Fundamental & Clinical Pharmacology

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1 Fundamental & Clinical Pharmacology ORIGINAL ARTICLE Keywords 7-NI, learning and memory, mice, nitric oxide, TRIM Received 8 February ; revised 1 April ; accepted 28 April Correspondence and reprints: oguzmutlu8@hotmail.com doi:.1111/j x Effects of nitric oxide synthase inhibitors 1-(2-trifluoromethylphenyl) imidazole (TRIM) and 7-nitroindazole (7-NI) on learning and memory in mice Oguz Mutlu a,b, Güner Ulak b, Catherine Belzung a a INSERM U-9 ERL CNRS 36, Université François Rabelais Tours; Faculté des Sciences et Techniques, Université François Rabelais Tours, Parc Grandmont, France b Department of Pharmacology, Faculty of Medicine, Kocaeli University, 4138, Kocaeli, Turkey ABSTRACT Nitric oxide (NO) plays an important role in hippocampal long-term potentiation (LTP), which is involved in memory processes. This led to the hypothesis that nitric oxide synthase (NOS) inhibitors will have disturbing effects on learning and memory. The aim of our study was to investigate the effects of the new selective neuronal and inducible NOS inhibitor 1- (2-trifluoromethylphenyl) imidazole (TRIM) ( 5 mg/ kg) on learning and memory and compare it to the nonselective NOS inhibitor 7-NI (15 45 mg/kg) using different behavioral tests in Swiss mice, thus clarifying the role of neuronal NOS (nnos) and endothelial NOS (enos) in cognitive processes. TRIM had no specific effect on either learning or memory parameters, while 7-NI ( mg/ kg) disturbed spatial memory in the probe trial of the Morris water maze test, which was performed on the last day of the test. No differences between TRIM and the control groups were observed, while 7-NI ( and 45 mg/kg) significantly disturbed memory in the novel object recognition test. In the social transmission of food preference test, both TRIM (5 mg/kg) and 7-NI (45 mg/kg) impaired hippocampal olfactory memory, but the total food consumption was also significantly decreased at these doses. In the passive avoidance test, TRIM did not disturb the performance, while memory impairment was observed, even with lower doses of 7-NI. All of these results suggest that TRIM has no clear effect on cognitive impairment compared to 7- NI and that inhibition of both nnos and enos are necessary for the deterioration of memory processes. INTRODUCTION Nitric oxide (NO) is known as a potent vasodilatator, atypical neurotransmitter and important signaling agent inside the cell. NO is synthesized from L-arginine by NO synthase (NOS) in response to Ca 2+ influx induced by the activation of N-methyl-D-aspartate (NMDA) receptors by excitatory amino acids [1,2]. The NOS enzyme has three isoforms: the endothelial (enos), the inducible (inos) and the neuronal (nnos) forms. nnos and enos are activated by binding of the calcium calmoduline complex to NOS, resulting in an increase in Ca 2+ concentration inside the cell [3,4]. Conversely, inos exerts its effect independent of Ca 2+ and is formed under pathological conditions such as ischemia, stroke, sepsis and inflammation. Previous studies showed that both neuronal (nnos) and endothelial (enos) isoforms are expressed in neurons [5,6]. In studies using knockout mice, enos was proposed to be a mediator of several neural processes [6,7]. NO has an important role in several behavioral, cognitive and emotional events such as depression-like behavior, anxiety behavior, 368

2 Effect of TRIM on memory 369 locomotion, aggression, tolerance, addiction and learning [8,9]. For example, it was shown that inhibition of NO-sensitive soluble guanylate cyclase has antidepressant, anxiolytic and antinociceptive effects in rats and mice [ 12]. It is postulated that NO affects the formation of longterm potentiation (LTP) [6,13] in hippocampus and long-term depression (LTD) [14] in cerebellum, which have important roles in learning and memory. LTP formation in the hippocampus constructs a background for synaptic plasticity, and it is widely accepted that LTP is related to learning and memory. Ca 2+ concentrations inside the cell increase following NMDA glutamate receptor activation during LTP formation, as does synaptic activity, which stimulates NO production [15]. Controversial results exist among the studies of the effects of NOS inhibitors on learning and memory. Some of these studies showed that drugs blocking NOS inhibited learning [16 18], while other studies did not support these findings [19,]. In addition, LTP, which is considered to have an important role in NO mediated learning, was blocked completely after hippocampal injection of NOS inhibitors [21,22]; other studies found only a partial inhibition [23,24], while some concluded that no effect was observed [25 27]. 1- (2-trifluoromethylphenyl) imidazole (TRIM) is an inhibitor of the neuronal and inducible isoforms of NOS, which shows very low activity against endothelial NOS []. TRIM exerts antidepressant, anxiolytic and antinociceptive effects in rodents and is an appropriate tool to clarify the role of nnos in the central nervous system [11]. On the other hand, 7-nitroindazole (7-NI) is a NOS inhibitor that does not show selectivity for any NOS isoform [28]. 7-NI has also exerted antidepressant and anxiolytic effects in behavioral tests in rodents [11,12]. Further, 7-NI has some amnesic effects in rodents. Systemic administration of 7-NI impaired spatial learning [16,29,] and object recognition [17] in rats and also had negative effects in the passive avoidance test in chicks [31]. Although there are some studies investigating the effects of TRIM on LTP formation, this is the first article investigating the effects of TRIM on learning and memory in different behavioral tests. The aim of this study was to investigate the effects of a selective neuronal and inducible NOS inhibitor, TRIM, on learning and memory and to compare TRIM to the nonselective NOS inhibitor 7-NI using different behavioral tests in Swiss mice, thus clarifying the role of nnos and enos in cognitive processes. The drugs were given before the retention trial in all tests used to explore the effects of drugs on retrieval of the memory. Chronic treatment of TRIM in the Morris water maze test is performed to examine whether there is an effect after subchronic injections on both spatial learning and memory. The tests chosen have different motivational aspects (aversive, neural and rewarding) and different set of situations representing very distinct principles. The tests used were (i) Morris water maze test (MWM), for evaluating hippocampal-dependent spatial memory [32,33]; (ii) Novel object recognition test (NOR), for evaluating hippocampal-dependent visual memory [34]; (iii) Social transmission of food preference test (STFP), for evaluating hippocampal-dependent olfactory memory [35]; (iv) Step-through passive avoidance test, for evaluating amygdala-dependent emotional memory [36]. MATERIALS AND METHODS Animals Male Swiss mice (Centre d Elevage Janvier, France) weighing g were used. Mice were kept 4 5 per cage at 21 ± 1.5 C under a 12-h light/dark cycle (light on at 8. PM). Tap water and food pellets were available ad libitum. The animals were kept for at least 1 week in the animal colony before entering experiments. All behavioral tests were carried out during scotophase. All procedures described here fully comply with French legislation on research involving animal subjects. This research protocol adhered to recommendations by the European Community Council for the Ethical Treatment of Animals (86/69/EEC). Morris water maze test The Morris water maze was a circular pool (9 cm diameter and cm height) that was filled to a depth of 14 cm with water (22 C) and rendered opaque by the addition of small black balls. The pool was located in a dimly lit, soundproof test room with a number of extramaze visual cues, including a white-black-colored poster on the wall, a halogen lamp, a camera and the experimenter. The maze was divided into four quadrants, and three equally spaced points were used as starting positions around the edge of the pool. The order of the release positions varied systematically throughout the experiment. An escape platform (6 cm diameter and 12 cm high) was located in one quadrant 1 cm above the water surface during the familiarization session and 1 cm below the water surface during the other sessions.

3 37 O. Mutlu et al. Videotracking conducted with a videocamera focused on the full diameter of the pool. Navigation parameters were analyzed using the ETHOVISION 3.1 video analysis system (Noldus, Amsterdam, Netherlands). The mice were trained in the Morris water maze during five daily sessions (S1, S2, S3, S4, S5). One familiarization and four acquisition sessions were performed in the Morris water maze test. During the familiarization session and acquisition phase of the experiment, each mouse was given three trials. The delay between the trials was 6 s, and a 1-day interval was used between the sessions. For each daily trial, the mouse was taken from the home cage and placed into the watermaze at one of three randomly determined locations with its head facing the center of the water maze. After the mouse had found and climbed on to the platform, the trial was stopped and the escape latency was recorded. If the mouse had not climbed onto the platform in 6 s, the trial ended automatically and the experimenter guided the mouse to the platform and an escape latency of 6 s was recorded. Twenty-four hours after the last acquisition session, a probe trial was used to assess the mouse spatial retention of the location of the hidden platform. During this trial, the platform was removed from the maze and the mouse was allowed to search the pool for 6 s. The percent of time spent in each quadrant was recorded. Novel object recognition test The protocol according to Ennaceur and Delacour [34] was modified. The apparatus consisted of a circular open field ( cm diameter and cm height) made of PVC with a black and white-striped cardboard pattern ( cm) nailed to one of the walls. The floor was divided into six peripheral sections and one central section of the same dimension. A bulb provided a constant illumination of about lux above the central section. The novel object recognition task procedure consisted of three trials: habituation, training and retention trials. Each mouse was individually habituated to the apparatus for 5 min in the absence of objects (habituation trial). Thirty min after the habituation trial, the mouse was placed in the apparatus for the training trial (T1) and two identical objects (moon or butterfly) were placed in a symmetrical position cm above the side wall. The order of objects used per subject per trial was determined randomly. The total time spent exploring the two objects was recorded for 5 min by the experimenter. Exploration of an object was defined as directing the nose to the object and/or touching it with the nose. After a predetermined retention interval of 1 h, the mouse was again placed in the apparatus for the retention trial (T2), but now with two dissimilar objects, a familiar one and a new one. The object not used in the training trial was used as the novel object in the retention trial. The animals were then allowed to explore freely for 5 min, and the time spent exploring each object was recorded. If recognition memory was intact, the mice were expected to spend more time exploring the novel object [34]. A ratio index (RI) was calculated as the time spent exploring the new object (N) divided by the total time exploring the objects (N+R) multiplied by. A higher ratio index was considered to reflect greater memory retention. Social transmission of food preference test Hippocampus-dependent nonspatial olfactory memory [35] was studied using the social transmission of food preference task. The experiment was conducted in three phases: (i) habituation to flavored food, (ii) interaction between demonstrator and observer mice and (iii) test of the food preference in the observer mice. Mice were housed at a ratio of 3 4 observer mice to one demonstrator. In the habituation phase, a demonstrator mouse was chosen from each cage. Demonstrators were housed singly in a cage separate from the colony for 3 h with free access to water but not food. At the end of the 3 h, each demonstrator was allowed to eat powdered ground chow scented with either cinnamon (1% w/w) or cocoa (2% w/w) for 2 h. The criterion for inclusion in the experiment was consumption of.2 g of chow. Each demonstrator was then placed back with its observer cagemates for min. Observers interacted with the demonstrator, including sniffing the scent around the muzzle and on the breath of the demonstrator mouse. After the interaction period, the demonstrator mouse was removed from the interaction cage and returned to its individual cage. The food preference of the observer mice was tested 24 h after the end of the interaction with the demonstrator. Five hours before the preference test, observer mice were caged individually, and 3 h before the test, food was removed from the observer s cage. The preference test consisted of presenting each observer with a pair of weighed food pellets in the individual cage for 2 h. Half the observers were tested with the cinnamonflavored cued food eaten by their demonstrator vs. the

4 Effect of TRIM on memory 371 novel cocoa-flavored food; the other half of the observers were tested with the cocoa-flavored cued food eaten by their demonstrator vs. the novel cinnamon-flavored food. The ratio of the weight of the cued food eaten to the weight of total food eaten was used as a measure of food preference. Passive avoidance test The apparatus consisted of a box with an illuminated part (L7X12.5Xh14 cm) and a dark part (L24X12. 5Xh14 cm), both equipped with a grid floor composed of steel bars (.3 cm diameter) spaced.9 cm apart. The inhibitory avoidance task consisted of two trials. In the acquisition trial, each mouse was placed in the illuminated compartment, which was lighted by a bright bulb ( lux). The intercompartment door was opened after a 6-s acclimation period. If the mouse stepped into the dark compartment (2/3 of the tail in the dark compartment), the door was closed by the experimenter and an inescapable foot shock (.25 ma/1 s) was delivered through the grid floor of the dark compartment. A cutoff time of 5 min was selected. The time taken to enter the dark compartment was recorded. Immediately after the shock, the mouse was returned back to the home cage. The retention trial started 24 h after the end of the acquisition trial. Each mouse was placed in the illuminated compartment as in the training trial. The door was opened after a -s acclimation period. The step-through latency in the retention trial (with a maximum s cutoff time) was used as the index of retention of the learned experience. Shock was not applied at the retention trial. Locomotor activity test The effect of drugs on locomotor activity of the animals was measured in the different groups of animals using the open field test. The apparatus was the same as the one used in the novel object recognition test. The number of transitions in the seven different sections of the open field was noted during 5 min. Experimental design During the acute injection of drugs in the Morris water maze, novel object recognition, social transmission of food preference and passive avoidance tests, 7-NI and TRIM were administered intraperitoneally (i.p.) and 5 min, respectively, before the retential trials. During the subchronic injections of TRIM in the Morris water maze test, TRIM was administered every day 5 min before starting each session and the probe trial. 7-NI and TRIM were administered and 5 min, respectively, before the open field test. The number of animals per group ranged from to19. Drugs 1- (2-trifluoromethylphenyl) imidazole was obtained from Tocris (Avonmouth, UK) and 7-NI was from Sigma (St. Louis, MO, USA). TRIM was dissolved in saline, and 7-NI was dissolved in saline mixed with 5% cremophor and % DMSO. All drugs were freshly prepared and given intraperitoneally (i.p.) in a volume of.1 ml per g body weight of mice. groups received the same volume of vehicle. of drugs were selected according to behavioral and neurochemical studies showing the drugs to have the intended effect [11,17,29,37]. Statistics One-way analysis of variance (ANOVA) was used as a parametric test when there was no significant difference between groups for normality of samples and homogeneity of variance, while the nonparametric Kruskal Wallis test was preferred in the other cases. The post hoc Tukey or Dunn s tests were performed for the comparison of the groups. The nonparametric Friedmann post hoc Dunn s test was used to evaluate the effect of subchronic injections of TRIM in the MWM test. Data are expressed as the mean values ± SEM. Differences were considered to be statistically significant when P was <.5. RESULTS Effect of NOS inhibitors in the morris water maze test Both the effects of TRIM and 7-NI in the Morris water maze were examined after acute injections, while only the effects of TRIM were assessed after chronic administration. The acute injections occurred before the probe trial, so we only present data from this trial, while we show data of the acquisition and probe trials in cases where the drug was injected chronically. After the acute injection of TRIM, the time spent (oneway ANOVA, F(3,36) =.45; P >.5) and the total distance travelled (Kruskal Wallis, H = 3.882, P >.5) in the target quadrant (the quadrant in which the platform was previously located) did not differ between the TRIM and saline groups (data not shown),

5 372 O. Mutlu et al. while acute administration of 7-NI produced a significant decrease in the time spent in the target quadrant at mg/kg (Kruskal Wallis post hoc Dunn s, H = 8.733, P <.5; Figure 1a) and shortened the total distance travelled in this quadrant at 45 mg/kg (one-way ANOVA post hoc Tukey, F(3,36) = 4.19, P <.1; Figure 1b). When we compared the average speed of the animals, we did not find any significant difference between the TRIM and saline groups (one-way ANOVA, F(3,36) = 2.51, P >.5; Figure 1c), while there was a significant difference between the 45 mg/kg 7-NI and saline groups (one-way ANOVA post hoc Tukey, F(3,36) = 5.55, P <.1; Figure 1d). To understand whether TRIM had any effect on learning and memory after chronic injections, TRIM was given before each acquisition session and there was a significant difference in escape latency (Kruskal Wallis post hoc Dunn s, H = 8.55, P <.5; Figure 2a), as well as in total distance travelled (Kruskal Wallis H =.497, P <.5; Figure 2b) in the third session at 5 and mg/kg doses. When we compared the effect of different doses of TRIM on escape latency and total distance travelled within all sessions, a significant difference was observed. The escape latency (Friedmann value (Fr) = 15,38; 24,46; 21,37; 32,72; P <.1 for control, TRIM, and 5 mg/kg groups, respectively) and the total distance travelled (Fr = 18,16; 23,44; 24,16; 28,24; P <.1 for saline, TRIM, and 5 mg/kg groups, respectively) were significantly altered during the sessions, and there was a significant difference when the first and fifth sessions were compared (Friedman post hoc Dunn s test, Figure 2a,b). There was no significant difference between the time spent (one-way ANOVA, F(3,36) =.31; P >.5) and the total distance travelled [F(3,36) =.89; P >.5] in the target quadrant in all chronic TRIM treated groups in the probe test (data not shown). Effect of NOS inhibitors in the novel object recognition test In the novel object recognition test, there was no significant difference between the TRIM and saline groups (one-way ANOVA, F(3,44) = 1.63, P >.5; Figure 3a), whereas 7-NI at and 45 mg/kg showed a significant effect on the recognition index when compared to the control group (Kruskal Wallis H = , P <.5 and P <.1 respectively; Figure 3b). Effect of NOS inhibitors in the social transmission of food preference test There was a significant difference between the 5 mg/kg TRIM and saline groups for the eaten cued food/total food (%) on the second day of the experiment (Kruskal Wallis H = , P <.1; Figure 4a), but the total food consumption was also significantly different between the 5 mg/kg TRIM and saline groups (Kruskal Wallis, H = , P <.1; Figure 4b). 7-NI at 45 mg/kg significantly decreased the total food eaten (one-way ANOVA post hoc Tukey, F(3,39) = 3.72, Time (s) (c) Speed (m/s) NI mg/kg (b) Total distance travelled (m) %.9 NaCI TRIM mg/kg TRIM mg/kg TRIM 5 mg/kg (d) Speed (m/s) # 7NI mg/kg 7NI mg/kg Figure 1 Effects of acute 1- (2-trifluoromethylphenyl) imidazole (TRIM) (,, 5 mg/kg; 5 min before testing) and 7-NI (15,, 45 mg/kg; min before testing) injected before the probe test of the Morris Water Maze. Time spent in the target quadrant after 7-NI. Total distance travelled in the target quadrant after 7-NI (b) and average speed after TRIM (c) or 7-NI (d). (n = per group). Results are expressed as mean ± SEM. P <.5 vs. control; P <.1 vs. control.

6 Effect of TRIM on memory (b) 25 Time spent for finding escape platform (s) %.9 NaCI TRIM mg/kg TRIM mg/kg TRIM 5 mg/kg # ## ## ## Total distance travelled (m) 15 5 $ %.9 NaCI TRIM mg/kg TRIM mg/kg TRIM 5 mg/kg # Sessions Sessions Figure 2 Effects of subchronic injection of 1- (2-trifluoromethylphenyl) imidazole (TRIM) (,, 5 mg/kg) before each acquisition session in the Morris Water Maze on: time spent and total distance travelled to find the platform during the acquisition session (b) (n = per group). Drugs were injected 5 min prior to testing for 5 days. Results are expressed as mean ± SEM. P <.5 TRIM 5 mg/kg vs. TRIM mg/kg; $ P <.5 TRIM 5 mg/kg vs. saline and TRIM mg/kg; # P <.5, ## P <.1 the first and the fifth sessions were compared for each drug group. 8 7 (b) RI Index 5 %.9 NaCI TRIM mg/kg TRIM mg/kg TRIM 5 mg/kg RI Index 6 5 7NI mg/kg Figure 3 Effects of 1- (2-trifluoromethylphenyl) imidazole (,, 5 mg/kg; n = 12 per group; or 7-NI (15,, 45 mg/kg; n = 12, 11, 12 respectively; (b) on ratio index (RI) in the novel object recognition test. Drugs were injected 5 and min, respectively, prior to testing. Results are expressed as mean ± SEM. P <.5, P <.1 vs. controls. P <.5; Figure 4d), and the eaten cued food/total food % was also significantly different between the 45 mg/kg 7-NI and control groups (Kruskal Wallis H = 9.8, P <.5; Figure 4c). No effect was observed with the other doses of 7-NI. Effect of NOS inhibitors in the step-through passive avoidance test There was no significant difference between the TRIM and saline groups in retention latency (Kruskal Wallis, H = 1.435, P >.5; Figure 5a), while 7-NI decreased the retention latency (T2) at all doses used (15,, 45 mg/kg; Kruskal Wallis, H = , P <.5; Figure 5b). Effect of NOS inhibitors on locomotion There was no significant effect of TRIM on locomotion (Kruskal Wallis, H = 2.199, P >.5; Figure 6a), while 7-NI disturbed this parameter at 45 mg/kg (Kruskal Wallis H = 8.4, P <.5; Figure 6b) in the open field test. DISCUSSION The main finding of this study is that the neuronal and inducible NOS inhibitor TRIM has no clear effect on learning and memory, while the nonselective NOS inhibitor 7-NI impairs memory in the tasks used. The effect of TRIM on both spatial learning and memory was

7 374 O. Mutlu et al. Cued food/total food eaten (%) (c) Total food consumption (g) %.9 NaCI TRIM mg/kg TRIM mg/kg TRIM 5 mg/kg %.9 NaCI TRIM mg/kg TRIM mg/kg TRIM 5 mg/kg (b) 1 Cued food/total food eaten (%) 8 6 (d) Total food consumption (g) 7NI mg/kg 7NI mg/kg Figure 4 Effects of 1- (2-trifluoromethylphenyl) imidazole (TRIM) (,, 5 mg/kg; n = 11, 13, 13, respectively) and/or 7-NI (15,, 45 mg/kg; n =, 12, ) in the social transmission of food preference test. (a, b) cued food/total food, respectively, after TRIM and after 7-NI, (c, d) total food consumption, respectively, after TRIM and 7-NI. Drugs were injected 5 (TRIM) and (7-NI) min prior to testing. Results are expressed as mean ± SEM. P <.5 vs. control; P <.1 vs. saline. Retention latency (s) %.9 NaCI TRIM mg/kg TRIM mg/kg TRIM 5 mg/kg (b) Retention latency (s) NI mg/kg Figure 5 Effect of 1- (2-trifluoromethylphenyl) imidazole (,, 5 mg/kg; n = 18, 17, 14, respectively; or 7-NI (15,, 45 mg/kg; n = 13, 13, 12, respectively; (b) on retention latency in the step-through passive avoidance test. Drugs were injected 5 and min, respectively, prior to testing. Results are expressed as mean ± SEM. P <.5 vs. control. evaluated while the effect of 7-NI on memory retrieval was evaluated in the MWM test. The effect of drugs on the retrieval of memory was investigated in all other tests. Indeed, TRIM did not elicit any effect in the Morris water maze, in the novel object recognition test or in the passive avoidance test, while 7-NI at mg/kg impaired spatial memory in the probe test and the Morris water maze, deteriorated object recognition at and 45 mg/ kg and impaired passive avoidance at all doses used. Both TRIM and 7-NI disturbed performance in the social transmission of food preference test, respectively, at 5 and 45 mg/kg doses, but there was also a significant difference in the amount of total food eaten at these doses, indicating that this amnesic effect might be nonspecific. Morris water maze test is a spatial, long-term memory test [32,33]. In rats trained in the MWM, the number of neurons containing NOS in the frontal cortex and the dentate gyrus was increased when compared with nontrained controls, suggesting that NOS inhibition may interfere with hippocampus-dependent memory [38]. The results of the MWM do not enable us to conclude a deterioration of spatial learning and memory by TRIM after acute and subchronic administration. On the other side, 7-NI disturbed spatial memory in the probe test after acute injection. This could, however, be because of some nonspecific effects unrelated to the synthesis of NO synthase, since at 45 mg/kg, the swimming speed and the locomotion of the animals were decreased.

8 Effect of TRIM on memory 375 Figure 6 Effects of 1- (2-trifluoromethylphenyl) imidazole (,, 5 mg/kg; n = 12 per group; or 7-NI (15,, 45 mg/kg; n = 12, 11, 12 respectively; (b) on locomotion of the animals in the open field test. Drugs were injected 5 and min, respectively, prior to testing. Results are expressed as mean ± SEM. P <.5 vs. control. 8 Locomotion %.9 NaCI TRIM mg/kg TRIM mg/kg TRIM 5 mg/kg (b) Locomotion NI mg/kg In the novel object recognition test, the failure to discriminate between familiar and novel objects can be related to either impaired memory of the object or the inability to use spatial information. In this situation, as in the MWM, TRIM did not induce any effect, while mice treated with and 45 mg/kg 7-NI sniffed the new object less than the familiar one. The effect of the highest dose of 7-NI might be unrelated to an amnesic action, as mice displayed an inhibition of locomotion. However, such an explanation does not apply to 7-NI at mg/kg, as this dose did not elicit sedation, so the impairment of performance observed at this dose can be attributed to learning and memory deficits in the NOR test. All together, these results emphasize the amnesic effect of 7-NI, while TRIM had no effect. In the novel object recognition test, it has been shown that AMPA and NMDA receptors are involved in the acquisition, consolidation and retrieval of recognition memory [39]. Because NOS inhibitors disturb LTP formation by NMDA receptor blockade, this might be the putative mechanism of the effects observed here. Social transmission of food preference is a hippocampal-dependent olfactory memory test [35]. Both TRIM and 7-NI impaired memory at the higher doses in the STFP test, but they also significantly decreased food consumption at these doses, suggesting that the amnesic effects are instead related to nonspecific processes. In the hippocampal-dependent MWM and NOR tests, 7-NI disturbed memory at mg/kg, an effect not observed in the STFP task. Different explanations of this discrepancy can be proposed. One explanation could be the different features of odor processing. Olfactory learning has no spatial components, as in the MWM and NOR tests. However, it could be that distinct brain processes underlie spatial and olfactory memory formation. Alternatively, the odor information used in the STFP test could be more difficult to forget. There are also some findings claiming that NO does not affect the retrieval of olfactory memory in adult sheep [] and that NO release is involved in the acquisition phase in an olfactory recognition test but does not affect postacquisition recall [41]. Therefore, NO inhibition can have an effect on the acquisition and consolidation of olfactory process, while it does not have a clear effect on olfactory memory retrieval, as seen in our study. Passive avoidance is an adaptive response to a stressful experience that serves as a measure of learning and memory [36]. In this situation, drugs were injected 24 h after giving electrical shock and just before the retention test so that we could study their effect on memory retrieval but not acquisition and consolidation. Giving the drug just before the electrical shock can cause some nonspecific effects (e.g. analgesic effect, pain perception, motility), which was not the case in our study. Deterioration in memory was seen, even at a low dose of 7-NI (15 mg/kg), while TRIM had no effect in this test. The difference between the effects of TRIM and 7-NI on cognitive functions could be related to the selectivity differences between the drugs, but also to the differences between the pharmacokinetic (absorption, protein binding, penetration to brain, biotransformation) and pharmacodynamic properties of indazole (7-NI) and imidazole (TRIM)-derivatives. In a passive avoidance test in chicks, it was determined that enos inhibition caused amnesic effects in both hemispheres, whereas nnos inhibition impaired memory only when administered to the left hemisphere. In recent studies, it was postulated that both enos and nnos activities are necessary for memory formation. In contrast to this claim, the formation of LTP in rodents could be related to the activation of only one NOS isoform, but not both [6]. In the same way, because of the localization in neuronal tissue (especially in cerebellum, hippocampus and striatum) in the mammalian brain, it is assumed that the nnos isoform is related to synaptic plasticity. However, this is not observed in all studies. enos was found to have greater immunostain-

9 376 O. Mutlu et al. ing than nnos in hippocampal CA1 pyramidal cells in both rats [5,42] and humans [43]. It was also shown that nnos is expressed in many cells, not just neurons, and that pyramidal neurons in the hippocampus also express enos [5]. Additionally, in a passive avoidance test in chicks, it was claimed that concerning LTP, the role of enos cannot be substituted by that of nnos [15]. Further, nnos is not the only isoform that could be responsible for memory formation. Son et al. [6] showed that LTP decreased only when both NO synthase isoforms were absent in the hippocampal CA1 area. All of these results support the claim that both isoforms have a role in LTP and can substitute for each other, but the mechanism underlying them and to what degree enos can compensate nnos is undetermined. These findings can explain why the nonselective NOS inhibitor 7-NI induced deterioration in memory retrieval, while the nnos selective NOS inhibitor TRIM had no effect on cognitive functions. One of the major problems with NO inhibitors or donors is the effect of these drugs on blood pressure [44]. It is difficult to dissociate neural effects of these drugs from their cardiovascular effects. Prickaerts et al. [17] published that mg/kg, but not mg/kg, of 7-NI increased mean arterial blood pressure. Interestingly, it has been shown that the disturbing effects of 7-NI were not correlated with the effects on blood pressure [17]. Meyer et al. [16] even reported no effect of 7-NI on systolic blood pressure at 65 mg/kg. In another study, Hölscher et al. [29] showed that 7-NI at mg/kg disturbed spatial learning without changing blood pressure in both the water maze and 8-arm radial maze tests. Further, others showed that both 7-NI and TRIM did not have a significant effect on blood pressure at the doses used in our study [16,29,45,46]. All together, these data indicate that one can exclude NOS inhibitor effects on blood pressure when considering the effects observed in our study. CONCLUSION The selective neuronal NOS inhibitor TRIM exerted no clear effect on learning and memory function, while the nonselective NOSI 7-NI significantly disturbed cognitive functions although we cannot completely exclude some side effects for 7-NI so further studies should be performed to enlighten the results of our study. These findings support the idea that nnos is not the only isoform that has a role in learning and memory processes, and it can be concluded that deterioration in memory, in some cases, needs the inhibition of both nnos and enos. REFERENCES 1 Garthwaite J. Glutamate, nitric oxide and cell-cell signalling in the nervous system. Trends Neurosci. 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