p.) or HA (8, 12 and 16 mg/kg, i.p.) on memory acquisition (panel A). Test session step-down latencies are expressed as median and quartile. In addition, exploratory behaviors including number of head dips (panel B), latency to head dip (panel C) and locomotor activity (panel D) were examined 5 min after memory testing. Each bar is mean+S.E.M. ***Pb 0.001, compared to pre-training saline/pre-testing saline.

L-NAME in the doses used had no effect on head-dip counts [F (3, 36) = 1.64, P > 0.05] (panel B), latency to head-dipping [F (3, 36) = 0.96, P >0.05] (panel C), locomotor activity [F (3, 36) = 1.02, P > 0.05] (panel D), number of rearings [F (3, 36) =1.45, P> 0.05], number of groomings [F (3, 36) = 1.83, P > 0.05] and number of defecations [F (3, 36) = 1.4, P >0.05]. In conclusion, L-NAME reversed HA-induced amnesia.

Fig. 2. Effects of pre-training injection of L-NAME on memory retrieval and exploratory behaviors in the presence and absence of HA. Panel A shows the effects of pre-training administration of L-NAME (5, 10 and 15 μg/mice, intra-CA1) or saline (1 μl/mice, intra-CA1) on animals that were trained under the influence of saline (10 ml/kg, i.p.; left panel) or HA (16 mg/kg, i.p.; right panel). Test session step-down latencies are expressed as median and quartile. In addition, exploratory behaviors, including number of head dips (panel B, left panel; dose response of L-NAME and right panel; effects of L-NAME on HA response), latency to head dip (panel C, left panel; dose response of L-NAME and right panel; effects of L-NAME on HA response) and locomotor activity (panel D, left panel; dose response of L-NAME and right panel; effects of L-NAME on HA response) were examined 5 min after memory testing. Each bar is mean+S.E.M. ***Pb 0.001 when compared to saline/saline group. ++Pb 0.01, +++Pb 0.001 when compared to HA/saline group.

3.4. Effects of pre-testing administration of L-NAME on memory retrieval and exploratory behaviors under the influence of HA treatment Fig. 3A (left panel) shows that the administration of different doses of L-NAME (5, 10 and 15 μg/mice, intra-CA1), 15 min before testing, did not alter memory retrieval [Kruskal–Wallis ANOVA, H (3) = 0.91, P> 0.05]. In addition, Fig. 3B, C and D (left panels) shows the effects of L-NAME on exploratory behaviors. One-way ANOVA and post hoc analyses revealed that L-NAME in the doses used also had no effect on head-dip counts [F (3, 36) = 0.44, P >0.05] (panel B), latency to head-dipping [F (3, 36) =0.63, P >0.05] (panel C), locomotor activity [F (3, 36) = 1.58, P > 0.05] (panel D), number of rearings [F (3, 36) = 1.32, P >0.5], number of groomings [F (3, 36) = 1.22, P> 0.05] and number of defecations [F (3, 36) = 1.01, P> 0.05]. In conclusion, the data

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rearings [F (3, 36) = 0.56, P> 0.05], number of groomings [F (3, 36) = 1.08, P >0.05] and number of defecations [F (3, 36) =0.9, P> 0.05]. In conclusion, L-NAME reversed HA-induced amnesia. 3.5. Effects of pre-training administration of L-arginine on memory acquisition and exploratory behaviors under the influence of HA treatment Fig. 4A (left panel) shows that the administration of different doses of L-arginine (3, 6 and 9 μg/mice, intra-CA1), 15 min before training, did not alter memory acquisition [Kruskal–Wallis ANOVA, H (3) = 1.15, P > 0.05]. In addition, Fig. 4B, C and D (left panels) shows the effects of L-arginine on exploratory behaviors. One-way ANOVA and post hoc analyses revealed that L-arginine in the doses used also had no effect on head-dip counts [F (3, 36) = 0.59,

Fig. 3. Effects of pre-testing injection of L-NAME on memory retrieval and exploratory behaviors in the presence and absence of HA. Panel A shows the effects of pre-testing administration of L-NAME (5, 10 and 15 μg/mice, intra-CA1) or saline (1 μl/mice, intra-CA1) on animals that were trained under the influence of saline (10 ml/kg, i.p.; left panel) or HA (16 mg/kg, i.p.; right panel). Test session step-down latencies are expressed as median and quartile. In addition, exploratory behaviors, including number of head dips (panel B, left panel; dose response of L-NAME and right panel; effects of L-NAME on HA response), latency to head dip (panel C, left panel; dose response of L-NAME and right panel; effects of L-NAME on HA response) and locomotor activity (panel D, left panel; dose response of L-NAME and right panel; effects of L-NAME on HA response) were examined 5 min after memory testing. Each bar is mean+S.E.M. ***Pb 0.001 when compared to saline/saline group. +Pb 0.05, ++Pb 0.01 and +++Pb 0.001 when compared to HA/saline group.

showed that sole injection of L-NAME (intra-CA1) had no effect on memory retrieval and anxiety-like behaviors Fig. 3A (right panel) also shows the effect of pre-testing administration of L-NAME on HA-induced amnesia. Mann–Whitney's U-test analysis revealed that different doses of L-NAME (5, 10 and 15 μg/mice) reversed memory impairment caused by pre-training administration of HA (16 mg/kg, i.p.) [Kruskal–Wallis ANOVA, H (3) =22.30, P b 0.001]. Fig. 3B, C and D (right panel) also shows the effects of L-NAME on exploratory behaviors induced by pre-training administration of HA. One-way ANOVA and post hoc analyses revealed that the highest dose of L-NAME decreased head-dip counts [F (3, 36)= 2.82, P b 0.05] (panel B) but did not alter other exploratory behaviors including latency to head-dipping [F (3, 36) = 1.12, P > 0.05] (panel C), locomotor activity [F (3, 36)= 1.78, P > 0.05] (panel D), number of

Fig. 4. Effects of pre-training injection of L-arginine on memory retrieval and exploratory behaviors in the presence and absence of HA. Panel A shows the effects of pre-training administration of L-arginine (3, 6 and 9 μg/mice, intra-CA1) or saline (1 μl/mice, intra-CA1) on animals that were trained under the influence of saline (10 ml/kg, i.p.; left panel) or HA (16 mg/kg, i.p.; right panel). Test session step-down latencies are expressed as median and quartile. In addition, exploratory behaviors, including number of head dips (panel B, left panel; dose response of L-arginine and right panel; effects of L-arginine on HA response), latency to head dip (panel C, left panel; dose response of L-arginine and right panel; effects of L-arginine on HA response) and locomotor activity (panel D, left panel; dose response of L-arginine and right panel; effects of L-arginine on HA response) were examined 5 min after memory testing. Each bar is mean + S.E.M. ***P b 0.001 when compared to saline/saline group.

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P > 0.05] (panel B), latency to head-dipping [F (3, 36) = 0.46, P > 0.05] (panel C), locomotor activity [F (3, 36) = 1.12, P > 0.05] (panel D), number of rearings [F (3, 36) = 2.25, P > 0.05], number of groomings [F (3, 36) = 1.51, P > 0.05] and number of defecations [F (3, 36) = 1.1, P > 0.5]. In conclusion, the data showed that sole injection of Larginine (intra-CA1) had no effect on memory acquisition and anxiety-like behaviors. Moreover, Fig. 4A (right panel) shows the effect of pre-training administration of L-arginine on HA induced amnesia. Mann–Whitney's U-test analysis revealed that the doses of L-arginine (3, 6 and 9 μg/mice) did not alter memory impairment caused by pre-training administration of HA (16 mg/kg, i.p.) [Kruskal–Wallis ANOVA, H (3) = 1.37, P >0.05]. Fig. 4B, C and D (right panel) also shows the effects of L-arginine on exploratory behaviors induced by pre-training administration of HA. One-way ANOVA and post hoc analyses revealed that L-arginine in the doses used had no effect on head-dip counts [F (3, 36) = 0.49, P >0.05] (panel B), latency to head-dipping [F (3, 36)= 0.67, P > 0.05] (panel C), locomotor activity [F (3, 36) = 0.87, P > 0.05] (panel D), number of rearings [F (3, 36) = 2.4, P > 0.05], number of groomings [F (3, 36) = 1.31, P >0.05] and number of defecations [F (3, 36)= 0.87, P >0.05]. In conclusion, L-arginine had no effect on HA-induced amnesia. 3.6. Effects of pre-testing administration of L-arginine on memory retrieval and exploratory behaviors under the influence of HA treatment Fig. 5A (left panel) shows that the administration of different doses of L-arginine (6 and 9 μg/mice, intra-CA1), 15 min before testing, did not alter memory retrieval [Kruskal–Wallis ANOVA, H (3) = 1.75, P >0.05]. In addition, Fig. 5B, C and D (left panels) shows the effects of L-arginine on exploratory behaviors. One-way ANOVA and post hoc analyses revealed that L-arginine in the doses used also had no effect on head-dip counts [F (3, 36) = 0.35, P >0.05] (panel B), latency to head-dipping [F (3, 36) = 0.51, P >0.05] (panel C), locomotor activity [F (3, 36) = 0.62, P > 0.05] (panel D), number of rearings [F (3, 36) = 2.2, P > 0.05], number of groomings [F (3, 36) = 0.92, P >0.05] and number of defecations [F (3, 36) = 0.66, P> 0.05]. In conclusion, the data revealed that sole injection of L-arginine (intra-CA1) had no effect on memory retrieval and anxiety-like behaviors. Moreover, Fig. 5A (right panel) indicates the effect of pre-testing administration of L-arginine on HA induced amnesia. Mann– Whitney's U-test analysis revealed that different doses of L-arginine (6 and 9 μg/mice) increased memory impairment caused by pre-training administration of HA (16 mg/kg, i.p.) [Kruskal–Wallis ANOVA, H (3) = 9.81, P b 0.01]. Fig. 5B, C and D (right panel) also shows the effects of L-arginine on exploratory behaviors induced by pre-training administration of HA. One-way ANOVA and post hoc analyses revealed that L-arginine (9 μg/mice) decreased head-dip counts [F (3, 36) = 4.71, P b 0.05] (panel B) but did not alter other exploratory behaviors such as latency to head-dipping [F (3, 36)= 1.89, P >0.05] (panel C), locomotor activity [F (3, 36) = 0.73, P > 0.05] (panel D), number of rearings [F (3, 36) =0.86, P> 0.05], number of groomings [F (3, 36) = 1.32, P > 0.05] and number of defecations [F (3, 36) = 0.82, P > 0.05]. In conclusion, L-arginine (6 μg/mice, intraCA1) increased HA-induced amnesia. 4. Discussion The present data indicated that pre-training injection of harmane (HA) attenuated memory retention in mice. Since the animals were given pre-training injections of the drug, both acquisition and early consolidation of memory processes could be influenced. Moreover, the effective doses of HA did not alter other behaviors, including number of head dipping, latency to head dip, locomotor activity, grooming, rearing and defecation, showing that the effect of harmane on memory is reliable. In agreement with these data, our previous

Fig. 5. Effects of pre-testing injection of L-arginine on memory retrieval and exploratory behaviors in the presence and absence of HA. Panel A shows the effects of pre-testing administration of L-arginine (3, 6 and 9 μg/mice, intra-CA1) or saline (1 μl/mice, intra-CA1) on animals that were trained under the influence of saline (10 ml/kg, i.p.; left panel) or HA (16 mg/kg, i.p.; right panel). Test session step-down latencies are expressed as median and quartile. In addition, exploratory behaviors, including number of head dips (panel B, left panel; dose response of L-arginine and right panel; effects of L-arginine on HA response), latency to head dip (panel C, left panel; dose response of L-arginine and right panel; effects of L-arginine on HA response) and locomotor activity (panel D, left panel; dose response of L-arginine and right panel; effects of L-arginine on HA response) were examined 5 min after memory testing. Each bar is mean + S.E.M. ***P b 0.001 when compared to saline/saline group. +P b 0.05 and ++P b 0.01 when compared to HA/saline group.

study had also shown that post-training administration of HA, in a dose lower than that used in this study, decreased memory consolidation, indicating different mechanism of drug effects on memory acquisition or memory consolidation processes [1]. Other researchers have also demonstrated that HA induces a variety of behaviors such as decreased locomotor activity, ataxia, catatonia, convulsions [45–48], and reinforcement of alcohol consumption in rats [49,50]. HA may also induce hallucination, excitation, feelings of elation, and euphoria, but would not alter short and long term memories [48]. There are reports indicating that low doses of β-carbolines may show some effects on non-spatial and non-aversive memory tasks, and enhance long term memory [51]. However, harmaline (a harmala alkaloid) can block both associative and motor learning [52,53]. The

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controversy may be due to the drug, the dose of the drug and the method used, which may influence the drug response. It has also been shown that there are minor differences in the structural parameters of the agents which lead to large differences in their affinities for different receptors. In vivo experiments have also indicated that tetrahydronorharmane exerts high affinity for the serotonergic sites and antagonizes the effects of dopamine-receptor stimulants [54,55], whereas HA, harmine, and harmaline have high affinity for muscarinic cholinergic and opiate receptors. It seems that the affinities of β-carbolines are highly dependent upon substitutions and ring saturation [6]. Bioavailability of HA is 19% in rats [56]. In human it rapidly will appear in the blood reaching a maximal plasma level at 21 min, with an AUC (area under the curve)[56]. Orally administered HA in rats (13%) is metabolized to harmine [56]. The systemic clearance (CL s) for harmine (103.2 ml/kg/ml) was two times greater than that for HA (52.2 ml/kg/ml). These results may show that HA is absorbed into the systemic circulation completely [56]. On the other hand, some investigations have shown that β-carboline alkaloids such as HA decrease the level of inducible nitric oxide synthase (iNOS) enzyme and induce a drastic decrease in serum nitric oxide (NO) level [18,19,57–61]. The nitrergic system (NO system) also exerts a key role in learning and memory formation. In order to get a deeper insight into the nature of HA-induced amnesia, the involvement of NO agents on impairment of avoidance response induced by HA was tested in the dorsal hippocampus (CA1). The results obtained in the present study showed that sole pre-training or pre-testing intra-CA1 microinjection of different doses of L-NAME (a NOS inhibitor) at the doses used caused no significant change in the step-down latencies and anxiety-like behaviors. Moreover, in mice trained under HA (16 mg/kg) influence, pretraining or pre-testing injection of similar doses of L-NAME improved the amnesia induced by pre-training injection of HA with no effect on anxiety-like behaviors, thus, the effect of drug on memory are reliable. The data also indicated that sole pre-training or pre-testing intra-CA1 microinjection of different doses of L-arginine (a NO precursor) in the doses used caused no significant change in step-down latencies and anxiety-like behaviors. On the other hand, pre-testing injection of a dose of L-arginine (6 μg/mouse), but not pre-training injection of the drug, increased amnesia induced by pre-training injection of HA (16 mg/kg) with no effect on anxiety-like behaviors. This also confirms the data are reliable on memory effect. In conclusion, the results may suggest that intraperitoneal administration of HA might lead to an increase in the level of NO in the CA1 which, in turn, might impair memory retrieval processes following HA administration. NO, as a biological messenger, play different roles in the cardiovascular, immune, and nervous systems. The messenger is produced by nitric oxide synthesis (NOS) enzyme [20,62]. Three isoforms of NOS including, neuronal (Type I), inducible (Type II), and endothelial (Type III) have, thus far, been reported [63]. Although all three types of isoforms are found in the brain, the neuronal NOS is the predominant one therein [20,63]. Also, the effects of NO on neurotransmission are mainly attributed to NO produced by neuronal NOS [64,65]. A study has shown that types I and III of NO isoforms are located in pyramidal neurons and interneurons of the hippocampus respectively [66]. It is accepted that NO signaling is required for formation of long-term memory (LTM) and disruption of NO signaling impairs LTM. There are numerous publications on this topic [67–69] and other studies on nNOS knockout mice [70,71]. Moreover it is shown that L-NAME caused impairment in step down passive avoidance task, while L-arginine reversed the effect of L-NAME [72] and L-arginine reversed cannabinoid-induced impairment in the same task [20]. On the other hand, other investigators have shown that NO and guanylate cyclase induced long term depression (LTD) in the hippocampus using low-frequency stimulation [73–75]. It has been proposed that excess NO may be involved in neurocircuitry disorder such as schizophrenia [76]. In general exceeding NO

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concentrations elicited mitochondrial dysfunction [77], neuronal damage [78] and level of NMDA-receptor-expression [79]. While NO has been indicated to affect memory processing by activating guanylate cyclase. Possibly this is not the only mechanism involved and there may evidence supporting the role of NO in a wide array of biochemical reactions [80]. For example it has been shown that systemic inhibition of iNOS activity prevented rats from the deficit in short-term memory [81]. In general the role of L-NAME in memory is very complex inasmuch as systemic administration of high doses of it usually impair memory, while at low doses facilitate memory or even antagonize memory impairments produced by NMDA hypofunction (i.e. MK-801 and ketamine, as NMDA receptor antagonists, induced performance deficits in different cognitive tasks) [76,82,83] or by DA hyperactivity (i.e. impairing effects of apomorphine in the novel object recognition test) [84]. Thus, the present results of the study are not surprising or unexpected. However, the actual action of low doses of NOS mechanism(s) on learning and memory is not completely clear; it seems that mild and transient use of NOS inhibitors induced neuroprotective effects, meanwhile, long-lasting application of these drugs induced neurotoxicity [85], indicating that the NO administration time and its local concentration are two important factors in NO biological action [76,85]. However, we did not test possible interaction of DA with NO in HA-induced amnesia, but one may propose the following explanations. Our previous study showed that block of dopamine D1 and D2 receptors by SCH23390 and sulpiride, respectively revered HA-induced amnesia [1]. Several investigations indicated that both too little and too much stimulation of DA impairs cognitive performance in both animal and human [86–88]. For instance, apomorphine (a DA D1/D2 receptors agonist) [89] influences on cognition ranging from memory facilitation [90–92] to memory impairments [90,93–98] both in humans and animals. Further, NO seems to be involved in attentional deficits produced by DA hyperfunction [31,99–101]. On the other hand, it is clear that there is a bidirectional interaction and relations between NO synthesis and DA release in the brain [102,103] thus one hypothesis in HA-induced amnesia may be due to DA and NO interaction. It has been proposed that NO can decrease [104,105] and increase the release of dopamine in the hippocampus [106,107]. Moreover, it has been reported that the activation of NMDA receptors, which have high expression in the hippocampal interneurons, increases the release of dopamine; this phenomenon is blocked by L-NAME [108]. The hippocampal CA1 plays an important role in learning and memory [28] and receives a dopaminergic input from the ventral tegmental area [28]. Two groups of dopamine receptors, D1 and D2, have been distinguished on the basis of pharmacological and biochemical data. The activation of dopamine D1 receptor stimulates adenylate cyclase while the activation of dopamine D2 inhibits it [28]. Also, several investigations have indicated that NO system is involved in the effects induced by drugs which regulate the tone of dopaminergic system. These drugs include morphine [43,109,110], apomorphine [111,112], ethanol [113–115] and nicotine [116,117]. Based on the MAO inhibitor properties of HA (which increases the tone of dopaminergic system) and also based on our present findings which indicated that the involvement of nitrergic system of CA1 in the HA induced amnesia, it can be suggested that peripheral administration of HA increases NO transmission in the CA1 and induces amnesia. Moreover, other investigations have reported that the injection of 7-nitroindazole (7-NI), a NOS inhibitor, increases the levels of dopamine metabolic in the extracellular space [15,16]. Another study also showed that 7-NI, but not L-NAME, decreased the activity of MAO enzyme (main effect of HA) and increased dopamine levels in the striatum [17]. Considering the increase in dopamine levels in the brain by HA shown by other investigations and also the recovery of memory impairment by the blockade of NOS shown in the present study, it could be suggested that the activation of the NOS by HA causes memory deficit, although some studies have showed that β-carboline alkaloids decrease the level of NO in the blood [57,58]. In conclusion,

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more investigations are yet required to get a deeper insight into the effects of the nitrergic system of CA1 upon HA-induced amnesia. Moreover, we tested locomotor and anxiety-like behavior on the same animals used for memory. Whether it may or may not be a proper way of testing for the locomotor activity and anxiety-like behavior remains to be more evaluated.

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