EFFECTS OF NITRIC OXIDE SYNTHASE INHIBITOR N G -NITRO-L-ARGININE METHYL ESTER ON SPATIAL AND CUED LEANING

Pergamon Neuroscience Vol. 83, No. 3, pp. 837 841, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved PII: S0306-4522(97)00457-0 0306 4522/98 $19.00+0.00 EFFECTS OF NITRIC OXIDE SYNTHASE INHIBITOR N G -NITRO-L-ARGININE METHYL ESTER ON SPATIAL AND CUED LEANING B. R. KNEPPER and D. D. KURYLO* Department of Psychology, Bowdoin College, Brunswick, ME 04011, U.S.A. Abstract An investigation was made of the effects of the nitric oxide synthase inhibitor N G -nitro-larginine methyl ester (L-NAME) on the acquisition and retention of two operantly conditioned discrimination tasks. Twenty Long Evans rats were conditioned to approach one of two spatial locations that was either held constant across trials (spatial task) or was associated with a visual cue (illuminated lamp) that was randomly assigned to one of the locations on each trial (cued task). Rats were assigned to one of two treatment groups in which they received intraperitoneal injections of either N G -nitro-l-arginine methyl ester or saline approximately 2 h before sessions on each day of training. Analysis was made of the trial-by-trial performance in order to identify the characteristics of learning under each condition. Assessment of learning acquisition was based on the number of trials required to reach a criterion of 80% correct responses, whereas retention was assessed by the number of trials to criterion on each day after the criterion was initially reached. Analysis indicated that treatment groups did not differ significantly on acquisition or retention of either the spatial or cued task. These results indicate that inhibition of nitric oxide synthase does not interfere with the learning or retention of basic operant tasks that involve simple spatial or visual analysis. Whereas results from biochemical and physiological investigations have suggested an impact of nitric oxide synthase on behavioural function, behavioural investigations indicate a limited impact of nitric oxide synthase inhibition on learning and memory. Although these results do not discount the role of nitric oxide synthase in a hippocampal mechanism, they illustrate that behavioural analysis should be made in the context of multiple interacting neural systems. Viewed with previous behavioural research on the effects of N G -nitro-l-arginine methyl ester, these results indicate that nitric oxide synthase inhibition results in impairment of certain forms of learning whereas other forms are preserved. 1998 IBRO. Published by Elsevier Science Ltd. Key words: L-NAME, nitric oxide, NOS, hippocampus. N G -nitro-l-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), has been shown to produce an inhibitory action on long-term potentiation. 8,13 Substantial NOS concentrations in hippocampal pyramidal cells further suggests its involvement in a hippocampal mechanism of synaptic plasticity. 4 Consistent with neurochemical investigations, animals treated with L-NAME have demonstrated learning deficits conventionally associated with hippocampal lesions. Systemic injections of L-NAME produced deficits on a submerged platform spatial learning task in rats. 3,6 Using a three panel runway, Ohno et al. 11 found that intrahippocampal injections of L-NAME in rats produced impairments on a working memory task, in which the location of correct panels were changed across sessions, but not on a reference memory task, in which correct panel locations remained constant across sessions. In addition to spatial learning deficits, L-NAME *To whom correspondence should be addressed. Abbreviations: L-NAME, N G -nitro-l-arginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase. was found to interfere with a conditioned eyeblink response in rabbits. 3 L-NAME had no effect on sensorimotor or motivational measures. 6 Although behavioural studies have begun to demonstrate that L-NAME inhibits some, but not all types of learning, the role of NOS in learning and memory needs to be further explored. In order to better characterize the nature of learning deficits that result from L-NAME treatment, the acquisition and retention of operantly conditioned behaviours by L-NAME-treated rats were examined in detail. Hippocampal lesions in rats have been shown to impair learning on tasks that rely on spatial information, whereas tasks that rely on nonspatial cues are preserved. 9 In order to examine whether L-NAME interferes with learning in a manner similar to hippocampal lesions, rats were conditioned on either a spatial learning or cued learning task. Trialby-trial examination was made within sessions as well as across sessions on successive days. Analysis indicated that L-NAME-treated rats did not differ significantly from control rats on either acquisition or retention of learning for either task tested. 837

838 B. R. Knepper and D. D. Kurylo Procedure Rats were water deprived for 22.5 h prior to testing. Testing occurred once a day for a 30 min session, after which rats received water ad libitum for 1 h. Rats from each treatment group participated in one of two experimental conditions: Spatial Learning or Cued Learning, with five rats in each condition. A trial was initiated by rats entering either of the two drinking wells. Trial by trial records were acquired throughout each session. The criterion for learning required 80% correct response for 20 consecutive trials, measured from the start of the series. Fig. 1. Schematic top view of the operant conditioning chamber. Each reward well contained an infra-red emitter detector pair that sensed the presence of the rat s head within the well, as well as a spout for water, from which rats could receive reward from inside the well. EXPERIMENTAL PROCEDURES Subjects Twenty Long Evans hooded rats (Charles River Laboratories) served as subjects. Measurements began at approximately 40 days-of-age, at which time rats weighed approximately 225 g. The animal housing facility maintained a 12 h light/dark cycle. Materials L-NAME-treated rats received N G -nitro-l-argininemethyl ester hydrochloride (Alexis Biochemical Corporation) dissolved in saline. Apparatus Behavioural measurements were made in an operant conditioning chamber (Fig. 1). The chamber was constructed of clear Plexiglas. The chamber was 20 cm high, and contained a front panel, 5 cm in width, flanked by two 10 cm-wide panels that were angled towards the back of the chamberby45. Each side panel possessed a 2.5 cm opening, centred 2.5 cm from the chamber floor, through which rats had access to an enclosed drinking well. Drinking wells extended 2.5 cm beyond the chamber walls. A small hole through which water was delivered was positioned on the back of each drinking well. Infra-red emitter/detector pairs, which monitored the placement of a rat s head within the drinking well, were mounted outside each well. Measured amounts of water (0.04 ml) were delivered to each well by means of solenoid driven valves. Circuitry for the two infra-red pairs and the two solenoids were interfaced via solid-state relay switches (CyberResearch CYRDIO) to a computer (DECpc Lpv 433dx). Data collection, trial events, and contingency algorithms were controlled by computer. Treatment groups Rats were divided equally into two treatment groups that received either L-NAME (75 mg/kg, dissolved in saline) or saline, delivered i.p. approximately 2 h before testing on each day of training. The L-NAME solution was prepared either the day of testing, or prepared on the previous day and kept frozen. Spatial learning. Rats received reward for placing their head into one of the two drinking wells, whereas placement into the alternative well had no consequences. The side assigned to each rat was counterbalanced across rats, and held consistent throughout the experiments. Rats generally reached criterion by the second or third day, and measurements were taken for five consecutive days. Room lights were on during each session. Cued learning. Two small lamps were positioned above each drinking well on the outside of the chamber. Lamps were clearly visible from inside the chamber. The session took place with room lights off. On each trial, one of the two lamps, assigned randomly, was illuminated. Rats received reward for placing their head into the well associated with the illuminated lamp. No consequences occurred for placing their head into the alternative drinking well. Rats reached criterion in approximately four to eight days. Measurements were acquired for 15 consecutive days. RESULTS Behaviour was quantified in several ways in order to determine level of activity, acquisition of learning, and retention of learning across days. Level of activity Level of activity was measured by the number of trials initiated in each session (Fig. 2A and B). For the Spatial Learning condition, an ANOVA indicated that the main effect of treatment group (F 1,8 =0.001; P>0.05) and the interaction of group by session (F 4,32 =1.48; P>0.05) were not significant. Similarly, for the Cued Learning condition, the main effect of treatment group (F 1,8 =0.10; P>0.05) and the group by session interaction (F 14,112 =0.44; P>0.05) did not reach significance. Learning acquisition Learning acquisition was indexed in two ways: number of trials to criterion (regardless of session) and percentage of correct trials within each session. For the Spatial Learning condition, the number of trials to criterion for the L-NAME group (mean=82.0, S.E.M.=42.4) and the saline group (mean=116.0, S.E.M.=45.0) did not differ significantly (t 8 =0.55; P>0.05). For the per cent correct across sessions (Fig. 3A), ANOVA indicated that the main effect of treatment group (F 1,8 =0.43; P>0.05) and the interaction of group by session (F 4,32 =0.29; P>0.05) were not significant.

Effects of L-NAME on learning 839 Fig. 2. Mean number of trials initiated in a 30 min session for each day of conditioning for the Spatial Learning (A) and Cued Learning (B) conditions. Error bars represent the S.E.M. Fig. 3. Mean percentage of correct responses for each day of conditioning for the Spatial Learning (A) and Cued Learning (B) conditions. For the Cued Learning condition, trials to criterion for the L-NAME group (mean=281.8, S.E.M.=40.0) and the saline group (mean=242.0, S.E.M.=37.2) did not differ significantly (t 8 =0.73; P>0.05). For the per cent correct across sessions (Fig. 3B), the main effect of treatment group (F 1,8 =2.49; P>0.05) and the interaction of group by session (F 14,112 =0.63; P>0.05) were not significant. Learning retention Learning retention was indexed by the number of trials to criterion on each day following the first session in which criterion was reached. Evidence of retention is reflected by a minimal number of trials to criterion in sessions after the task was learned. With some exceptions, rats reached criterion within the first few trials on the sessions following acquisition of the task, indicating that learning was retained across days (Fig. 4A and B). An exception occurred in the Spatial Learning condition in which a rat from the saline group did not reach criterion on the day following acquisition. The other exceptions occurred in the Cued Learning condition, in which a rat from the saline group required 41 trials to reach criterion on the day following acquisition, a rat from the saline group did not reach criterion on three days following acquisition, and a rat from the L-NAME group did not reach criterion on the day following acquisition. For rats who did not reach criterion on days following the initial acquisition of learning, the total number of trials for that session were used for analysis. For the Spatial Learning condition, the main effect of treatment group (F 1,8 =1.33; P>0.05) and the interaction of group by session (F 1,8 =0.87; P>0.05) was not significant. Similarly, for the Cued Learning condition, the main effect of treatment group (F 1,8 =0.13; P>0.05) and the interaction of group by session (F 5,40 =0.18; P>0.05) was not significant. DISCUSSION Neurochemical and physiological investigations have associated nitric oxide (NO) with neural function. 2,14 Although the role of NO in neural

840 B. R. Knepper and D. D. Kurylo Fig. 4. Number of trials to criterion (80% correct for 20 consecutive trials) on each day following the initial acquisition of learning for the Spatial Learning (A) and Cued Learning (B) conditions. In most cases, rats reached criterion within the first few trials, indicating the retention of learning across days. Extensive S.E.M. (error bars) for day 1 of Spatial Learning and days 1 3 of Cued Learning reflect subjects who did not reach criterion on days following initial acquisition, in which case the total number of trials for that session were used. mechanisms is currently under investigation, several lines of evidence associate NO with a hippocampal mechanism of long-term potentiation, suggesting a role in memory. Consistent with neurochemical analyses are behavioural measurements in which memory deficits are observed in animals treated with either L-NAME or with other NOS inhibitors. 1,7 Preservation of learning capacities observed here may reflect the nature of the tasks that were used. Normal acquisition of the visually cued task is consistent with previous investigations in which rats with hippocampal lesions retained the ability to use visual and tactile cues in a radial maze task. 9 Furthermore, retention of both the spatial and visual tasks is consistent with hippocampal lesion studies, 10 as well as behavioural measurements of the effects of L-NAME for watermaze 3 and three-panel runway tasks. 11 In this regard, a hippocampal mechanism is not critical for the retention of previously acquired information. Alternatively, preservation of the acquisition of the spatial task used here may reflect task complexity. Hippocampal lesions may disrupt the ability to process relationships among stored information, whereas processing stimuli individually remains intact. 5 The singular association of the two-alternative spatial discrimination used here may differ in this regard from task requirements of the water maze paradigm. Although processing spatial relationships underlies learning in both cases, the two-alternative condition used here may not rely on a hippocampal mechanism. Further investigation, however, is required to determine the degree to which a hippocampal mechanism contributes to the tasks used here. In addition to learning function, the role of NO in other neural mechanisms remains uncertain. Activity measurements made here indicate that L-NAME does not interfere with general motor function, or related factors such as motivation. Previous research has indicated that L-NAME does affect other physiological function, such as an increase in blood pressure. 12 Retention of performance found here indicates that hypertensive response to L-NAME does not play a role in performing these tasks, possibly lending support to the notion that deficits found in previous investigations of NOS inhibition were not due to changes in blood pressure and blood flow in the brain. These results are consistent with previous behavioural studies in which NOS inhibition impaired selective aspects of learning. These data also indicate that a lack of NOS in other regions of the brain does not interfere with the task requirements used here. In this regard, the interaction of multiple neural systems, to which a hippocampal mechanism contributes, underlies the context in which behavioural analyses should be made. CONCLUSIONS These results indicate that inhibition of NOS does not interfere with the learning or retention of basic operant tasks that involve simple spatial or visual analysis. Whereas results from biochemical and physiological investigations have suggested an impact of NOS on behavioural function, behavioural investigations indicate a limited impact of NOS inhibition on learning and memory. Although these results do not discount the role of NOS in a hippocampal mechanism, they illustrate that behavioural analysis should be made the context of multiple interacting neural systems. Viewed with previous behavioural research on the effects of L-NAME, these results indicate that NOS inhibition results in impairment of

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