Effects of the Nitric Oxide Synthase Inhibitor L-NAME on Recognition and Spatial Memory Deficits Produced by Different NMDA Receptor Antagonists in the Rat

1 ,1 Antonios Boultadakis and Nikolaos Pitsikas*

1 Department of Pharmacology, School of Medicine, University of Thessaly, Larissa, Greece

There is consistent experimental evidence that noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor, such as

ketamine, MK-801, and phencyclidine (PCP), impair cognition and produce psychotomimetic effects in rodents. Nitric oxide (NO) is

considered as an intracellular messenger in the brain. The implication of NO in learning and memory is well documented. This study was

designed to investigate the ability of the NO synthase inhibitor L-NAME to antagonize recognition and spatial memory deficits produced

by the NMDA receptor antagonists, MK-801 and ketamine, in the rat. L-NAME (1–3 mg/kg) counteracted MK-801- (0.1 mg/kg) and

ketamine (3 mg/kg)-induced performance impairments in the novel object recognition task. L-NAME (10 mg/kg) attenuated ketamine

(15 mg/kg)-induced spatial working memory and retention deficits in the radial water maze paradigm. L-NAME, applied at 3 mg/kg,

however, disrupted rodents’ performance in this spatial memory task. The present findings indicate (1) that L-NAME is sensitive to

glutamate hypofunction produced by other than PCP NMDA antagonists such as MK-801 and ketamine and (2) that L-NAME alone

differentially affects rodents’ spatial memory.

Neuropsychopharmacology (2010) 35, 2357–2366; doi:10.1038/npp.2010.109; published online 21 July 2010

Keywords: L-NAME; MK-801; ketamine; recognition memory; spatial memory; rat

INTRODUCTION de Lima et al, 2005). Owing to these psychotomimetic properties, NMDA receptor antagonists are widely used as Schizophrenia is a serious mental disorder that affects up to models of schizophrenia in animals (Geyer and Markou, 1994). 1% of the population worldwide. Cognitive deficits in Nitric oxide (NO), a soluble, short-lived, and freely schizophrenic patients are core features of the illness, and diffusible gas, is an important intracellular messenger in the predict vocational and social disabilities for patients brain (Garthwaite, 1991). Reportedly, NO is involved in the (Freedman, 2003). Numerous studies have indicated that mechanisms of synaptic plasticity in the hippocampus the function of the glutamatergic system, in particular (O’Dell et al, 1991; Haley et al, 1992) and is important in N-methyl-D-aspartate (NMDA) receptors, might be com- cognition (Prast and Philippu, 2001). promised in schizophrenia. Exposure to noncompetitive A plethora of experimental data indicates the implication NMDA receptor antagonists like phencyclidine (PCP) or of NO in schizophrenia (Bernstein et al, 2005). In this ketamine produces behavioral symptoms that resemble context, an abnormal distribution of nitrergic neurons in both positive and negative symptoms of schizophrenia in the frontal and temporal lobes of schizophrenic patients has healthy individuals (Javitt and Zukin, 1991; Krystal et al, been observed, which may reflect that the normal pattern of 1994) and exacerbates symptoms in schizophrenic patients neuronal migration during development of the cerebral (Malhotra et al, 1997; Lahti et al, 2001). In addition, cortex may be affected in these patients (Akbarian et al, ketamine, PCP, or MK-801 induce schizophrenia- 1993). In addition, experimental evidence suggests that like symptoms, including cognitive deficits, in rodents polymorphisms in the neuronal nitric oxide synthase (Tricklebank et al, 1989; Verma and Moghaddam, 1996; (nNOS) gene are associated with schizophrenia and prefrontal cortex (PFC) function in schizophrenic patients *Correspondence: Dr N Pitsikas, Department of Pharmacology, (Reif et al, 2006). Contradictory results were reported School of Medicine, University of Thessaly, Biopolis, 411-10 Larissa, however, concerning the levels of NO metabolites in the Greece, Tel: + 30 2410 685535, Fax: + 30 2410 685552, serum of schizophrenic patients. Either high (Taneli et al, E-mail: [email protected] 2004; Yilmaz et al, 2007) or low (Ramirez et al, 2004) Received 7 May 2010; revised 25 June 2010; accepted 25 June 2010 concentrations of NO metabolites have been reported. Effects of L-NAME on NMDA blockade A Boultadakis and N Pitsikas 2358 Because both alterations in the synaptic organization of December 1987; NIH Guide for Care and Use of Laboratory the brain (Roberts et al, 2005) and neurotransmitter deficits Animals, NIH publication no. 85-23, 1985). (Costa et al, 2004) are key features of schizophrenia, excess NO might contribute to disturbed neurocircuitry in this Novel Object Recognition Test disease. Reportedly, exceeding NO concentrations are associated with neuronal damage (Dawson et al, 1991), The test apparatus and procedure have been extensively mitochondrial dysfunction (Das et al, 1998), and impair- described earlier (Boultadakis et al, 2010). Before testing, ment of the NMDA-receptor-mediated neurotransmission for 3 consecutive days, rats were allowed to explore the (Clinton et al, 2003). As a whole, these data suggest that apparatus for 2 min. Testing consisted of a session of two reduced NOS activity in schizophrenia may be neuropro- 2-min trials. During the ‘sample’ trial (T1), two identical tective. Therefore, efforts are made to develop NOS samples (objects) were placed in two opposite corners of the inhibitors as possible therapeutic tools for schizophrenia apparatus in a random manner. A rat was placed in the (Bernstein et al, 2005). middle of the apparatus and was left to explore these two A number of animal studies have shown that diverse NOS identical objects. After T1, the rat was put back in its home inhibitors antagonized PCP-induced psychotomimetic effects cage and an intertrial interval (ITI) was given. Subsequently, (Johansson et al, 1997; Klamer et al, 2001, 2004, 2005a, b). In the ‘choice’ trial (T2) was performed. During T2, a new this context, it has been proposed that NOS inhibition object (N) replaced one of the samples presented in T1, selectively amends the behavioral effects of PCP and not those therefore, the rats were reexposed to two objects: the of MK-801 (Klamer et al, 2005a). In addition, that application familiar (F) and the new (N). of the nonselective NOS inhibitor L-NAME counteracted PCP- The times spent by rats in exploring each object during induced acquisition deficits in a spatial learning task in the rat T1 and T2 were recorded manually by using a stopwatch. has also been reported (Wass et al, 2006a, b). From this measure a series of variables was then calculated: Under our experimental conditions, we have observed the total time spent in exploring the two identical objects in that L-NAME (1–3 mg/kg) antagonized delay-dependent T1, and that spent in exploring the two objects, (F) and (N) deficits in the novel object recognition memory task in in T2. The discrimination between (F) and (N) during T2 the rat (Boultadakis et al, 2010). At the moment, it is not was measured by comparing the time spent in exploring clear if and how L-NAME is able to counteract recognition the (F) with that spent in exploring the (N). As this time memory deficits produced by NMDA hypofunction. In this may be biased by differences in overall levels of exploration context, we have observed that two NMDA receptor a discrimination index (D) was then calculated; D ¼ NÀ blockers with different profile in terms of potency or F/N + F. D is the discrimination ratio and represents pharmacokinetic properties, MK-801 and ketamine, dis- the difference in exploration time expressed as a proportion rupted animals’ performance in the novel object recognition of the total time spent exploring the two objects in T2 paradigm (Pitsikas et al, 2006, 2008). (Cavoy and Delacour, 1993). In addition, motor activity of Taken the above evidences into account, the first aim of each animal expressed as total number of steps during each our study was to evaluate the efficacy of L-NAME in trial was also recorded. antagonizing MK-801- and ketamine-induced performance deficits in a recognition memory task in the rat. For this Radial Water Maze Test purpose, the novel object recognition task was selected. This test is a nonrewarded paradigm based on the spontaneous Spatial reference and spatial working memories were exploratory behavior of rats (Ennaceur, 2010). Subse- studied using an eight-arm radial water maze procedure quently, the efficiency of L-NAME to counteract ketamine- modified from Jarrard et al (1984) and Pitsikas and Algeri induced detrimental effects on spatial memory was assessed (1992). The test apparatus has been described before by using a radial water maze task (Pitsikas et al, 2007). (Pitsikas et al, 2007). Each trial involved placing the rat in the water, facing the wall, at the end of one of the seven unbaited arms (start MATERIALS AND METHODS positions). Each rat was allowed 90 s to escape onto the Animals platform. If it failed to escape within this time, it was guided to the platform. When the rat found the escape platform it Different groups of male (3-month-old) Wistar rats was allowed to stay there for 30 s. Then the rat was placed (Hellenic Pasteur Institute, Athens, Greece), weighing 250– again into the maze for its next trial, which used the same 300 g, were used in this study. The animals were housed in platform location (eg, channel 1) and a different starting Makrolon cages (47.5 Â 20.5 Â 27 cm), three per cage, in a position as the previous trial. This was repeated until the rat regulated environment (21±11C, 50–55% relative humidity, found the platform or 90 s had elapsed whichever occurred 12 : 12 h light/dark cycle, white lights on from 0700 to 1900 first. Each animal received a daily session of two trials for 4 hours), with free access to food and water. Experiments consecutive days. were conducted in the room where only these animals Choices of arms (defined by whole body entry) were where housed, and took place during the light cycle between recorded manually for each trial. The order of entry into the 1000 and 1400 hours. maze arms was recorded so that the total number of errors Procedures involving animals and their care were could be calculated. Errors were quantified in terms of conducted in conformity with the international guidelines reference and working memory incorrect errors (Jarrard in compliance with national and international laws and et al, 1984). Reference memory errors were the number of policies (EEC Council Directive 86/609, JL 358, 1, 12 first entries into a channel that had never contained a

Neuropsychopharmacology Effects of L-NAME on NMDA blockade A Boultadakis and N Pitsikas 2359 platform. Working memory incorrect errors were the recognition task. Rats were randomly divided into eight number of repeated entries into an arm that had never experimental groups (10 rats per group) as follows: vehicle contained a platform (repeated entries into a reference + vehicle; vehicle + L-NAME 1 mg/kg; vehicle + L-NAME memory arm thus, reflecting the combined effects of failures 3 mg/kg; vehicle + L-NAME 10 mg/kg; ketamine 3 mg/kg + of both working and reference memory) (Okaichi et al, vehicle; ketamine 3 mg/kg + L-NAME 1 mg/kg; ketamine 1989). These parameters were averaged for each daily block 3 mg/kg + L-NAME 3 mg/kg; and ketamine 3 mg/kg + of trials and for each rat, whose daily performance was thus L-NAME 10 mg/kg. To study the effects of compounds on characterized. post-training memory components (storage and/or retrie- On day 5, the rats performed a single spatial probe trial val), we administered the compounds just after T1. For this (Gage et al, 1984). This trial consisted of removing the study, the 1-h ITI was selected as at this delay condition platform from the baited arm of the radial water maze and recognition memory is still intact in the vehicle-treated rat allowing the rat to swim for 60 s in search of it. The time (Bartolini et al, 1996) and impairments associated with spent in each of the eight channels of the pool was treatment with ketamine (hypermotility, stereotypies, calculated as a percentage over 60 s. ataxia) (Verma and Moghaddam, 1996) were not observed (Pitsikas et al, 2008). Drugs Radial water maze task. Preliminary results have shown L -NAME, ( + )-MK-801 maleate (Sigma, St Louis, MO, USA) that rats treated with L-NAME 3 mg/kg showed a lower and ketamine hydrochloride were dissolved in saline (NaCl performance regarding the vehicle-treated animals. There- 0.9%) and administered intraperitoneally (i.p.) in a volume fore, the effects exerted by 3 mg/kg L-NAME on rats’ spatial L of 1 ml/kg. Doses of -NAME were chosen on the basis of a memory and the ability of L-NAME to antagonize ketamine- study in which they were effective against learning induced performance deficits in the radial water maze were impairments and did not produce adverse side effects subsequently assessed in two separate studies. (Boultadakis et al, 2010). The doses of MK-801 (0.1 mg/kg) and ketamine (3 mg/kg) were selected based on previous findings in which they were found to impair rat perfor- Experiment 3: effects of L-NAME (3 mg/kg) on animals’ mance in the novel object recognition task without performance in the radial water maze task. Rats were producing side effects (Pitsikas et al, 2006, 2008). For the randomly divided into two experimental groups (10 rats per radial water maze test, the dose of ketamine (15 mg/kg) was group) as follows: vehicle and L-NAME 3 mg/kg. L-NAME and chosen based on a previous report in which it was found to vehicle were injected every day 60 min before starting testing. impair place learning in a water maze task (Wesierska et al, 1990). For all studies, control animals received isovolu- Experiment 4: effects of L-NAME in antagonizing metric amounts of the vehicle (NaCl 0.9%). ketamine-induced performance deficits in the radial water maze task. Animals were randomly divided into six experimental groups (10 rats per group) as follows: vehicle Experimental Protocol + vehicle; vehicle + L-NAME 1 mg/kg; vehicle + L-NAME Experiment 1: effects of L-NAME in antagonizing 10 mg/kg; ketamine 15 mg/kg + vehicle; ketamine 15 mg/kg MK-801-induced performance deficits in the novel object + L-NAME 1 mg/kg; and ketamine 15 mg/kg + L-NAME recognition task. Rats were randomly divided into eight 10 mg/kg. Control rats were treated with the vehicle 60 experimental groups (10 rats per group) as follows: vehicle + and 40 min before starting the daily testing. Animals were vehicle; vehicle + L-NAME 1 mg/kg; vehicle + L-NAME 3 mg/ treated with L-NAME and vehicle 60 and 40 min, respec- kg; vehicle + L-NAME 10 mg/kg; MK-801 0.1 mg/kg + vehicle; tively, before starting testing. Rats received vehicle and MK-801 0.1 mg/kg + L-NAME 1 mg/kg; MK-801 0.1 mg/kg + ketamine 60 and 40 min, respectively, before starting the L-NAME 3 mg/kg; and MK-801 0.1 mg/kg + L-NAME 10 mg/ daily testing. Rats received L-NAME and ketamine 60 and kg. To study the effects of compounds on post-training 40 min, respectively, before starting the daily session. memory components (storage and/or retrieval), we adminis- Ketamine administration (10–30 mg/kg) is associated with tered compounds immediately after T1. It has been reported behavioral symptoms, such as hypermotilty and stereotypy, that treatment with MK-801 (0.1 mg/kg) induces hypermo- that reach the peak effect 15 min after treatment (Wesierska tility (Verma and Moghaddam, 1996; Homayoun et al,2004) et al, 1990; Verma and Moghaddam, 1996). On the basis of and produce a sustained increase in stereotypy that peaked these evidences, we performed the daily administration of 40 min after injection and returned to baseline 2 h later ketamine 40 min before testing as at this condition ketamine (Homayoun et al, 2004). Therefore, it is quite possible that does not induce side effects. these factors may interfere with learning performance. There is also experimental evidence that recognition memory abilities in the young rat remained intact when a delay Statistical Analysis condition up to 6 h was used (Bartolini et al, 1996). To avoid Data are expressed as mean±SEM. In experiments 1 and 2, the possibility that rats’ performance is influenced by these results were analyzed using the two-way analysis of variance already mentioned side effects due to the treatment with (ANOVA). Post hoc comparisons were performed by the MK-801, we selected the 3-h ITI. Tukey’s t-test. In experiment 3, reference and working memory incorrect errors were evaluated by the two-way Experiment 2: effects of L-NAME in antagonizing ANOVA with a split-plot design (between–within subjects). ketamine-induced performance deficits in the novel object Post hoc comparisons were performed by the Tukey’s t-test.

Neuropsychopharmacology Effects of L-NAME on NMDA blockade A Boultadakis and N Pitsikas 2360 Spatial probe trial data are calculated as median and suggesting that animals treated with ketamine showed higher interquartile ranges. When comparing among groups the locomotor activity levels than the other experimental groups percentage of time spent in the previously reinforced arm, (Table 2). Total exploration levels were not different among Wilcoxon’s nonparametric test was used. the various experimental groups (Table 2). In experiment 4, the effects of drugs on daily performance Concerning D data, a significant main effect of ketamine (reference and working memory) were analyzed by the (F(1, 72) ¼ 15.8, po0.01), of L-NAME (F(3, 72) ¼ 3.4, three-way ANOVA (two between, one within subjects). po0.05) and a significant interaction between ketamine Significant interactions between ketamine, L-NAME, and and L-NAME (F(3, 72) ¼ 3, po0.05) was evidenced. Post hoc trials were further analyzed by comparing ketamine- and analysis has shown that animals treated with the vehicle + L-NAME-treated and -nontreated rats at each level sepa- ketamine and the ketamine + L-NAME 10 mg/kg were unable rately for the 4-day periods, using the two-way ANOVA with to discriminate between the familiar and the novel object as a split-plot design. Post hoc comparisons were performed compared with the other groups (po0.05, Figure 2). by the Tukey’s t-test. Spatial probe data were analyzed by Kruskall–Wallis nonparametric test followed by the Experiment 3: Effects of L-NAME (3 mg/kg) on Animals’ Newman–Keuls post hoc test. A p-value o0.05 was Performance in the Radial Water Maze Task considered significant (Kirk, 1968). Reference memory. Data are presented in Figure 3a. An overall ANOVA showed that both the vehicle- and the RESULTS L-NAME-treated animals made progressively fewer reference memory errors over days (F(3, 54) ¼ 5.47, po0.01). In Experiment 1: Effects of L-NAME in Antagonizing MK-801-Induced Performance Deficits in the Novel addition, the L-NAME 3 mg/kg-treated animals committed Object Recognition Task significantly more reference memory errors than their vehicle-treated counterparts (F(1, 18) ¼ 8.48, po0.01). A two-way ANOVA of motor activity results showed a main effect either of MK-801 (F(1, 72) ¼ 18.4, po0.01) or of Working memory. Results are illustrated in Figure 3b. L-NAME (F(3, 72) ¼ 2.93, po0.05) indicating that rats that Statistical analysis of the working memory data evidenced received both these compounds showed higher motility levels as compared to the other groups (Table 1). Total

exploration levels were not different among the various Vehicle+vehicle 0.6 Vehicle+L-NAME (1 mg/kg) experimental groups (Table 1). Vehicle+L-NAME (3 mg/kg) Data for index D (Figure 1) revealed a significant main Vehicle+L-NAME (10 mg/kg) 0.5 MK-801 (0.1 mg/kg)+vehicle effect of MK-801 (F(1, 72) ¼ 24.2, po0.01), of L-NAME MK-801+L-NAME (1 mg/kg) MK-801+L-NAME (3 mg/kg) (F(3, 72) ¼ 4.8, po0.01) and a significant MK-801 Â L- 0.4 MK-801+L-NAME (10 mg/kg) NAME interaction (F(3, 72) ¼ 3.8, p ¼ 0.014). Post hoc comparisons have shown that rats treated with the vehicle 0.3 + MK-801 and the MK-801 + L-NAME 10 mg/kg were unable 0.2 + to discriminate between the familiar and the novel object as * 0.1 compared to the rest of groups (po0.05). Discrimination index D 0.0

Experiment 2: Effects of L-NAME in Antagonizing Figure 1 Novel object recognition task. Vehicle, MK-801, and L-NAME Ketamine-Induced Performance Deficits in the Novel were injected i.p., just after T1. The 3-h ITI was used. Results are expressed Object Recognition Task as mean±SEM. Discrimination index D performance expressed by different groups of rats during T2. *po0.05 vs all the other groups + A two-way ANOVA of motility results showed only a (except the MK-801 + L-NAME 10 mg/kg group); po0.05 vs all the other significant main effect of ketamine (F(1, 72) ¼ 29.8, po0.01), groups (except the MK-801 + vehicle group).

Table 1 Effects of MK-801 and L-NAME on Rats’ Performance in the Novel Object Recognition Task

Group N T2 motor activity (number of steps) T2 exploration time (s) mean±SEM mean±SEM

Vehicle+vehicle 10 47.5±1.8 12.5±0.3

Vehicle+L-NAME (1 mg/kg) 10 41.8±5.2 12.2±0.4

Vehicle+L-NAME (3 mg/kg) 10 35.5±2.1 11.6±1.3

Vehicle+L-NAME (10 mg/kg) 10 44.4±3.9 12.5±0.6 MK-801 (0.1 mg/kg)+vehicle 10 58.6±3.5 11.9±0.5

MK-801+L-NAME (1 mg/kg) 10 55.4±3.7 14.2±0.8

MK-801+L-NAME (3 mg/kg) 10 47.2±5.1 12.4±0.9

MK-801+L-NAME (10 mg/kg) 10 54.1±3.6 12.4±0.4

Abbreviation: N, number of rats. Compounds were injected intraperitoneally immediately after T1.

Neuropsychopharmacology Effects of L-NAME on NMDA blockade A Boultadakis and N Pitsikas 2361 Table 2 Effects of Ketamine and L-NAME on Rats’ Performance in the Novel Object Recognition Task

Group N T2 motor activity (number of steps) T2 exploration time (s) mean±SEM mean±SEM

Vehicle+vehicle 10 45.4±2.6 12.7±0.7

Vehicle+L-NAME (1 mg/kg) 10 46.9±4.5 14.2±1

Vehicle+L-NAME (3 mg/kg) 10 46.4±5.2 13.6±0.7

Vehicle+L-NAME (10 mg/kg) 10 45.1±4.9 14.1±1.3 Ketamine (3 mg/kg)+vehicle 10 67.6±1.7 14.5±0.8

Ketamine+L-NAME (1 mg/kg) 10 57.8±3.3 13.3±0.5

Ketamine+L-NAME (3 mg/kg) 10 58.7±3.3 12.7±0.8

Ketamine+L-NAME (10 mg/kg) 10 59.1±4.3 13.2±0.9

Abbreviation: N, number of rats. Compounds were injected intraperitoneally immediately after T1.

Vehicle+vehicle Additional analyses of rats’ performance in this spatial Vehicle+L-NAME (1 mg/kg) Vehicle+L-NAME (3 mg/kg) reference memory task were carried out by split- 0.6 Vehicle+L-NAME (10 mg/kg) plot ANOVAs. The ketamine + vehicle-treated animals Ketamine (3 mg/kg)+vehicle Ketamine+L-NAME (1 mg/kg) committed significantly more reference memory errors 0.5 Ketamine+L-NAME (3 mg/kg) Ketamine+L-NAME (10 mg/kg) than their vehicle + vehicle-treated cohorts (F(1, 18) ¼ 40.6, 0.4 po0.01). L-NAME, at any dose, failed to counteract this ketamine-induced reference memory impairment. 0.3 Animals treated with 1 mg/kg L-NAME + ketamine made L 0.2 + more reference memory errors than the vehicle + -NAME * 1 mg/kg-treated rats (F(1, 18) ¼ 7.3, p ¼ 0.014). Choice accu-

Discrimination index D 0.1 racy abilities, in terms of number of reference memory errors, of the 10 mg/kg L-NAME + ketamine-treated rats and 0.0 those animals that received vehicle + L-NAME 10 mg/kg were Figure 2 Novel object recognition task. Vehicle, ketamine, and L-NAME not different. were injected i.p., just after T1. The 1-h ITI was used. Results are expressed as mean±SEM. Discrimination index D performance expressed by Working memory. Results are presented in Figure 4b. different groups of rats during T2. *po0.05 vs all the other groups + Overall ANOVA did not show a significant three-way (except the ketamine + L-NAME 10 mg/kg group); po0.05 vs all the other groups (except the ketamine + vehicle group). ketamine Â L-NAME Â days interaction, neither a significant two-way interaction between ketamine and days and between L-NAME and days. Interestingly, a significant two- that either the vehicle- or the L-NAME-treated animals way interaction between ketamine and L-NAME was made progressively fewer working memory incorrect errors evidenced (F(2, 216) ¼ 3.64, po0.05) indicating that the over days (F(3, 54) ¼ 9.87, po0.01). Moreover, the L-NAME rates of learning were different among the various groups. 3 mg/kg-treated rats made a significantly higher number of In addition, a significant main effect of ketamine working memory incorrect errors than their vehicle-treated (F(1, 216) ¼ 28.1, po0.01), of days (F(3, 216) ¼ 8, po0.01) cohorts (F(1, 18) ¼ 4.4, p ¼ 0.05). but not of L-NAME was evidenced. Further analyses of performance of the animal in this Spatial probe trial. Probe analysis comparing percentage of spatial working memory paradigm were carried out by split- time spent in the previously reinforced quadrant showed an plot ANOVAs. Animals that received ketamine + vehicle effect of treatment. The 3 mg/kg L-NAME group spent committed significantly more working memory incorrect significantly less time in the previously baited channel than errors than their vehicle + vehicle counterparts (F(1, 18) ¼ the control group (po0.05, Figure 3c). 17.9, po0.01). Rats that received 10 mg/kg L-NAME + ketamine made significantly fewer working memory incorrect errors than their counterparts treated with ketamine + vehicle Experiment 4: Effects of L-NAME in Antagonizing (F(1, 18) ¼ 8.35, po0.01)AQ. In addition, these rats perfor- Ketamine-Induced Performance Deficits in the Radial mance, in terms of working memory incorrect errors, was not Water Maze Task different than that showed by their vehicle cohorts (10 mg/kg Reference memory. Data are presented in Figure 4a. Overall L-NAME + vehicle-treated rats). Animals treated with 1 mg/kg ANOVA did not show a significant three-way ketamine Â L-NAME + ketamine made a higher number of working L-NAME Â days interaction, neither a significant two-way memory incorrect errors than the vehicle + L-NAME 1 mg/kg- interaction between ketamine and L-NAME, between treated rats (F(1, 18) ¼ 13.8, po0.01). ketamine and days, and between L-NAME and days. A significant main effect of ketamine (F(1, 216) ¼ 25.1, Spatial probe trial. Probe analysis comparing percentage of po0.01), of days (F(3, 216) ¼ 11.1, po0.01) but not of time spent in the previously reinforced quadrant showed an L-NAME however was revealed. effect of treatment (H ¼ 25.8, po0.01, Figure 4c). Animals

Neuropsychopharmacology Effects of L-NAME on NMDA blockade A Boultadakis and N Pitsikas 2362 8 Vehicle attenuated this ketamine-induced impairment (po0.05 vs L-NAME (3 mg/kg) ketamine + vehicle-treated animals). 7

6 DISCUSSION 5 The major findings of this study have shown that the NMDA 4 receptor antagonists MK-801 and ketamine induced performance deficits in different cognitive tasks in the rat. 3 These cognitive impairments could be antagonized by the NOS inhibitor L-NAME. 2

1 Novel Object Recognition Task Number of reference memory errors 0 Our study results are in line with previous studies in which 0 1 2 3 45post-training administration of both MK-801 and ketamine Days disrupted rats’ performance in the novel object recognition task (Pitsikas et al, 2006, 2008). A single post-training 7 Vehicle injection of L-NAME (1 and 3, but not 10 mg/kg) attenuated L-NAME (3 mg/kg) 6 either the MK-801- or the ketamine-induced performance deficits in this recognition memory paradigm. MK-801, 5 ketamine and L-NAME influenced rats’ performance during retention, seemingly reflecting a modulation of post- 4 training mnemonic processes (storage and/or retrieval of information). 3 Compounds were administered systemically, thus, it cannot be excluded that nonspecific factors (attentional, 2 sensorimotor deficits) might have influenced animals’ performance. All animals that received these two NMDA 1 receptor antagonists showed higher levels of motility, but not of general exploration during T2, regarding their 0 Number of working memory incorrect errors 012345 control cohorts. Treatment with L-NAME did not antag- Days onize this NMDA blockade-induced hyperactivity but successfully counteracted recognition memory deficits. This 60 pattern of results implies that the effects of the compounds Vehicle on rats’ cognitive performance were unrelated to the extent L-NAME (3 mg/kg) 50 of motility and exploratory behavior. Present results are in agreement with our previous findings showing that L-NAME did not impair recognition 40 memory but it reversed delay-dependent impairments in the novel object recognition task, especially at lower doses 30 (Boultadakis et al, 2010).

(%) of time * 20 Radial Water Maze Task

Data have shown that L-NAME (1 and 10 mg/kg) alone did 10 not disrupt animals’ spatial learning abilities. A per se effect of L-NAME was observed when this NOS inhibitor was 0 delivered at the dose of 3 mg/kg, however. Those animals’ Baited arm spatial learning abilities were poorer as compared to those Figure 3 Radial water maze task. Vehicle and L-NAME were injected i.p., shown by the vehicle + vehicle-treated subjects. Rats that every day 60 min before testing. (a) Spatial reference memory. Results are received ketamine performed less efficiently than the expressed as mean±SEM. Number of errors cumulating over days by vehicle + vehicle and the L-NAME (1 and 10 mg/kg) + different groups of rats. (b) Spatial working memory. Results are expressed as mean±SEM. Number of errors cumulating over days by different groups vehicle-treated rats suggesting that this NMDA receptor of rats. (c) Percentage of total time spent in the previously reinforced arm antagonist disrupted both spatial reference and spatial of the radial water maze by different groups of rats. Results are expressed working memory. The higher dose of L-NAME (10 mg/kg) as medians. *po0.05 vs the vehicle-treated animals. attenuated ketamine-induced working, but not reference, memory deficits. Both ketamine + L-NAME groups expressed a lower performance as compared to the respective treated with ketamine + vehicle and ketamine + L-NAME cohorts treated with L-NAME + vehicle. 1 mg/kg spent less time in the previously baited channel as When the ability of rats to use the extramaze cues compared to the respective control populations (Newman– surrounding the radial water maze was evaluated (spatial Keuls post hoc test, po0.05). L-NAME, at 10 mg/kg, probe trial) all control rats persistently swam more in the

Neuropsychopharmacology Effects of L-NAME on NMDA blockade A Boultadakis and N Pitsikas 2363 previously reinforced arm of the maze, whereas L-NAME L-NAME (10 mg/kg) attenuated this ketamine-produced 3 mg/kg + vehicle, ketamine + vehicle, and ketamine + disrupting effect. L-NAME 1 mg/kg-treated animals were unable to do so. The radial water maze test evaluates rodents’ spatial memory. The procedure used in this study offers the possibility to assess in the same animal simultaneously two aspects of memory (reference and working; Jarrard et al, Vehicle+vehicle 1984; Pitsikas and Algeri, 1992). This within-subject and Vehicle+L-NAME (1 mg/kg) Vehicle+L-NAME (10 mg/kg) within-task design is advantageous because each dissocia- 9 Ketamine (15 mg/kg)+vehicle tion is obtained from the same animal at the same time, Ketamine+L-NAME (1 mg/kg) minimizing many sources of variability such as task Ketamine+L-NAME (10 mg/kg) 8 magnitude of reinforcement, motivational, perceptual, and 7 motor processes (Olton, 1983). 6 Our results of the radial water maze paradigm are consistent with previous reports in which the effects of 5 L-NAME (10 mg/kg) on glutamatergic hypofunction were 4 assessed (Wass et al, 2006b, a). The outcome of these studies 3 was that treatment with this NOS inhibitor antagonized PCP-induced spatial reference, spatial working memory, but 2 not retention deficits in a modified Morris water maze 1 procedure (Wass et al, 2006b, a).

Number of reference memory errors 0 A potential issue when using NOS inhibitors relates to its 012345 hypertensive properties. It is difficult, therefore, to quantify Days how and at what extent these cardiovascular effects might have specifically affected animals’ cognitive performance.

Vehicle+vehicle Reportedly, NOS inhibitors injected peripherally induce a Vehicle+L-NAME (1 mg/kg) nearly maximal hypertensive effect at 10 mg/kg (Rees et al, Vehicle+L-NAME (10 mg/kg) 1990). Under our experimental conditions, L-NAME, Ketamine (15 mg/kg)+vehicle Ketamine+L-NAME (1 mg/kg) administered systemically at 10 mg/kg, attenuated ketamine- 8 Ketamine+L-NAME (10 mg/kg) induced performance deficits in the radial water maze task. This implies that the effects of this compound on rats’ 7 cognitive performance were unrelated to its potential 6 hypertensive action.

4 NMDA Hypofunction and Nitric Oxide

3 Pharmacological blockade of NMDA receptors has been proposed as a relevant model of schizophrenia in humans 2 and animals. (Javitt and Zukin, 1991; Geyer and Markou, 1994; Krystal et al, 1994). The NMDA antagonist treatment 1 model has excellent face validity for cognitive symptoms of 0 schizophrenia because an exposure to these drugs impairs Number of working memory incorrect errors 012345 PFC-dependent cognitive functions in a manner similar to Days schizophrenia (Javitt and Zukin, 1991; Krystal et al, 1994). PCP, ketamine, and MK-801 produce their psychotomimetic Vehicle+vehicle effects by blocking NMDA receptors located on GABA Vehicle+L-NAME (1 mg/kg) interneurons, resulting in decreased firing of GABAergic 60 Vehicle+L-NAME (10 mg/kg) Ketamine (15 mg/kg)+vehicle inhibitory neurons and, thereby, increased excitability in Ketamine+L-NAME (1 mg/kg) limbic circuits (Olney et al, 1991; Moghaddam et al, 1997). 50 Ketamine+L-NAME (10 mg/kg) This disinhibitory action elicits an increase in terms of neuronal activity and excessive glutamate and dopamine 40

30 + Figure 4 Radial water maze task. Vehicle, ketamine, and L-NAME were injected i.p., every day 40 and 60 min, respectively, before testing. (a) Spatial

(%) of time reference memory. Results are expressed as mean±SEM. Number of 20 + # errors cumulating over days by different groups of rats. (b) Spatial working * memory. Results are expressed as mean±SEM. Number of errors 10 cumulating over days by different groups of rats. (c) Percentage of total time spent in the previously reinforced arm of the radial water maze by different groups of rats. Results are expressed as medians. *po0.05 vs the 0 vehicle + vehicle-treated animals; + po0.05 vs the respective control # Baited arm populations; po0.05 vs ketamine + L-NAME 10 mg/kg-treated rats.

Neuropsychopharmacology Effects of L-NAME on NMDA blockade A Boultadakis and N Pitsikas 2364 release in the PFC and limbic regions (Moghaddam et al, It has been reported that a wide range of translational 1997; Lorrain et al, 2003; Razoux et al, 2007). PCP-induced effects including deficits in preattentive NOS inhibitors may exert both excitatory and inhibitory information processing, nonassociative learning, selective effects on NMDA-receptor-associated events. The dual attention, spatial memory can all be prevented by interfer- action of NO as mediator and negative modulator of ing with the production of NO (Johansson et al, 1997; glutamate neurotransmission at the NMDA receptor com- Klamer et al, 2001, 2004, 2005a, b). These findings suggest plex gives this system a unique role in regulating the that the schizophrenia-like behavioral effects of PCP in balance of excitatory and inhibitory influences. It is this rodents are, at least in part, mediated by an increase in NO balance of excitatory and inhibitory processes that is likely activity (Palsson et al, 2009). This is supported by a recent to be disrupted in schizophrenia, as proposed by the study showing an NO-dependent increase in cyclic GMP glutamatergic hypofunction (Dawson et al, 1991). (cGMP) signaling, a main effector of NO in the brain, in the Our findings indicate that L-NAME, for the first time to mouse PFC following PCP administration (Fejgin et al, our knowledge, antagonized both MK-801- and ketamine- 2008). In this context, it has been found that L-NAME induced recognition memory deficits, suggesting that this attenuated sensorimotor gating deficits caused by PCP, by NOS inhibitor is also effective against NMDA blockade reducing PCP-induced increase in cGMP production in the produced by the action of NMDA antagonists other than PFC of the mouse brain (Fejgin et al, 2008). This mechanism PCP. Furthermore, L-NAME was found to attenuate spatial might be a plausible explanation for the beneficial effects of memory deficits produced by ketamine in the rat. The latter, low doses of L-NAME on memory impairments related to support and extend previous results in which spatial glutamatergic hypofunction. Further studies however are memory deficits induced by another NMDA antagonist required to elucidate this important issue. (PCP) were counteracted by this NOS inhibitor (Wass et al, 2006b, a). Collectively, the present results further suggest an involvement of NO in the NMDA hypofunction-induced Concluding Remarks cognitive deficits. In summary, studies presented herein show that L-NAME Contrary to the expectations, when L-NAME was was capable in antagonizing MK-801 and ketamine-induced administered alone at 3 mg/kg, it impaired rats’ spatial but memory deficits and this beneficial action exerted by not recognition memory abilities. It remains uncertain if L-NAME on cognition was observed at a different dose this dual effect of L-NAME on memory was related to the range. This, in turn, indicates that L-NAME is sensitive to task specificity, the type of memory studied, and/or the glutamatergic hypofunction produced by other than PCP different pharmacological design used. NMDA receptor antagonists such as MK-801 and ketamine. The radial water maze is a negatively reinforced paradigm In addition, the present results show that L-NAME alone assessing spatial memory. The novel object recognition test differentially affects rodents’ spatial memory. The latter is a task that does not involve at all the learning of a rule as suggest a careful consideration of the balance between it is based on the spontaneous exploratory behavior of neurodegenerative and neuroprotective effects produced by rodents evaluating recognition memory (Ennaceur, 2010). this NOS inhibitor as L-NAME might constitute a potential In the novel object recognition task rats received a single candidate for the treatment of schizophrenia. injection of compounds just after the ‘sample’ trial T1. Conversely, in the radial water maze paradigm, treatment was applied once per day 60 min before starting the daily ACKNOWLEDGEMENTS testing, for 5 consecutive days. Interestingly, this impairing effect of L-NAME on spatial This study was supported by a grant of the Research memory, was not observed at the ‘side’ doses of 1 and Committee of the University of Thessaly (no. 3689) to NP. 10 mg/kg revealing thus, an U-shaped dose–effect curve. At present, the biological bases of U-shaped dose–response relationship are unknown, although receptor fatigue or DISCLOSURE tachyphylaxis (Day, 1979) has been suggested as potential The authors hereby declare that no financial support or mechanisms (Martinez, 1986). Moreover, L-NAME has also compensation has been received from any individual been reported to function as a partial agonist in the NO or corporate entity over the past 3 years for research or system, antagonizing or stimulating NO synthesis in professional service and there are no personal financial different tissues (Archer and Hampi, 1992). Further studies holdings that could be perceived as constituting a potential are needed however to clarify this important issue. conflict of interest. The mechanism(s) of action of low doses of NOS inhibitors on learning and memory is not yet clarified and is matter of investigation. 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