Behavioural Brain Research

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1 Behavioural Brain Research 201 (2009) Contents lists available at ScienceDirect Behavioural Brain Research journal homepage: Research report The role of hippocampal nitric oxide (NO) on learning and immediate, short- and long-term memory retrieval in inhibitory avoidance task in male adult rats Hooman Eshagh Harooni a,b, Nasser Naghdi b,, Hoori Sepehri a, Ali Haeri Rohani a a Dept. of Physiology, School of Biology, College of Science, University of Tehran, Tehran, Iran b Dept. of Physiology and Pharmacology, Pasteur Institute of Iran, Pasteur Ave., Tehran 13164, Iran article info abstract Article history: Received 14 September 2008 Received in revised form 2 February 2009 Accepted 9 February 2009 Available online 21 February 2009 Keywords: Hippocampus Nitric oxide l-name l-arginine Inhibitory avoidance task Rat There is impressive amount of evidence suggesting the involvement of nitric oxide (NO) in hippocampal synaptic plasticity and consequently learning and memory. Hippocampus is a brain region which is widely implicated in several types of learning and memory formation, including inhibitory avoidance learning. Since the CA1 region of hippocampus has shown nitric oxide synthase (NOS) activity, inhibition of the NOS enzymes can modulate hippocampal function, hence affecting memory processes. Therefore, we conducted series of experiments to further investigate the role of NO on inhibitory avoidance short- and long-term memory in rats. For this purpose, male Wistar rats were divided into 15 groups (n = 10), and bilaterally implanted with guide cannulae aimed at the CA1 region of hippocampus. Animals received pre-training, post-training and pre-retrieval injections of vehicle (saline) or different doses of l-name (5, 10 and 15 g/0.5 l/side) or l-arginine (alone or in combination with l-name), tested for immediate, short- and long-term memory retention in an inhibitory avoidance task. Our results indicated that stepthrough latency (STL) of short- and long-term memory retention test was significantly reduced in l-name treated rats (15 g/0.5 l for immediate and short-term memory; 10 g/0.5 l for long-term memory), as compared to that of control group. Results also revealed that, l-arginine produced no any significant effect on STL, however could reverse the effect of l-name on memory. Our results also showed that, blocking of NO signaling immediately after training had no effect on either short- or long-term memory, indicating that NO release only during training, and not during consolidation, plays a role in memory formation. Together, our findings suggest that NO synthase inhibition by l-name can induce impairments in immediate, short- and long-term memories of inhibitory avoidance task, and these impairments are dependent on the learning and memory processes at which NOS inhibited Published by Elsevier B.V. 1. Introduction Nitric oxide (NO) is an intercellular retrograde messenger, originally described as endothelial relaxation factor, which has been shown to involve in several physiological processes such as hippocampal long-term potentiation (LTP) [3,13,14], synaptic plasticity and consequently learning and memory [2,7,13]. Nitric oxide is synthesized from l-arginine, in the presence of NADPH and O 2,bya series of isoenzymes of the family of nitric oxide synthases (NOS s). At least, three distinct isoforms of NOS [endothelial (enos), neuronal (nnos) and inducible (inos)] have been identified in the central nervous system (CNS) [34,86]. In addition, several studies have shown that nnos and enos are abundant in brain areas such as the hippocampus (particularly in the CA1 regions), which is widely implicated in learning and memory processes [25,27,78,84]. Corresponding author. Tel.: ; fax: addresses: nnaghdi@yahoo.com, nnaghdiir@yahoo.com (N. Naghdi). Previous studies revealed that NO has modulatory effects on different learning and memory processes including inhibitory (passive) avoidance [56,73,83,90], and spatial learning [22,55,62,87]. For instance, using a local or intracerebroventricular (i.c.v.) administration of a constitutive NOS inhibitor, induced impairments in spatial learning [46,73], object recognition [12] and inhibitory avoidance [32,73] tasks in rats. Furthermore NOS inhibitors impaired memory performance in one-trial passive avoidance in chicks [43] and spontaneous alternations in mice [87]. Other studies, however, showed no effects of NOS inhibition on learning and memory processes. For example, Bannerman et al. [4] and Blokland et al. [11] have reported that, systemic or intrahippocampal injections of NOS inhibitors did not impair spatial learning in water maze task. Bohme et al. also reported that l-name treatment left shock-avoidance learning unaffected in rats [13]. Together, previous studies suggested that NO plays important roles in certain forms of learning and memory. However the precise mechanism(s) through which NO may affect learning and memory processes is poorly understood. Experiments investigating the role of hippocampal nitric oxide in memory processes have shown that /$ see front matter 2009 Published by Elsevier B.V. doi: /j.bbr

2 H.E. Harooni et al. / Behavioural Brain Research 201 (2009) NOS inhibition in the rat hippocampus impaired memory retention of an inhibitory avoidance learning task [7,32,44] and induced a state-dependent performance deficit in an object recognition task [12]. In the other hand, studies using systemic or i.c.v. administration of l-name have been shown to impair learning on tasks that rely on spatial information such as T-maze [46], radial arm maze [13] and Morris water maze [20] tasks, thereby suggesting a hippocampal dependent mechanism [20,41,57]. The majority of NO-mediated physiological processes are thought to result from activation of guanylate cyclase (GC) and in turn cgmp activation of protein kinase G (PKG) [18,29,31]. This is also assumed to be a prominent pathway by which NO acts in memory processes [8,9,52]. For example, using a step-down inhibitory avoidance task for rats, Bernabeu et al. found that infusion of a GC inhibitor (LY83583) into the rat hippocampus immediately after training impaired retention, while, pre-training intrahippocampal injection of a cgmp analogue (8-bromo-cGMP) enhanced retention of passive avoidance performance, when tested 24 h after training [8,9]. In a study Katzof et al. reported that l-name treatment in aplysia, induced impairments in immediate and short-term memory and attenuated long-term memory [53]. It has yet to be elucidated whether NO is involved in the formation of both shortand long-term memory in a hippocampal dependent learning task, and which memory processes might be affected by the inhibition of NO synthesis. In the present study, we examined the effects of pre-training, post-training and pre-retrieval intrahippocampal injections of NOS inhibitor l-name, on learning and immediate, short- and long-term memory, using step-through inhibitory avoidance task in male adult rats. Also, we examined the effect of l-arginine (7.8 g/0.5 l/side) on memory impairment induced by a molar equivalent dose of l-name (10 g/0.5 l/side). 2. Materials and methods 2.1. Animals Adult male Wistar rats ( g) were obtained from the breeding colony of Pasteur Institute of Iran. Rats were housed three per cage in a temperature (23 ± 1 C) controlled room that was maintained on a 12:12 reversed light cycle (light on at 07:00 a.m.). Rats had unrestricted access to food and water in their home cage Surgical procedures Approximately 7 8 days prior to initiation of the behavioral experiments, the rats were anesthetized with a mixture of ketamine (100 mg/kg, ip) and xylazine (25 mg/kg, ip) and were bilaterally implanted with cannulae (23-gauge) aimed at site immediately above the CA1 (AP: 3.8 mm from Bregma, ML: ±2.2 mm from midline, and DV: 2.7 mm from the skull surface) according to the atlas of Paxinos and Watson [68]. Two screws were inserted into the skull and cannulae fixed to them with dental cement Microinjection procedure Intrahippocampal injections were made via guide cannulae with injection needles (30-gauge) that were connected by polyethylene tubing to 10- l Hamilton microsyringe. The injections (0.5 l total volume) were delivered over 2 min with a syringe pump, and the injection needles (extending 0.5 mm from the end of the guide cannulae) were left in place an additional minute before they were slowly withdrawn Behavioral testing Inhibitory avoidance Apparatus. The step-through PA apparatus consisted of a lighted chamber (30 cm 20 cm 20 cm) made up of transparent plastic and of a dark chamber (30 cm 20 cm 20 cm) with walls and ceiling made up of dark opaque plastic. A rectangular opening (8 cm 8 cm) was located between the two chambers and could be closed by an opaque guillotine door. The floor of both chambers was made of stainless steel rods (2 mm diameter) spaced 1 cm apart. The floor of the dark compartment could be electrified. The apparatus was placed in an acoustically insulated room, kept in standard condition Procedure. The step-through type of passive avoidance task was used to examine the short- and long-term memory based on the negative reinforcement, as previously described [38]. A day before initiation of the tests, animals were handled and adapted to the testing room. For adaptation to the apparatus, on the day one 30 min before training, rats were placed in the lighted chamber and were allowed to explore for 30 s, and then guillotine door was raised. After the entering of the rats to the dark chamber, guillotine door was lowered and the rats remained there for 30 s. Following the habituation of all animals, the first rat again placed into the lighted chamber for 10 s, the door was lifted, and the crossover latency was recorded. The door was closed behind it and a shock was delivered (1 ma, 5-s duration). Immediate memory was tested in 2 min immediately after training. In this test, animals were allowed to be in light compartment during first 2 min after receiving the shock, while the guillotine door was left open. The number of entries into the dark compartment during 2 min was measured as an index for immediate memory. Retention tests were performed 90 min and 24 h (day 2) after training to assess short- and longterm memory, respectively. The rats were placed in the lighted chamber, 10 s later the door was opened, and the step-through latency (STL) and the time-spent in the dark compartment were recorded, up to 600 s. For short-term memory, STL was recorded up to 300 s and rats were removed from the apparatus after the first entry into dark compartment. During this session the electric shocks were not applied to the grid floor Experimental protocol Experiment 1: pre-training injection of l-name The aim of this experiment was to assess the effect of pre-training injections of l-name into the CA1 region of hippocampus on learning in inhibitory avoidance task. Forty rats were divided into four groups (n = 10) that received vehicle (saline) or different doses of l-name (5, 10 or 15 g/0.5 l/side) 25 min before training in the inhibitory avoidance task. Rats were tested for memory retention at three times, immediately, 90 min and 24 h after training to measure immediate, shortand long-term memory, respectively. Step-through latency, time-spent in and number of entries into dark compartment during the retrieval tests, were recorded for statistical analysis Experiment 2: post-training injection of l-name The aim of this experiment was to assess the effect of post-training injections of l-name on consolidation of inhibitory avoidance task. Forty rats were divided into four groups (n = 10) and received intrahippocampal injections of vehicle (saline) or different doses of l-name (5, 10, 15 g/0.5 l/side) immediately after training. Rats were tested for memory retention at three times, immediately, 90 min and 24 h after training. Step-through latency, time-spent in and number of entries into dark compartment during the retrieval tests, were recorded for statistical analysis Experiment 3: pre-test injection of l-name The aim of this experiment was to assess the effect of per-test injection of l-name into the CA1 region of hippocampus on retrieval of long-term memory traces of inhibitory avoidance task. Forty rats were divided into four groups (n =10) that received vehicle (saline) or different doses of l-name (5, 10, 15 g/0.5 l/side) 25 min before the long-term memory test (24 h after training). Step-through latency, time-spent in and number of entries into dark compartment during the retrieval tests, were recorded for statistical analysis Experiment 4: pre-test injection of l-arginine The aim of this experiment was to assess the effect of intrahippocampal injection of l-arginine on memory in inhibitory avoidance task. l-arginine was injected alone or in concomitant with the l-name, min before retrieval of long-term memory (24 h after training). Step-through latency, time-spent in and number of entries into dark compartment during the retrieval tests, were recorded for statistical analysis Statistical analysis Results are expressed as mean ± S.E.M. of step-through latencies and timespent in and number of entries into dark chamber of inhibitory avoidance task. The data were analyzed using ordinary one-way analysis of variances (ANOVA). Where appropriate, ANOVA s were followed by Tukey Kramer multiple comparisons test to determine between group differences. P < 0.05 was considered as level of statistical significance in all comparisons Histology Following behavioral testing, animals were deeply anesthetized, decapitated and the brains were removed and fixed in formalin. For histological examination of cannulae and injection placement in CA1 area, 100- m thick sections were taken and cannulae and injection tracks were examined for each side with light microscopy. Only data obtained from animals whose cannulae and injections were exactly placed in CA1 region were used to analysis.

3 168 H.E. Harooni et al. / Behavioural Brain Research 201 (2009) Results One-way ANOVA showed that the latency to enter dark compartment at training trials were not significantly different between all groups (data is not shown), so that indicated the uniformity of animals at the time of training Experiment 1: pre-training injection of l-name Immediate memory test Fig. 1-A shows a significant difference in immediate memory test between l-name treated rats and control group [f(3,35) = 3.55; P = 0.024]. Pre-training administration of l-name (15 g/0.5 l/side) significantly increased (P < 0.05) the number of entries into the dark compartment during 2 min after training. This result indicated that pre-training NOS inhibition can hinder learning processes by increasing the number of entries into dark chamber to reach to the criteria Short-term memory test (90 min after training) The data from short-term memory was analyzed using one-way ANOVA. There was a main significant difference between groups treated pre-training with intrahippocampal injection of l-name [f(3,35) = 7.24; P = 0.000]. Post hoc comparisons showed significant difference (P <0.01) on STL of short-term memory test in group received l-name 15 g/0.5 l/side as compared to control group (Fig. 1-B). Since the trial for short-term memory was finished as the animal stepped into the dark chamber, and then animal was removed from the apparatus, the data about time-spent in dark compartment was not available in this session. This result indicated that pre-training hippocampal NOS inhibition, could impair acquisition of short-term memory when tested 90 min after training Long-term memory test (24 h after training) One-way ANOVA followed by multiple comparisons was used to analyze step-through latency of retention test between groups. There was not a main significant effect of treatment on step-through latency [f(3,35) = 2.50; P = 0.074] and time-spent in dark chamber [f(3,35) = 1.12; P = 0.353] during long-term memory retrieval, as compared to control group. However, we have found that, as the higher doses of l-name were applied, the more tendency toward longer stay in dark compartment (lower STL) has been shown, Fig. 1. (A) The effect of pre-training injection of l-name on immediate memory in inhibitory avoidance task. Columns show the means ± S.E.M. of step-through into dark chamber during 2 min immediately after training. *P < 0.05 significant difference vs. control (saline) group. (B) The effect of pre-training injection of l-name on acquisition in inhibitory avoidance learning. Columns show mean ± S.E.M. of stepthrough latency of short-term memory test, which was performed at 90 min after training. **P < 0.01 significant difference vs. control (saline) group. though failed to reach the significant criteria (Table 1). These results indicate that hippocampal NOS inhibition during acquisition of inhibitory avoidance learning, attenuated but did not block memory, when tested 24 h after training Experiment 2: post-training injection of l-name One-way ANOVA revealed no any significant difference in STL of either short- [f(3,33) = 0.31; P = 0.816] or long-term [f(3,33) = 0.52; Table 1 The effects of pre-training, post-training and pre-retrieval injections of l-name and pre-test injection of l-arginine and l-arg + l-name on inhibitory avoidance task. Experiments Doses ( g/0.5 l) Number of rats Immediate memory (mean number of entries into the dark chamber) Short-term memory latency (s) mean ± S.E.M. (90 min after training) Long-term memory (s) mean ± S.E.M. (24 h after training) Step-through latency Time-spent in dark chamber 1. Pre-training Control ± ± ± ± ± ± ± ± ± * 125 ± 41.6 ** ± ± Post-training Control ± ± ± ± ± ± ± ± ± ± ± ± Pre-test Control ± ± ± ± ± ± ± ± 76.4 * ± 73.3 * ± ± ± 41.9 l-arg ± ± ± 60.7 l-arg + l-name ± ± ± 52.2 Saline + saline ± ± ± 25.9 * P < 0.05 indicates significant difference vs. control group. ** P < 0.01 indicates significant difference vs. control group.

4 H.E. Harooni et al. / Behavioural Brain Research 201 (2009) Fig. 2. (A) The effect of pre-test injection of l-name on memory retrieval in inhibitory avoidance learning. Columns show mean ± S.E.M. of step-through latency of retention test which was performed 24 h after training. *P < 0.05 indicates significant difference vs. control (saline) group. Drugs were injected 25 min before retention test which was performed 24 h after training. (B) The effect of intrahippocampal injection of l-name on memory retrieval in inhibitory avoidance learning. Columns show mean ± S.E.M. of time-spent in dark chamber. *P < 0.05 indicates significant difference vs. control (saline) group. Drugs were injected 25 min before retention test which was performed 24 h after training. P = 0.666] memory retention test between l-name treated and control groups (Table 1). The data analysis also showed no significant difference on time-spent in dark compartment during long-term memory retention [f(3,33) = 0.18; P = 0.902]. These results indicated that post-training intrahippocampal injection of l-name did not affect memory consolidation Experiment 3: pre-test injection of l-name Step-through latencies for long-term memory in inhibitory avoidance learning were analyzed also using one-way ANOVA. There was a main significant effect of treatment on STL between groups [f(3,35) = 2.58; P = 0.048] (Fig. 2-A). Post hoc multiple comparisons revealed significant decrease in STL of the group received pre-test l-name (10 l/0.5 l/side) into the CA1 of hippocampus, as compared to the saline treated group (P < 0.05). Data analysis also showed a significant increase on time-spent in dark chamber during performance of long-term memory test, in group received l-name (10 g/0.5 l/side) as compared to control group [f(3,35) = 5.12; P = 0.004] (Fig. 2-B). These results indicated that, hippocampal NOS inhibition before memory test could impair retrieval of memory traces in inhibitory avoidance task, when tested 24 h after training Experiment 4: pre-test injection of l-arginine Results of this experiment showed that l-arginine (7.8 g/0.5 l/side) did not affect retrieval of memory per se. Fig. 3. (A) The effect of intrahippocampal injections of l-arginine (7.8 g/0.5 l; an equimolar dose to l-name) and l-arg + l-name on inhibitory avoidance memory. Columns show mean ± S.E.M. of step-through latency of retention test which was performed 24 h after training. Drugs were injected pre-retrieval (before retention test). There is no significant difference by coadministration of l-arginine and l-name vs. control group. *P < 0.05 indicates significant difference vs. control group. (B) The effect of intrahippocampal injections of l-arginine (7.8 g/0.5 l; an equimolar dose to l-name) and l-arg + l-name on inhibitory avoidance memory. Columns show mean ± S.E.M. of time-spent in dark chamber. Drugs were injected pre-retrieval (before retention test; 24 h after training). *P < 0.05 indicates significant differences vs. l-arg + l-name and control groups. However it has been shown that l-arginine pre-treatment to l-name (10 g/0.5 l/side) before test, could restore the memory retrieval in second day. One-way ANOVA revealed a significant difference in STL [f(3,34) = 2.78; P < 0.05] and time-spent in dark chamber [f(3,34) = 4.37; P < 0.05] for l-name vs. control group (Fig. 3-A) and l-name vs. l-arginine + l-name and control group (Fig. 3-B), respectively. Results of this experiment indicated that retrieval of memory was not blocked by the simultaneous treatment with l-name and the NO donor, so that l-arginine reverses the memory impairment induced by l-name on inhibitory avoidance task. 4. Discussion The effects of pre-training, post-training and pre-retrieval intrahippocampal injections of nitric oxide synthase inhibitor (l- NAME) on immediate, short- and long-term memory was assessed using step-through inhibitory (passive) avoidance learning and memory task. Pre-training injection of l-name blocked the formation of two separable memory processes: (1) very short-term (immediate) memory and (2) short-term memory (90 min after training). The treatment also attenuated, but did not block, longterm memory (24 h after training). Post-training administration of l-name (5, 10 and 15 g/( l side) had no significant effect and

5 170 H.E. Harooni et al. / Behavioural Brain Research 201 (2009) pre-retrieval injection of l-name (10 g/0.5 l/side) significantly impaired retrieval of memory traces, when tested 24 h after training. Together, in the present study, NO synthesis inhibition by intraca1 injection of l-name induced impairments in immediate, short- and long-term memories which were dependent on the processes at which NO synthesis was inhibited. The results of our experiments also indicated that short- and long-term memory in inhibitory avoidance task are differentially modulated by hippocampal NOS inhibition, since, while long-term memory was not blocked, short-term memory of the same animals impaired by pre-training administration of l-name (15 g/0.5 l/side). On the other hand, retrieval processes was significantly impaired only when the drug was injected pre-retention test. These results indicate that NO is involved in the retrieval processes to recall a previously formed memory. Moreover, our experiments revealed that NO synthesis may not be involved in memory consolidation, since, animals which received post-training injections of l-name, still showed a permanent avoidance response on the retention tests, comparable to that of control group. In another experiment, intrahippocampal injection of NO donor, l-arginine, was tested on inhibitory avoidance memory. l-arginine did not significantly affect memory retention per se, although simultaneous treatment with l-name it could restore memory impairment induced by l-name, to a level, comparable to control group, confirming that the effect of l-name was attributable to its effects as a competitive inhibitor of l-arginine in the production of NO rather than to possible effects at other sites. In consistence with our study, there are also several lines of evidence pointing to a role for NO in different learning and memory tasks, including inhibitory avoidance learning [6,7,32,42 44,58,73,75,83], spatial learning [13,20,41,71,73,88], olfactory learning [13,79] and object recognition task [23,72]. In such experiments, inhibition of neuronal NO production by NO synthase inhibitors such as l-name or other NOS inhibitors, has also been reported to disrupt the memory processes in animals. However, some negative findings have also been reported, that failed to confirm such effects [4,11,13]. Systemic injection of NOS inhibitors have been shown to disrupt inhibitory avoidance learning in rats [59,89]. Pre-training and pre-retrieval IP injections of l-name impaired learning and memory but post-training injection did not significantly affect memory processes (Yildirim et al., 2004) [89]. This is a very similar result to our experiments in which intrahippocampal injections of l-name impaired acquisition and retrieval but not consolidation of memory. In consistence to these results, it has been shown that hippocampal NO is involved in several learning tasks. In a three panel running task, Bohme GA et al. (1993) [13] showed that NO synthesis inhibition impaired working memory. Further evidence to support hippocampal effects of NOS inhibition on inhibitory avoidance tasks has come from researches by Bernabeu et al. [7], Fin et al. [32], and Huang and Lee [44]. In these series of experiments memory retrieval was impaired by hippocampal NOS inhibition. In 1998, Blockland and Pickarets have reported that there is a state-dependent memory impairment caused by hippocampal NOS inhibition. Negative findings by Blockland et al. (1999) [11] and Bohme et al. [13] have shown that NOS inhibition did not affect learning in Morris water maze and inhibitory avoidance tasks, respectively. There are some possible explanations, describing the effect of NO synthase inhibitors on learning and memory processes, as will be discussed in the following paragraphs. To date, several behavioral studies have been carried out to explore the effects of NO in different learning tasks, and there is a general consensus about the substantial involvement of NO in the performance of the tasks [63]. In addition to the large amount of behavioral studies which investigated and suggested the role of NO in memory processes, several electrophysiological and neurochemical researches have also been done to further clarify the mechanisms underlying NO action. Soon after the discovery of NO synthesis in brain tissue, it has been suggested that NO acts as a retrograde messenger which influences synaptic transmission in the presynaptic cell and promotes synaptic plasticity [2,15,25,36,45,63,67]. LTP is a form of activitydependent synaptic plasticity that is widely believed to participate in learning and memory processes [10]. Evidence supporting a role for the mechanisms of LTP in learning is based, in part, on comparable effects of drugs on LTP and on learning [13,48]. Moreover, it has been proposed that STM and LTM processes are mediated by mechanisms of synaptic plasticity referred to as short-term (STP) and long-term potentiation [19]. On the other hand, there is considerable evidence suggesting the involvement of NO/GC pathway in hippocampal long-term potentiation [1,16,18,39,40,60,65,91,92]. In consistent, previous studies showed a complete block of long-term potentiation in the hippocampus after application of NOS inhibitors [14,15,28,67,80]. However, despite the evidence in favor of a role for NO in LTP, blockade of LTP by NOS inhibitors is controversial. For example, there are other studies which do not confirm the role of NO in hippocampal synaptic plasticity, since they have found only partial blockade of hippocampal LTP [21,37] while yet others did not find any effect using NOS inhibitors [5,17,24,66]. Discrepant results, however, may be due to the stimulation technique used so as to elicit LTP [69]. Since inhibitory avoidance learning is highly dependent on hippocampal function [49,77], it is suggested that the most related and known mechanism, underlying short- and long-term memory, may be a blockade of the hippocampal STP or LTP and consequently reduced hippocampal synaptic plasticity during the learning or memory retrieval of the task. Although, it may be the possible mechanism contributing to the results of present study, the question of whether NO synthase inhibition is producing a learning impairment through an effect on LTP, is still the subject of challenge. Previous evidences revealed that some of the intracellular second messengers have potential importance to mediating the effects of NO on substituted cell. It is thought that under physiological conditions NO exerts its role mainly via activation of soluble guanilate cyclase (sgc) and cyclic guanosine monophosphate (cgmp) production [26,33,35]. More importantly, a number of behavioral studies have jointly implicated NO and GC in memory processing. For example, Kemenes et al. [54] suggested that NO and GC were both involved in long-term memory for an appetitive single-trial associative conditioning task [54]. NO and GC have also been implicated in avoidance tasks by Bernabeu et al. [7 9] and Izquierdo et al. [52] who found that both the NOS inhibitor nitro-arginine (NO-Arg) and GC inhibitor (LY83583) effectively impaired retention for a singletrial step-down inhibitory avoidance task [7 9,52]. Another intracellular protein which is both activated by NO and involved in memory processing, is mono (ADP-ribosyl) transferase [30]. Evidence suggests that ADP-ribosylation may be an important alternative mechanism through which NO acts during hippocampal LTP [81]. This idea is substantiated further by finding that the CA1 region of hippocampus possesses an ADP-ribosytransferase activity that can be enhanced by NO and attenuated by inhibitors of mono ADP-ribosylation [81]. Edwards and Rickard [30] have shown that, inhibition of ADPribosyltransferase, by medione bisulphate and novobiocin caused an memory impairment from 120 min post-training [30], while in the same task, inhibition of NOS resulted in a memory impairment from 40 min post-training [42,43,76]. This may indicate that NO play a later function in memory formation which involves mono (ADP-ribosyl) tranferase activation.

6 H.E. Harooni et al. / Behavioural Brain Research 201 (2009) In addition to the cellular processes which can be directly affected by NO to influence memory, studies suggest an indirect effect of NO on memory processes, through modulating neurotransmitters and neuropeptides which have been involved in synaptic plasticity and learning. Previous findings indicated that NO modulates transmission mediated by glutamate, GABA, acetylcholine (Ach), noradrenaline, dopamine and serotonin [62,69]. Almost all of these transmitters are abundant in most parts of the brain and represent putative role in the cognitive processes. Acetylcholine, One of the promising transmitters to influence learning and memory, is another candidate to mediate the effects of NO in several brain regions, including cerebral cortex and hippocampus. Kopf and Baratti [58], demonstrated that the impairment of passive avoidance task retention induced in mice by NO synthesis inhibition can be reversed by physostigmine and oxotremorine, a finding that suggests the involvement of a cholinergic mechanism [58]. This hypothesis is further supported by Kopf et al. [59], reported that inhibition of NO synthesis, caused both an impairment of memory retention in an inhibitory avoidance task, and a decrease in cortical Ach release, which are presumably related [59]. In vivo and in vitro studies with NO donors and NOS inhibitors suggest that, in various brain areas and the spinal cord, NO enhances glutamate outflow [61,82]. Moreover, NO-induced Ach release from nucleus accumbens is abolished by glutamate receptor antagonists. Therefore, glutamate seems to play a role for the NO evoked Ach release [70]. Since there are important cholinergic innervations and because of the importance of glutamate transmission in the hippocampus, reduced glutamate and ACh release, may be responsible for (at least some parts of) memory impairments, caused by intraca1 injections of an NOS inhibitor, l-name. Together, these evidences indicated that NO serve s as a modulator to facilitate synaptic transmission, that may be similarly occur during processes known as learning and memory. Thus, it could be reasonable to speculate that, inhibition of NO synthase in hippocampus, a brain region which frequently implicated in cognitive processes, would result in impairment of performance in the task mainly depend on integrity and functionality of the hippocampus. However, since opposing findings also exist, some of the NO-mediated effects should be interpreted cautiously. Together with previous studies, our findings suggest that short- and longterm memory for inhibitory avoidance task are possibly established through partially independent molecular pathways in the hippocampus [19,47,50 52,64,74,85]. In conclusion, our experiments demonstrated that locally inhibition of NO synthase by l-name caused memory impairments in both acquisition and retrieval processes on inhibitory avoidance task in male rats. This finding also suggest that, NO is involved in learning and performance on both short- and long-term memory retention of hippocampally dependent task. Since l-name is not a specific inhibitor of NOS, it could be generally accepted that NOS activity was inhibited in the CA1 region, though non-specific effects cannot be excluded. 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