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Effects of Nicergoline on Age-Related Decrements in Radial Maze Performance and Acetylcholine Levels
Brain Research Bulletin, Vol. 43, No. 3, pp. 305–311, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00
PII S0361-9230(97)00010-5
Effects of Nicergoline on Age-Related Decrements in Radial Maze Performance and Acetylcholine Levels ROBERT A. MCARTHUR,1 NICOLA CARFAGNA, LAURA BANFI, STEFANO CAVANUS, MARIA ANTONIETTA CERVINI, RUGGERO FARIELLO AND CLAES POST CNS Preclinical Research, Milan Pharmacia & Upjohn, SpA Viale Pasteur, 10 I-20014, Nerviano, (MI), Italy [Received 27 August 1996; Revised 11 December 1996; Accepted 12 December 1996]
ABSTRACT: The effects of chronic oral administration of nicergoline (5.0 mg/kg; bid) on locomotor activity, eight-arm radial maze performance plus striatal, cortical, and hippocampal acetylcholine (ACh) levels were examined in young and aged Wistar rats. Chronic nicergoline administration did not modify either the locomotor activity or radial maze learning in young rats. Young rats learned the radial maze procedure rapidly and improved their performance throughout the successive training sessions. Radial maze performance in young rats was characterised by very few arm reentries. Aged rats were hypoactive and did not explore or enter the radial maze arms, and consequently performed poorly in the radial maze throughout the training sessions. Nicergoline treatment did not significantly modify locomotor activity in aged rats. Aged rats treated with nicergoline also performed poorly initially but improved with repeated training in the radial maze. This improvement was associated with an increasing number of arms being entered and very few arm reentries. Reduced acetylcholine (ACh) levels were also associated with age. Aged rats had significantly reduced levels of ACh in the striatum and cortex, but not the hippocampus as compared to young rats. Nicergoline treatment did not change ACh levels in young rats, but substantially restored the reduced ACh levels in aged rats. These results indicate that nicergoline is an effective cognitive enhancer in a learning model of agerelated deficits and that these results may be related to changes in the cholinergic system. Q 1997 Elsevier Science Inc.
[ 9,49 ] ) . Destruction of the ascending cholinergic projections from the septal area to the hippocampus or from the nucleus basalis of Meynert to the cortex ( e.g., [ 5,12 ] ) , produce severe cognitive impairments in animals that can be ameliorated by cholinergic treatments ( cf. [17,18 ] ) . In humans, memory impairment can be improved by pharmacological treatment that restores cholinergic activity ( e.g., [11] ) . In the clinic, moreover, the cholinesterase inhibitor, tetrahydroaminoacridine ( tacrine ) , has been shown to improve the memory impairments in AD patients [ 25,44 ] , and is approved for partial and transient relief of memory dysfunctions in the United States. This drug, however, has shown limited efficacy ( e.g., [15,42 ] ) and is associated with hepatotoxicity [ 47 ] . Nicergoline (1,6-dimethyl-8b-(Bromoisonicotinoyloxymethyl)10a-methoxyergoline) is an ergoline derivative with a variety of electrophysiological and biochemical effects on the CNS (cf [36]) such as improved cerebral blood flow, glucose uptake, and consumption [3,27]. These effects have been related to amelioration of memory deficits in different animal models including ischaemic gerbils and aged rats [27,41], or scopolamine-treated mice [26,46]. In humans, the effects of nicergoline have been related to improvements in vigilance and memory in the elderly [38], presenile subjects [51], and in hypoxia [39]. These changes in cerebral blood flow, glucose uptake, and consumption formed the basis for the initial use of nicergoline as a cerebral metabolic enhancer. Nicergoline has also been shown to affect the cholinergic system. Matsuoka and his colleagues [ 28 ] have shown that nicergoline inhibits acetylcholinesterase activity both in vitro and in vivo. The interaction with the cholinergic system was related to the potentiation of rat cortical cell excitation induced by ACh. Moreover, nicergoline reversed age-related declines of choline acetyltransferase activity and brain muscarinic receptor density [ 32 ] , as well as hippocampal ACh release [ 6 ] . These results suggested that cognitive enhancing properties of the compound is at least in part explained by modulation of cholinergic activity. To test this hypothesis, the aim of this investigation was to evaluate the effect of nicergoline on learning performance in young and aged rats in the radial arm maze and to assess concomitant biochemical changes in the cholinergic system.
KEY WORDS: Aging, Locomotor activity, Brain regions, Rat strains, Radial maze.
INTRODUCTION Primary degenerative dementia of Alzheimer’s type ( AD ) and multinfarct dementia account for a majority of the dementing processes of adulthood [13,19 ] . In both of these conditions, memory deficits are early and prominent symptoms that contribute substantially to the patient’s disability. Neuroanatomical and neurochemical correlates of the cognitive disorders implicate cholinergic systems in the brain. In particular, the basal forebrain-cortical and hippocampal projections have been subject of considerable study over the last decade ( cf 1
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Animals In two separate experiments two groups of young (38–40 days) and aged (20 months) Wistar rats (Iffa Credo) were used. The rats were kept in an animal house maintained at a constant temperature (207C) and relative humidity (60%), with a 12-h light–dark cycle (lights on 0600 to 1800 h). Drugs and Dosing Procedure Nicergoline (Pharmacia & Upjohn, SpA, Nerviano, MI, Italy) was dissolved in distilled water by grinding the compound together with an equimolar weight of tartaric acid crystals. Fresh solutions were prepared twice daily and administered orally. The nicergoline-treated animals received 5 mg/kg (free base) in a volume of 2 ml, while the vehicle-treated animals received 1.5 mg/kg of tartaric acid twice daily. This dose of nicergoline, route, and length of administration was chosen on the basis of previous studies by Moretti and colleagues [31] examining changes in neurotransmitters systems in young and aged rats. During weekends, the animals received a single oral administration of either nicergoline (10 mg/kg) or tartaric acid (3 mg/kg). Behavioural training and testing took place between 0900 to 1300 h. Animals from each experimental group were distributed across testing periods to control for diurnal variations. Dosing took place immediately after behavioural testing and between 1630 to 1700 h. Experiment 1: The Effects of Chronic Nicergoline Treatment on Locomotor Activity and Radial Maze Performance of Young Rats Two groups of young Wistar rats were treated twice daily either with tartaric acid vehicle (n Å 11) or with nicergoline (5.0 mg/kg; PO, n Å 13) for a total of 11 weeks. The locomotor activity of these rats was measured at weekly intervals of chronic dosing with nicergoline or vehicle in Digiscan activity monitors (Omnitech Electronics, Columbus, OH). After 4 weeks of chronic dosing with either nicergoline or vehicle, rats were gradually food deprived to 85% of their body weight for 1 week before being trained daily in an eight-arm radial maze for 6 weeks. Weekly locomotor activity measures were carried out immediately after radial maze training. The rats were allowed to feed freely for 4 h after behavioural testing. These rats were sacrificed immediately after the end of the radial maze training. Horizontal locomotor activity was measured in four observation cages (40 1 40 1 30 cm) equipped with infrared photoelectric cells. Each rat was placed into the observation cage and the frequency of crossing the infrared beam (horizontal activity) within 15 min was counted. The eight-arm radial maze consisted of an octagonal central platform (28 cm wide) with eight equally spaced arms. The arms were identical in dimensions (65 long 1 7 wide 1 9 cm deep). Extramaze visual cues were placed in the laboratory and their position was kept constant throughout the experiment. Each trial consisted of placing the rat in the central platform and allowing it to explore each of the arms freely until a total of eight arm entries within 10 min were made. Correct choices, i.e., entries into arms that had not been previously entered were rewarded with two 45 mg food pellets (Bio-Serv Dustless Precision Pellets, Bio-Serv, Frenchtown, NJ) placed in a food cup (4 cm i.d. 1 3 cm deep) at the end of each arm. Performance was assessed according to the number of correct choices out of eight within each 10-min session. Errors were scored as reentries into arms and/or missed opportunities to complete eight choices within the time allotted.
Experiment 2: The Effects of Chronic Nicergoline Treatment on Eight-Arm Radial Maze Performance and Open-Field Activity of Young and Aged Rats In a separate experiment, additional groups of eight young and aged rats were also treated twice daily with nicergoline (5 mg/kg; PO) or tartaric acid vehicle for a total of 12 weeks. As in Experiment 1, these rats were also gradually food deprived to 85% of their body weight for 1 week after 4 weeks of chronic dosing with either nicergoline or vehicle. They were then trained daily in an eight-arm radial maze for 6 weeks. Young, nicergoline-treated rats were not trained in the radial maze or examined behaviourally, as the results of the first experiment indicated that chronic nicergoline treatment had no effect either on locomotor activity or on radial maze performance in young rats. This group, however, was dosed, food deprived, and handled exactly as the other three groups for subsequent biochemical evaluation. Open-Field Activity Immediately after the 13th radial maze training trial, the locomotor activity of the three groups of rats was measured in a square Plexiglas box (55 1 55 1 60 cm) open field. The white floorboard was divided by black lines into 16 equal squares. The rat was allowed to explore freely for 3 min. The number of total squares crossed was scored. Biochemical Analysis At the end of radial arm training, the rats were allowed to feed freely for an additional week, during which twice daily dosing with nicergoline or tartaric acid vehicle was maintained. On the 12th week of chronic treatment, the rats were sacrificed by focused microwave irradiation (5 kW, 0.8 s). To determine the cerebral content of ACh, the brain was removed and the striatum, hippocampus, and cortex were dissected on ice-chilled glass plate and frozen on dry ice according to a modification of the method of Glowinski and Iversen [16]. Tissue samples were stored at 0807C until ACh determinations could be made, usually within 24 h of dissection. Tissue samples were homogenised by sonication in 20 mM sodium phosphate (pH 5.3). After centrifugation at 12,500 1 g for 10 min, 10 ml aliquots of supernatant were injected directly into the HPLC column. ACh levels were measured according to the method described by Potter et al. [35]. The HPLC system used consisted of a Waters pump (model 590), an automatic and refrigerated sample injector ‘‘WISP717’’ (Waters Association, Framinghan, MA), a BAS ACh/Ch analytical column kit (MF-8910) incorporating a prepacked enzyme reactor, an electrochemical detector with a working electrode of platinum at /500 mV (BAS, model LC-4B), and a Spectra Physics digital integrator recorder (San Jose, CA). The mobile phase was 20 mM sodium phosphate buffer (pH 8.5) that was pumped at a flow rate of 0.7 ml/min. Quantification of ACh was performed by comparison with a known amount of standard. Statistical Analysis All data are expressed as means ( { SEM). Data were analysed by analyses of variance (ANOVA) and the significance of the difference between means was evaluated according to various multiple comparison methods [24] (see figure captions for more detailed explanations of statistical analyses). RESULTS The Effects of Chronic Nicergoline Treatment on Locomotor Activity and Radial Maze Performance of Young Rats The locomotor activity of young Wistar rats during chronic administration of nicergoline did not change significantly with
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NICERGOLINE AND RADIAL MAZE PERFORMANCE
FIG. 1. The effects of chronic oral nicergoline administration (5.0 mg/ kg; bid) on horizontal locomotor activity (A) and radial arm maze learning (B) in young rats. Horizontal locomotor activity was measured by Digiscan for 15 min once weekly (mean counts per 15 min ( SEM) for 10 weeks of chronic dosing. Daily radial maze training began after 5 weeks of chronic dosing with nicergoline and continued for an additional 6 weeks. The radial maze training data has been expressed as the mean number of correct choices ( {SEM) made over 30 10-min sessions starting from sessions 1 to 30 and (mean number of correct choices { SEM) binned in blocks of five daily training sessions. Locomotor and radial maze data were analysed by two-way split-plot ANOVAs comparing treatment by time (weeks of administration or blocks of training trials).
respect to that of their vehicle-treated controls (Fig. 1A). The only significant result observed was that locomotor activity decreased over time, F (10, 220) Å 3.26, p õ 0.001. Chronic nicergoline treatment did not alter the rate at which young rats learned the radial maze procedure, as shown by the data in Fig. 1(B). Within the first block of five training trials both vehicleand nicergoline-treated rats were responding with five to six correct choices. This performance improved with repeated training, F(5, 110) Å 6.96, p õ 0.001. Nicergoline treatment did not affect radial maze performance, F (1, 22) Å 1.56, NS, nor did nicergoline treatment interact with repeated training, F (5, 110) Å 0.51, NS.
307 The data shown in Fig. 3 refers to the mean number of baited arms not entered during a 10-min training session in the radial maze, or reentered arms more than once. Young vehicle-treated rats missed very few opportunities to enter the baited arms (Fig. 3A). The errors committed by vehicle-treated aged rats on radial maze learning were due mainly to the aged rats remaining within the central hub of the maze and not entering the baited arms during the daily 10-min training sessions. Nicergoline-treated aged rats steadily reduced the number of baited arm not entered over the course of the 30 daily training sessions. This reduction in not entering baited arms over sessions is reflected by the significant interaction between treatment and session, F(58, 609) Å 3.23, p õ 0.001. As the aged vehicle-treated rats were not entering baited arms, this lack of exploration was reflected in the lack of arms being reentered (Fig. 3B). Indeed, over the course of the 30 training sessions, only two of the aged vehicle-treated rats reentered more than two arms within a session. Young vehicle-treated rats, very rarely reentered more than two arms within a training session, although during the initial sessions between one to four arms were reentered by this group of rats. During the initial training sessions, aged, nicergoline-treated rats were committing errors of omission in a manner similar to their vehicle-treated age controls, i.e., not entering baited arms. However, as the number of arms not entered by this group of rats declined by the 13th training session, the number of reentries increased. The number of reentries of nicergoline-treated aged rats did not exceed those made by young vehicle-treated rats. The changes in the number of reentries by young vehicle-treated and aged nicergoline-treated rats over sessions is reflected by the significant interaction between treatment and session, F (58, 609) Å 3.38, p õ 0.001. The Effects of Chronic Nicergoline Treatment on Open-Field Activity of Young and Aged Rats Vehicle-treated aged rats tended to remain within the central hub of the maze and failed to enter rather than reentering baited
The Effects of Chronic Nicergoline Treatment on Radial Maze Performance of Young and Aged Rats The rapid learning of young rats of the radial maze procedure shown in Fig. 1 was confirmed. Young vehicle-treated rats made between five and six correct choices within the first block of five training sessions, and reached a stable responding of seven correct choices by the 21st training session (Fig. 2). Vehicle-treated aged rats, in contrast, hardly made more than two to three correct responses. Indeed, the number of correct responses remained at 0 until the 22nd session, and then improved slightly. Overall, the difference between aged and young rats was highly significant, F(2, 21) Å 59.5, p õ 0.001. Nicergoline improved the poor radial maze performance by aged rats as indicated by the interaction between nicergoline treatment and training sessions, F (10, 105) Å 4.28, p õ 0.001. Although initiating from the same baseline level as their vehicletreated, age-matched controls, nicergoline-treated aged rats began rapidly to improve in this task over successive sessions.
FIG. 2. The effect of chronic oral nicergoline treatment on maze performance in young and aged rats. The radial maze training data has been expressed as the mean number of correct choices ( { SEM) made over 30 10-min sessions starting from sessions 1 to 30. The radial maze data were analysed by two-way split-plot ANOVAs comparing treatment by blocks of five training sessions. The significance of differences between means was evaluated by Tukey’s multiple comparison method where a p õ 0.05 (significantly different from young, vehicle-treated control performance during trials 1–5) and **p õ 0.01 (significantly different from young, vehicle-treated control performance during each training trial block).
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FIG. 3. The effect of chronic oral nicergoline treatment on the mean ( {SEM) number of arms not entered during daily 10-min training sessions (A) and number of arms reentered (B) made over 30 10-min sessions starting from sessions 1 to 30. The radial maze data were analysed by two-way split-plot ANOVAs.
arms within the 10-min session. The locomotor activity of the aged rats was measured in an open field to simulate the central hub of the radial maze. This assessment was carried out immediately after the 13th radial maze training session, during which a significant difference in maze performance was already noted between the two groups of aged rats. As expected, a significant age effect was observed, F (2, 21) Å 8.07, p õ 0.003. While young control animals crossed an average of 46.1 ( { 7.3) squares within 3 min, vehicle-treated aged rats crossed significantly fewer squares (16.8 { 3.0; Tukey’s q Å 5.58, p õ 0.01). Nicergoline-treated aged rats also crossed significantly less squares (27.0 { 4.6; Tukey’s q Å 3.64, p õ 0.05) than the vehicle-treated young controls, although more squares than their vehicle, aged controls. This difference in motility from vehicletreated aged controls was not different, however (Tukey’s q Å 1.94, NS).
The effects of chronic treatment with nicergoline on locomotor activity and radial arm maze performance in young and aged rats were examined and the results may be summarised as follows: (1) aged rats show an impairment in radial maze learning. This impaired performance improved in response to chronic nicergoline treatment. Impaired radial maze performance can be mainly accounted for by a decrease in errors due to not entering baited arms. (2) Aged rats were hypoactive on open-field locomotor activity measures. Nicergoline treatment was found not to alter locomotor activity significantly in either young or aged rats. (3) Brain ACh levels in the striatum and cortex, but not the hippocampus, of aged rats were significantly reduced when compared to ACh levels in these brain areas of young rats. Agerelated reductions in ACh levels were substantially restored by chronic nicergoline treatment. Furthermore, chronic nicergoline treatment did not alter brain ACh levels in young rats. Nicergoline improved radial arm maze performance of aged animals (Figs. 2 and 3). However, even though the results are clear, the interpretation of this result needs to consider the fact that vehicle-treated, age-matched controls were hypoactive through out the experiment. The question, therefore, arises whether nicergoline’s effects upon radial maze performance in the aged rat were due to enhancement of cognition, or indirectly to a stimulation of locomotion, or other factors such as motivation or attention. Being able to distinguish between the pharmacological effects of a putative cognitive enhancer upon cognition rather than on other factors in this context, therefore, becomes increasingly important. This distinction can be done both in terms of consistency of results in other models (cf. [34]), and through a process of exclusion of other factors (cf. [40]). Regarding the first point, nicergoline has been shown to have effective cognitive enhancing properties in a variety of different models of cognitive impairment such as ischaemia or aged rats [27,41] or pharmacologically induced deficits [26,46]. Age-related impairments on locomotion, motivation, or other sensory-motor variables are potentially confounding factors in studies of aging on cognitive processes (cf. [20], for a review). Gage and his colleagues [14], for example, noted that aged rats not only show impaired locomotor activity, but have impaired coordination or exploration as well. While it may appear self-
The Effects of Chronic Nicergoline Treatment on Acetylcholine Levels of the Striatum, Cortex, and Hippocampus of Young and Aged Rats Vehicle-treated aged rats had significantly reduced levels of striatal, F (1, 25) Å 13.97, p õ 0.001, and cortical, F(1, 22) Å 5.53, p õ 0.03, ACh levels (Fig. 4). Young, vehicle-treated rats had mean ACh levels of 1167.0 ( { 140.1) and 511.9 ( { 29.4) ng/mg protein in the striatum and cortex, respectively. In contrast, aged, vehicle-treated rats had 729.4 ( { 65.5) and 347.0 ( { 19.9) ng/mg protein. No substantial difference in hippocampal ACh levels was noted in aged, vehicle-treated rats as compared to young, vehicle-treated controls (421.6 { 16.0 and 351.6 { 22.7 ng/mg protein), however. Nicergoline did not change ACh levels in young rats, but substantially restored the reduced ACh levels in aged rats. Striatal and cortical ACh levels of nicergolinetreated aged rats were comparable to those of young, vehicletreated rats (1114.2 { 45.4 and 476.7 { 19.4).
FIG. 4. The effect of chronic oral nicergoline treatment on brain acetylcholine levels in young and aged Wistar rats (n Å 8 per group). The data are expressed as ng/mg of protein (mean { SEM) and were analysed by separate two-way ANOVAs for each brain area. The significance of differences between means were evaluated by Tukey’s method for multiple comparisons where a p õ 0.01 vs. young-vehicle treated rats; *p õ 0.05 vs. aged-vehicle treated rats and **p õ 0.01 vs. aged-vehicle treated rats.
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NICERGOLINE AND RADIAL MAZE PERFORMANCE evident that the lack of movement affects learning, however, the exact relationship of locomotor impairments on cognitive process can be dissociated. Willig and his colleagues [50] demonstrated that while aged rats have impaired locomotor activity and impaired radial arm maze performance, spontaneous alternation, and object discrimination, they are nonetheless unimpaired in delayed reinforced alternation. This may be explained in terms of differences in motivation and/or familiarity with an environment. Moretti and colleagues [31] had shown previously that chronic oral administration of 5.0 mg/kg of nicergoline for 6–7 weeks did not affect either noradrenaline or serotonin metabolism, while dopamine turnover was significantly enhanced in mesolimbic areas, but not striatum of rats. Furthermore, the enhanced mesolimbic dopamine turnover was greater in aged than young rats. These changes in dopamine metabolism by nicergoline could conceivably modify motivated behaviour (e.g., [2]) and/or locomotion. The results of the first experiment (Fig. 1A) demonstrated that nicergoline did not stimulate locomotor activity in young rats. Previous studies in our laboratory with aged Sprague–Dawley rats chronically treated with nicergoline (unpublished results) have indicated that aged rats show significantly reduced horizontal locomotor activity compared to young rats (1228 { 224 vs. 3085 { 378 horizontal activity counts within 15 min). Nine weeks of chronic nicergoline treatment did not affect locomotor activity in either aged (1568 { 194) or young (2242 { 374) rats. It would appear unlikely, therefore, that the improved radial maze performance shown by nicergoline-treated aged rats was due to direct stimulation of locomotor activity in these rats. Indeed, open-field locomotor activity in the rats trained in the radial maze measured after the 13th training session did not reveal any significant differences in locomotor activity between vehicle-treated and nicergolinetreated aged rats. On the other hand, clear differences in radial maze performance was evident between these two groups. Aged rats did not explore the maze, and consequently failed to learn that the end of each arm contained a food reward. Notwithstanding, the lack of ability of nicergoline to stimulate locomotor activity directly in rats, maze exploration could have been stimulated by changes in other behaviours such as attention. Clinical trials with nicergoline and studies with normal, healthy subjects indicate that nicergoline has pronounced effects upon attentional processing. EEG changes such as increases in slow wave delta/theta activity, and decreases in fast wave alpha/beta activity is characteristic in the elderly and are associated with impaired vigilance [37]. These authors [38] further observed that nicergoline dose dependently decreased delta and theta activity while alpha and beta activity was increased in healthy elderly subjects. These EEG changes were related to an improvement in sentence recall. Further studies in demented patients with dementia indicate that nicergoline acts upon attentional processing. Arrigo et al. [1], for example, observed in a double-blind, placebo-controlled crossover study, that patients with mild to moderate dementia showed that nicergoline not only decreased delta and increased beta activity, but improved mental alertness and a lessening of indifference to the environment, as measured by the Sandoz clinical assessment geriatric scale (SCAG). Zappoli and colleagues [51], on the other hand, also observed that nicergoline improved cognitive dysfunction and mood as measured by the SCAG rating scale as well as improved reaction time and event-related potential considered to reflect vigilance, attention, and concentration in patients with initial presenile idiopathic cognitive decline. It is possible, therefore, that nicergoline may have improved attentional processing in the aged rats and stimulated maze exploration.
309 The lack of exploration of the radial maze by aged rats may be considered as an inability to establish a long-term or reference memory of the procedure, i.e., learning that at the beginning of each training session all arms of the maze are baited with food, and that it has to enter an arm to obtain this reward. Even though this study was not designed to examine differential effects of nicergoline on reference (unvarying, procedure-specific information) or working (trial-specific, i.e., remembering which of the arms had been entered previously to avoid reentering an arm in which there is no longer any food) memory, the separation of errors due to a lack of entering the arms of the maze during training sessions (Fig. 3A), and those due to reentering arms (Fig. 3B) is, nevertheless, useful to understand the mechanisms by which nicergoline improved radial maze performance in aged rats. Nicergoline reduced the number of arms not entered during successive training sessions in aged rats thus increasing the probability of these rats to learn and establish a reference memory of the procedure. This a very clear effect and reflects the improved overall performance in the radial maze (Fig. 2). Working memory errors, i.e., the reentries into arms during the training session, on the other hand, declined steadily in young rats to reach an asymptote of one arm reentry by the 13th training session. Nicergoline-treated aged rats also reached this asymptote by the 13th training trial in spite of not entering two of eight baited arms per training session by at the end of the training procedure. These results suggest that nicergoline improved working memory aspects of the radial maze procedure as well. A regional-specific biochemical deficit in aged rats was indicated by significant decreases of ACh levels in the cortex and striatum, but not the hippocampus (Fig. 3). These effects of aging on cortical and striatal ACh levels agree with those shown by Meek and his colleagues [29] or Weiler [48]. The lack of changes in the hippocampus may be ascribed to possible strain differences (cf. [21]). These findings suggest the possibility of a substantial functional decline in cholinergic transmission with age, which may contribute to the impairment of radial maze performance. While nicergoline has only negligible affinity for muscarinic cholinergic binding sites [30], and chronic administration does not affect muscarinic binding site number [32], or basal ACh release [6] in young adult rats, a different profile is observed in aged rats. Chronic treatment with nicergoline is associated with restored ACh levels (present study), and basal ACh release in aged rats [6], as well as restoring choline acetyltransferase activity and muscarinic receptor density in the brain of aged rats [32]. These corresponding neurochemical changes in aged rats are consistent with other observations of concomitant changes in neurochemical indices of forebrain cholinergic neurone function and parameters of impaired learning and memory in aged rats [4,22,43]. The changes in both radial maze performance and ACh levels by nicergoline indicate that at least one of the mechanisms through which nicergoline improved performance is by modulation of the cholinergic system. However, recent studies have also indicated that nicergoline modulates transduction systems that could contribute to the improvements in radial maze performance observed in this study. Chronic administration of nicergoline increases cortical phosphoinositide turnover in young Wistar rats and potentiates noradrenaline- or carbachol-induced inositol triphosphate accumulation [7]. Di Luca and colleagues [10] have also shown that nicergoline enhances protein kinase C translocation to the membrane compartment in striatum and hippocampus. These changes in transduction systems are associated with synaptic plasticity, cognition, and are impaired in aged rats and patients with Alzheimer’s disease [8,23,33,45] and could be
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also be related to the behavioural changes in aged rats treated chronically with nicergoline in this study. In conclusion, chronic nicergoline treatment improved radial maze performance in aged rats and reversed age-associated changes in cortical and striatal ACh levels in these rats. The associated changes in learning and brain ACh levels suggests a cholinergic mechanism of action for nicergoline. However, nicergoline’s effects on transducer systems may also contribute to the overall changes in radial maze performance in aged rats. The improvement in radial maze performance was associated with enhanced maze exploration. It is unlikely that nicergoline directly stimulated locomotor activity in these rats, and that the changes in radial maze performance was secondary to increased activity. Nicergoline has been shown to improve vigilance and attention clinically. It is conceivable that the improved radial maze performance in aged rats by nicergoline could have been influenced by changes in attention in these rats. Finally, nicergoline’s ability to increase dopamine turnover in mesolimbic areas could alter motivated or incentive learning in these rats. It is clear that further studies would be required to determine the effects of nicergoline upon motivation and attention to characterise the cognitive-enhancing effect of nicergoline more fully. ACKNOWLEDGEMENTS
The authors wish to acknowledge the participation of all the members of the CNS preclinical research who contributed throughout this experiment, Dr. Maurizio Rocchetti for his excellent statistical advice, and to give special thanks to Professor Trevor Archer and Dr. Erik Wong for their comments and suggestions during the preparation of the manuscript. The authors affirm that these studies were carried out in accordance with the principles of the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health (NIH Publication No. 85-23, revised 1985).
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NICERGOLINE AND RADIAL MAZE PERFORMANCE 29. Meek, J. L.; Bertilsson, L.; Cheney, D. L.; Zsilla, G.; Costa, E. Aging-induced changes in acetylcholine and serotonin content of discrete brain nuclei. J. Gerontol. 2:129–131; 1977. 30. Moretti, A.; Carfagna, N.; Caccia, C.; Carpentieri, N.; Amico, A.; Marchi, G.; Trunzo, F. Neurochemical effects of ergoline derivatives. In: Heidrich, H., ed. Proof of therapeutical effectiveness of nootropic and vasoactive drugs. Berlin: Springer Verlag; 1986:103– 110. 31. Moretti, A.; Carfagna, N.; Caccia, C.; Carpentieri, M. Effect of ergolines on neurotransmitter systems in the rat brain. Arch. Int. Pharmacodyn. Ther. 294:33–45; 1988. 32. Ogawa, N.; Asanuma, M.; Hirata, H.; Konodo, Y.; Kawada, Y.; Mori, A. Cholinergic deficits in aged rat brain are corrected with nicergoline. Arch. Gerontol. Geriatr. 16:103–110; 1993. 33. Olds, J. L.; Anderson, M. L.; McPhie, D. L.; Staten, L. D.; Alkon, D. L. Imaging of memory-specific changes in the distribution of protein kinase C in the hippocampus. Science 245:866–869; 1989. 34. Porsolt, R.; Roux, S.; Lene`gre, A. Critical issues in cognition enhancing drug research. In: Palomo, T.; Archer, T.; Beninger, R., eds. Strategies for studying brain disorders, vol. 2. London: Farrand Press; 1994:285–299. 35. Potter, P. E.; Meek, J. L.; Neff, N. H. Acetylcholine and choline in neuronal tissue measured by HPLC with electrochemical detection. J. Neurochem. 41:188–194; 1983. 36. Rossi, A.; Battaglia, A.; Novellini, R.; Buonamici, M.; Carfagna, N.; Orlando, N.; Pamparana, F. Nicergoline: Pharmacological properties and clinical studies in aging brain. In: Aging brain and dementia: New trends in diagnosis and therapy. New York: Alan R. Liss, Inc; 1990:549–578. 37. Saletu, B.; Gru¨nberger, J. Memory dysfunction and vigilance: Neurophysiological and psychpharmacological aspects. Ann. NY Acad. Sci. 444:406–427; 1985. 38. Saletu, B.; Anderer, A.; Gru¨nberger, J. Topographic brain mapping of EEG after acute application of ergotalkaloids in the elderly. Arch. Gerontol. Geriatr. 11:1–22; 1990. 39. Saletu, B.; Gru¨nberger, J.; Lintzmayer, L.; Anderer, A. Brain protection of nicergoline against hypoxia: EEG brain mapping and psychometry. J. Neural. Transm. [P-D Sect]. 2:305–325; 1990.
311 40. Sarter, M.; Hagan, J.; Dudchenko, P. Behavioral screening for cognitive enhancers: From indiscriminate to valid testing: Part 1. Psychopharmacology (Berlin) 107:144–159; 1992. 41. Schindler, U.; Rush, D. K.; Fielding, S. Nootropic drugs: Animal models for studying effects on cognition. Drug Dev. Res. 4:567– 576; 1984. 42. Schneider, L. S. Clinical pharmacology of aminoacridines in Alzheimer’s disease. Neurology 43(Suppl. 4):S64–S79; 1993. 43. Strong, R.; Hicks, P.; Hsu, L.; Bartus, R. T.; Enna, S. J. Age-related alterations in the rodent brain cholinergic system and behavior. Neurobiol. Aging 1:59–63; 1980. 44. Summers, W. K.; Majovski, L. V.; Marsh, G. M.; Tachiki, T.; Kling, A. Oral tetrahydroaminoacridine in long-term treatment of senile dementia; Alzheimer type. N. Engl. J. Med. 315:1241–1245, 1986. 45. Sunayashiki–Kusuzaki, K.; Lester, D. S.; Schreurs, B. G.; Alkon, D. L. Associative learning potentiates protein kinase C activation in synaptosomes of the rabbit hippocampus. Proc. Natl. Acad. Sci. USA 90:4286–4289; 1993. 46. Voronina, T. A.; Nerobkova, L. N.; Garibova, T. L.; Dikova, M.; Nikolov, R.; Nikolova, M.; Markina, N. V. Effect of nicergoline on learning and memory. Methods Find. Exp. Clin. Pharmacol. 10:431– 435; 1988. 47. Watkins, P. B.; Zimmerman, H. J.; Knapp, M. J.; Gracon, S. I.; Lewis, K. W. Hepatotoxic effects of tacrine administration in patients with Alzheimer’s disease. JAMA 271:992–998; 1994. 48. Weiler, M. Acetylcholine release from striatal slices of young adult and aged Fisher 344 rats. Neurobiol. Aging 11:401–407; 1990. 49. Whitehouse, P. J.; Price, D. L.; Struble, R. G.; Clark, A. W.; Coyle, J. T.; Delong, M. R. Alzheimer’s disease and senile dementia: Loss of neurons in the basal forebrain. Science 215:1237–1239; 1982. 50. Willig, F.; Palacios, A.; Monmaur, P.; M’Harzi, M.; Laurent, J.; Delacour, J. Short-term memory, exploration and locomotor activity in aged rats. Neurobiol. Aging 8:393–402; 1987. 51. Zappoli, R.; Arnetoli, G.; Paganini, M.; Battaglia, A.; Novellini, R.; Pamparana, F. Topographic bit-mapped event-related neurocognitive potentials and clinical status in patients with primary presenile mental decline chronically treated with nicergoline. Curr. Ther. Res. 49:1078–1097; 1991.
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PII S0361-9230(97)00010-5
Effects of Nicergoline on Age-Related Decrements in Radial Maze Performance and Acetylcholine Levels ROBERT A. MCARTHUR,1 NICOLA CARFAGNA, LAURA BANFI, STEFANO CAVANUS, MARIA ANTONIETTA CERVINI, RUGGERO FARIELLO AND CLAES POST CNS Preclinical Research, Milan Pharmacia & Upjohn, SpA Viale Pasteur, 10 I-20014, Nerviano, (MI), Italy [Received 27 August 1996; Revised 11 December 1996; Accepted 12 December 1996]
ABSTRACT: The effects of chronic oral administration of nicergoline (5.0 mg/kg; bid) on locomotor activity, eight-arm radial maze performance plus striatal, cortical, and hippocampal acetylcholine (ACh) levels were examined in young and aged Wistar rats. Chronic nicergoline administration did not modify either the locomotor activity or radial maze learning in young rats. Young rats learned the radial maze procedure rapidly and improved their performance throughout the successive training sessions. Radial maze performance in young rats was characterised by very few arm reentries. Aged rats were hypoactive and did not explore or enter the radial maze arms, and consequently performed poorly in the radial maze throughout the training sessions. Nicergoline treatment did not significantly modify locomotor activity in aged rats. Aged rats treated with nicergoline also performed poorly initially but improved with repeated training in the radial maze. This improvement was associated with an increasing number of arms being entered and very few arm reentries. Reduced acetylcholine (ACh) levels were also associated with age. Aged rats had significantly reduced levels of ACh in the striatum and cortex, but not the hippocampus as compared to young rats. Nicergoline treatment did not change ACh levels in young rats, but substantially restored the reduced ACh levels in aged rats. These results indicate that nicergoline is an effective cognitive enhancer in a learning model of agerelated deficits and that these results may be related to changes in the cholinergic system. Q 1997 Elsevier Science Inc.
[ 9,49 ] ) . Destruction of the ascending cholinergic projections from the septal area to the hippocampus or from the nucleus basalis of Meynert to the cortex ( e.g., [ 5,12 ] ) , produce severe cognitive impairments in animals that can be ameliorated by cholinergic treatments ( cf. [17,18 ] ) . In humans, memory impairment can be improved by pharmacological treatment that restores cholinergic activity ( e.g., [11] ) . In the clinic, moreover, the cholinesterase inhibitor, tetrahydroaminoacridine ( tacrine ) , has been shown to improve the memory impairments in AD patients [ 25,44 ] , and is approved for partial and transient relief of memory dysfunctions in the United States. This drug, however, has shown limited efficacy ( e.g., [15,42 ] ) and is associated with hepatotoxicity [ 47 ] . Nicergoline (1,6-dimethyl-8b-(Bromoisonicotinoyloxymethyl)10a-methoxyergoline) is an ergoline derivative with a variety of electrophysiological and biochemical effects on the CNS (cf [36]) such as improved cerebral blood flow, glucose uptake, and consumption [3,27]. These effects have been related to amelioration of memory deficits in different animal models including ischaemic gerbils and aged rats [27,41], or scopolamine-treated mice [26,46]. In humans, the effects of nicergoline have been related to improvements in vigilance and memory in the elderly [38], presenile subjects [51], and in hypoxia [39]. These changes in cerebral blood flow, glucose uptake, and consumption formed the basis for the initial use of nicergoline as a cerebral metabolic enhancer. Nicergoline has also been shown to affect the cholinergic system. Matsuoka and his colleagues [ 28 ] have shown that nicergoline inhibits acetylcholinesterase activity both in vitro and in vivo. The interaction with the cholinergic system was related to the potentiation of rat cortical cell excitation induced by ACh. Moreover, nicergoline reversed age-related declines of choline acetyltransferase activity and brain muscarinic receptor density [ 32 ] , as well as hippocampal ACh release [ 6 ] . These results suggested that cognitive enhancing properties of the compound is at least in part explained by modulation of cholinergic activity. To test this hypothesis, the aim of this investigation was to evaluate the effect of nicergoline on learning performance in young and aged rats in the radial arm maze and to assess concomitant biochemical changes in the cholinergic system.
KEY WORDS: Aging, Locomotor activity, Brain regions, Rat strains, Radial maze.
INTRODUCTION Primary degenerative dementia of Alzheimer’s type ( AD ) and multinfarct dementia account for a majority of the dementing processes of adulthood [13,19 ] . In both of these conditions, memory deficits are early and prominent symptoms that contribute substantially to the patient’s disability. Neuroanatomical and neurochemical correlates of the cognitive disorders implicate cholinergic systems in the brain. In particular, the basal forebrain-cortical and hippocampal projections have been subject of considerable study over the last decade ( cf 1
To whom requests for reprints should be addressed.
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Animals In two separate experiments two groups of young (38–40 days) and aged (20 months) Wistar rats (Iffa Credo) were used. The rats were kept in an animal house maintained at a constant temperature (207C) and relative humidity (60%), with a 12-h light–dark cycle (lights on 0600 to 1800 h). Drugs and Dosing Procedure Nicergoline (Pharmacia & Upjohn, SpA, Nerviano, MI, Italy) was dissolved in distilled water by grinding the compound together with an equimolar weight of tartaric acid crystals. Fresh solutions were prepared twice daily and administered orally. The nicergoline-treated animals received 5 mg/kg (free base) in a volume of 2 ml, while the vehicle-treated animals received 1.5 mg/kg of tartaric acid twice daily. This dose of nicergoline, route, and length of administration was chosen on the basis of previous studies by Moretti and colleagues [31] examining changes in neurotransmitters systems in young and aged rats. During weekends, the animals received a single oral administration of either nicergoline (10 mg/kg) or tartaric acid (3 mg/kg). Behavioural training and testing took place between 0900 to 1300 h. Animals from each experimental group were distributed across testing periods to control for diurnal variations. Dosing took place immediately after behavioural testing and between 1630 to 1700 h. Experiment 1: The Effects of Chronic Nicergoline Treatment on Locomotor Activity and Radial Maze Performance of Young Rats Two groups of young Wistar rats were treated twice daily either with tartaric acid vehicle (n Å 11) or with nicergoline (5.0 mg/kg; PO, n Å 13) for a total of 11 weeks. The locomotor activity of these rats was measured at weekly intervals of chronic dosing with nicergoline or vehicle in Digiscan activity monitors (Omnitech Electronics, Columbus, OH). After 4 weeks of chronic dosing with either nicergoline or vehicle, rats were gradually food deprived to 85% of their body weight for 1 week before being trained daily in an eight-arm radial maze for 6 weeks. Weekly locomotor activity measures were carried out immediately after radial maze training. The rats were allowed to feed freely for 4 h after behavioural testing. These rats were sacrificed immediately after the end of the radial maze training. Horizontal locomotor activity was measured in four observation cages (40 1 40 1 30 cm) equipped with infrared photoelectric cells. Each rat was placed into the observation cage and the frequency of crossing the infrared beam (horizontal activity) within 15 min was counted. The eight-arm radial maze consisted of an octagonal central platform (28 cm wide) with eight equally spaced arms. The arms were identical in dimensions (65 long 1 7 wide 1 9 cm deep). Extramaze visual cues were placed in the laboratory and their position was kept constant throughout the experiment. Each trial consisted of placing the rat in the central platform and allowing it to explore each of the arms freely until a total of eight arm entries within 10 min were made. Correct choices, i.e., entries into arms that had not been previously entered were rewarded with two 45 mg food pellets (Bio-Serv Dustless Precision Pellets, Bio-Serv, Frenchtown, NJ) placed in a food cup (4 cm i.d. 1 3 cm deep) at the end of each arm. Performance was assessed according to the number of correct choices out of eight within each 10-min session. Errors were scored as reentries into arms and/or missed opportunities to complete eight choices within the time allotted.
Experiment 2: The Effects of Chronic Nicergoline Treatment on Eight-Arm Radial Maze Performance and Open-Field Activity of Young and Aged Rats In a separate experiment, additional groups of eight young and aged rats were also treated twice daily with nicergoline (5 mg/kg; PO) or tartaric acid vehicle for a total of 12 weeks. As in Experiment 1, these rats were also gradually food deprived to 85% of their body weight for 1 week after 4 weeks of chronic dosing with either nicergoline or vehicle. They were then trained daily in an eight-arm radial maze for 6 weeks. Young, nicergoline-treated rats were not trained in the radial maze or examined behaviourally, as the results of the first experiment indicated that chronic nicergoline treatment had no effect either on locomotor activity or on radial maze performance in young rats. This group, however, was dosed, food deprived, and handled exactly as the other three groups for subsequent biochemical evaluation. Open-Field Activity Immediately after the 13th radial maze training trial, the locomotor activity of the three groups of rats was measured in a square Plexiglas box (55 1 55 1 60 cm) open field. The white floorboard was divided by black lines into 16 equal squares. The rat was allowed to explore freely for 3 min. The number of total squares crossed was scored. Biochemical Analysis At the end of radial arm training, the rats were allowed to feed freely for an additional week, during which twice daily dosing with nicergoline or tartaric acid vehicle was maintained. On the 12th week of chronic treatment, the rats were sacrificed by focused microwave irradiation (5 kW, 0.8 s). To determine the cerebral content of ACh, the brain was removed and the striatum, hippocampus, and cortex were dissected on ice-chilled glass plate and frozen on dry ice according to a modification of the method of Glowinski and Iversen [16]. Tissue samples were stored at 0807C until ACh determinations could be made, usually within 24 h of dissection. Tissue samples were homogenised by sonication in 20 mM sodium phosphate (pH 5.3). After centrifugation at 12,500 1 g for 10 min, 10 ml aliquots of supernatant were injected directly into the HPLC column. ACh levels were measured according to the method described by Potter et al. [35]. The HPLC system used consisted of a Waters pump (model 590), an automatic and refrigerated sample injector ‘‘WISP717’’ (Waters Association, Framinghan, MA), a BAS ACh/Ch analytical column kit (MF-8910) incorporating a prepacked enzyme reactor, an electrochemical detector with a working electrode of platinum at /500 mV (BAS, model LC-4B), and a Spectra Physics digital integrator recorder (San Jose, CA). The mobile phase was 20 mM sodium phosphate buffer (pH 8.5) that was pumped at a flow rate of 0.7 ml/min. Quantification of ACh was performed by comparison with a known amount of standard. Statistical Analysis All data are expressed as means ( { SEM). Data were analysed by analyses of variance (ANOVA) and the significance of the difference between means was evaluated according to various multiple comparison methods [24] (see figure captions for more detailed explanations of statistical analyses). RESULTS The Effects of Chronic Nicergoline Treatment on Locomotor Activity and Radial Maze Performance of Young Rats The locomotor activity of young Wistar rats during chronic administration of nicergoline did not change significantly with
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FIG. 1. The effects of chronic oral nicergoline administration (5.0 mg/ kg; bid) on horizontal locomotor activity (A) and radial arm maze learning (B) in young rats. Horizontal locomotor activity was measured by Digiscan for 15 min once weekly (mean counts per 15 min ( SEM) for 10 weeks of chronic dosing. Daily radial maze training began after 5 weeks of chronic dosing with nicergoline and continued for an additional 6 weeks. The radial maze training data has been expressed as the mean number of correct choices ( {SEM) made over 30 10-min sessions starting from sessions 1 to 30 and (mean number of correct choices { SEM) binned in blocks of five daily training sessions. Locomotor and radial maze data were analysed by two-way split-plot ANOVAs comparing treatment by time (weeks of administration or blocks of training trials).
respect to that of their vehicle-treated controls (Fig. 1A). The only significant result observed was that locomotor activity decreased over time, F (10, 220) Å 3.26, p õ 0.001. Chronic nicergoline treatment did not alter the rate at which young rats learned the radial maze procedure, as shown by the data in Fig. 1(B). Within the first block of five training trials both vehicleand nicergoline-treated rats were responding with five to six correct choices. This performance improved with repeated training, F(5, 110) Å 6.96, p õ 0.001. Nicergoline treatment did not affect radial maze performance, F (1, 22) Å 1.56, NS, nor did nicergoline treatment interact with repeated training, F (5, 110) Å 0.51, NS.
307 The data shown in Fig. 3 refers to the mean number of baited arms not entered during a 10-min training session in the radial maze, or reentered arms more than once. Young vehicle-treated rats missed very few opportunities to enter the baited arms (Fig. 3A). The errors committed by vehicle-treated aged rats on radial maze learning were due mainly to the aged rats remaining within the central hub of the maze and not entering the baited arms during the daily 10-min training sessions. Nicergoline-treated aged rats steadily reduced the number of baited arm not entered over the course of the 30 daily training sessions. This reduction in not entering baited arms over sessions is reflected by the significant interaction between treatment and session, F(58, 609) Å 3.23, p õ 0.001. As the aged vehicle-treated rats were not entering baited arms, this lack of exploration was reflected in the lack of arms being reentered (Fig. 3B). Indeed, over the course of the 30 training sessions, only two of the aged vehicle-treated rats reentered more than two arms within a session. Young vehicle-treated rats, very rarely reentered more than two arms within a training session, although during the initial sessions between one to four arms were reentered by this group of rats. During the initial training sessions, aged, nicergoline-treated rats were committing errors of omission in a manner similar to their vehicle-treated age controls, i.e., not entering baited arms. However, as the number of arms not entered by this group of rats declined by the 13th training session, the number of reentries increased. The number of reentries of nicergoline-treated aged rats did not exceed those made by young vehicle-treated rats. The changes in the number of reentries by young vehicle-treated and aged nicergoline-treated rats over sessions is reflected by the significant interaction between treatment and session, F (58, 609) Å 3.38, p õ 0.001. The Effects of Chronic Nicergoline Treatment on Open-Field Activity of Young and Aged Rats Vehicle-treated aged rats tended to remain within the central hub of the maze and failed to enter rather than reentering baited
The Effects of Chronic Nicergoline Treatment on Radial Maze Performance of Young and Aged Rats The rapid learning of young rats of the radial maze procedure shown in Fig. 1 was confirmed. Young vehicle-treated rats made between five and six correct choices within the first block of five training sessions, and reached a stable responding of seven correct choices by the 21st training session (Fig. 2). Vehicle-treated aged rats, in contrast, hardly made more than two to three correct responses. Indeed, the number of correct responses remained at 0 until the 22nd session, and then improved slightly. Overall, the difference between aged and young rats was highly significant, F(2, 21) Å 59.5, p õ 0.001. Nicergoline improved the poor radial maze performance by aged rats as indicated by the interaction between nicergoline treatment and training sessions, F (10, 105) Å 4.28, p õ 0.001. Although initiating from the same baseline level as their vehicletreated, age-matched controls, nicergoline-treated aged rats began rapidly to improve in this task over successive sessions.
FIG. 2. The effect of chronic oral nicergoline treatment on maze performance in young and aged rats. The radial maze training data has been expressed as the mean number of correct choices ( { SEM) made over 30 10-min sessions starting from sessions 1 to 30. The radial maze data were analysed by two-way split-plot ANOVAs comparing treatment by blocks of five training sessions. The significance of differences between means was evaluated by Tukey’s multiple comparison method where a p õ 0.05 (significantly different from young, vehicle-treated control performance during trials 1–5) and **p õ 0.01 (significantly different from young, vehicle-treated control performance during each training trial block).
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FIG. 3. The effect of chronic oral nicergoline treatment on the mean ( {SEM) number of arms not entered during daily 10-min training sessions (A) and number of arms reentered (B) made over 30 10-min sessions starting from sessions 1 to 30. The radial maze data were analysed by two-way split-plot ANOVAs.
arms within the 10-min session. The locomotor activity of the aged rats was measured in an open field to simulate the central hub of the radial maze. This assessment was carried out immediately after the 13th radial maze training session, during which a significant difference in maze performance was already noted between the two groups of aged rats. As expected, a significant age effect was observed, F (2, 21) Å 8.07, p õ 0.003. While young control animals crossed an average of 46.1 ( { 7.3) squares within 3 min, vehicle-treated aged rats crossed significantly fewer squares (16.8 { 3.0; Tukey’s q Å 5.58, p õ 0.01). Nicergoline-treated aged rats also crossed significantly less squares (27.0 { 4.6; Tukey’s q Å 3.64, p õ 0.05) than the vehicle-treated young controls, although more squares than their vehicle, aged controls. This difference in motility from vehicletreated aged controls was not different, however (Tukey’s q Å 1.94, NS).
The effects of chronic treatment with nicergoline on locomotor activity and radial arm maze performance in young and aged rats were examined and the results may be summarised as follows: (1) aged rats show an impairment in radial maze learning. This impaired performance improved in response to chronic nicergoline treatment. Impaired radial maze performance can be mainly accounted for by a decrease in errors due to not entering baited arms. (2) Aged rats were hypoactive on open-field locomotor activity measures. Nicergoline treatment was found not to alter locomotor activity significantly in either young or aged rats. (3) Brain ACh levels in the striatum and cortex, but not the hippocampus, of aged rats were significantly reduced when compared to ACh levels in these brain areas of young rats. Agerelated reductions in ACh levels were substantially restored by chronic nicergoline treatment. Furthermore, chronic nicergoline treatment did not alter brain ACh levels in young rats. Nicergoline improved radial arm maze performance of aged animals (Figs. 2 and 3). However, even though the results are clear, the interpretation of this result needs to consider the fact that vehicle-treated, age-matched controls were hypoactive through out the experiment. The question, therefore, arises whether nicergoline’s effects upon radial maze performance in the aged rat were due to enhancement of cognition, or indirectly to a stimulation of locomotion, or other factors such as motivation or attention. Being able to distinguish between the pharmacological effects of a putative cognitive enhancer upon cognition rather than on other factors in this context, therefore, becomes increasingly important. This distinction can be done both in terms of consistency of results in other models (cf. [34]), and through a process of exclusion of other factors (cf. [40]). Regarding the first point, nicergoline has been shown to have effective cognitive enhancing properties in a variety of different models of cognitive impairment such as ischaemia or aged rats [27,41] or pharmacologically induced deficits [26,46]. Age-related impairments on locomotion, motivation, or other sensory-motor variables are potentially confounding factors in studies of aging on cognitive processes (cf. [20], for a review). Gage and his colleagues [14], for example, noted that aged rats not only show impaired locomotor activity, but have impaired coordination or exploration as well. While it may appear self-
The Effects of Chronic Nicergoline Treatment on Acetylcholine Levels of the Striatum, Cortex, and Hippocampus of Young and Aged Rats Vehicle-treated aged rats had significantly reduced levels of striatal, F (1, 25) Å 13.97, p õ 0.001, and cortical, F(1, 22) Å 5.53, p õ 0.03, ACh levels (Fig. 4). Young, vehicle-treated rats had mean ACh levels of 1167.0 ( { 140.1) and 511.9 ( { 29.4) ng/mg protein in the striatum and cortex, respectively. In contrast, aged, vehicle-treated rats had 729.4 ( { 65.5) and 347.0 ( { 19.9) ng/mg protein. No substantial difference in hippocampal ACh levels was noted in aged, vehicle-treated rats as compared to young, vehicle-treated controls (421.6 { 16.0 and 351.6 { 22.7 ng/mg protein), however. Nicergoline did not change ACh levels in young rats, but substantially restored the reduced ACh levels in aged rats. Striatal and cortical ACh levels of nicergolinetreated aged rats were comparable to those of young, vehicletreated rats (1114.2 { 45.4 and 476.7 { 19.4).
FIG. 4. The effect of chronic oral nicergoline treatment on brain acetylcholine levels in young and aged Wistar rats (n Å 8 per group). The data are expressed as ng/mg of protein (mean { SEM) and were analysed by separate two-way ANOVAs for each brain area. The significance of differences between means were evaluated by Tukey’s method for multiple comparisons where a p õ 0.01 vs. young-vehicle treated rats; *p õ 0.05 vs. aged-vehicle treated rats and **p õ 0.01 vs. aged-vehicle treated rats.
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NICERGOLINE AND RADIAL MAZE PERFORMANCE evident that the lack of movement affects learning, however, the exact relationship of locomotor impairments on cognitive process can be dissociated. Willig and his colleagues [50] demonstrated that while aged rats have impaired locomotor activity and impaired radial arm maze performance, spontaneous alternation, and object discrimination, they are nonetheless unimpaired in delayed reinforced alternation. This may be explained in terms of differences in motivation and/or familiarity with an environment. Moretti and colleagues [31] had shown previously that chronic oral administration of 5.0 mg/kg of nicergoline for 6–7 weeks did not affect either noradrenaline or serotonin metabolism, while dopamine turnover was significantly enhanced in mesolimbic areas, but not striatum of rats. Furthermore, the enhanced mesolimbic dopamine turnover was greater in aged than young rats. These changes in dopamine metabolism by nicergoline could conceivably modify motivated behaviour (e.g., [2]) and/or locomotion. The results of the first experiment (Fig. 1A) demonstrated that nicergoline did not stimulate locomotor activity in young rats. Previous studies in our laboratory with aged Sprague–Dawley rats chronically treated with nicergoline (unpublished results) have indicated that aged rats show significantly reduced horizontal locomotor activity compared to young rats (1228 { 224 vs. 3085 { 378 horizontal activity counts within 15 min). Nine weeks of chronic nicergoline treatment did not affect locomotor activity in either aged (1568 { 194) or young (2242 { 374) rats. It would appear unlikely, therefore, that the improved radial maze performance shown by nicergoline-treated aged rats was due to direct stimulation of locomotor activity in these rats. Indeed, open-field locomotor activity in the rats trained in the radial maze measured after the 13th training session did not reveal any significant differences in locomotor activity between vehicle-treated and nicergolinetreated aged rats. On the other hand, clear differences in radial maze performance was evident between these two groups. Aged rats did not explore the maze, and consequently failed to learn that the end of each arm contained a food reward. Notwithstanding, the lack of ability of nicergoline to stimulate locomotor activity directly in rats, maze exploration could have been stimulated by changes in other behaviours such as attention. Clinical trials with nicergoline and studies with normal, healthy subjects indicate that nicergoline has pronounced effects upon attentional processing. EEG changes such as increases in slow wave delta/theta activity, and decreases in fast wave alpha/beta activity is characteristic in the elderly and are associated with impaired vigilance [37]. These authors [38] further observed that nicergoline dose dependently decreased delta and theta activity while alpha and beta activity was increased in healthy elderly subjects. These EEG changes were related to an improvement in sentence recall. Further studies in demented patients with dementia indicate that nicergoline acts upon attentional processing. Arrigo et al. [1], for example, observed in a double-blind, placebo-controlled crossover study, that patients with mild to moderate dementia showed that nicergoline not only decreased delta and increased beta activity, but improved mental alertness and a lessening of indifference to the environment, as measured by the Sandoz clinical assessment geriatric scale (SCAG). Zappoli and colleagues [51], on the other hand, also observed that nicergoline improved cognitive dysfunction and mood as measured by the SCAG rating scale as well as improved reaction time and event-related potential considered to reflect vigilance, attention, and concentration in patients with initial presenile idiopathic cognitive decline. It is possible, therefore, that nicergoline may have improved attentional processing in the aged rats and stimulated maze exploration.
309 The lack of exploration of the radial maze by aged rats may be considered as an inability to establish a long-term or reference memory of the procedure, i.e., learning that at the beginning of each training session all arms of the maze are baited with food, and that it has to enter an arm to obtain this reward. Even though this study was not designed to examine differential effects of nicergoline on reference (unvarying, procedure-specific information) or working (trial-specific, i.e., remembering which of the arms had been entered previously to avoid reentering an arm in which there is no longer any food) memory, the separation of errors due to a lack of entering the arms of the maze during training sessions (Fig. 3A), and those due to reentering arms (Fig. 3B) is, nevertheless, useful to understand the mechanisms by which nicergoline improved radial maze performance in aged rats. Nicergoline reduced the number of arms not entered during successive training sessions in aged rats thus increasing the probability of these rats to learn and establish a reference memory of the procedure. This a very clear effect and reflects the improved overall performance in the radial maze (Fig. 2). Working memory errors, i.e., the reentries into arms during the training session, on the other hand, declined steadily in young rats to reach an asymptote of one arm reentry by the 13th training session. Nicergoline-treated aged rats also reached this asymptote by the 13th training trial in spite of not entering two of eight baited arms per training session by at the end of the training procedure. These results suggest that nicergoline improved working memory aspects of the radial maze procedure as well. A regional-specific biochemical deficit in aged rats was indicated by significant decreases of ACh levels in the cortex and striatum, but not the hippocampus (Fig. 3). These effects of aging on cortical and striatal ACh levels agree with those shown by Meek and his colleagues [29] or Weiler [48]. The lack of changes in the hippocampus may be ascribed to possible strain differences (cf. [21]). These findings suggest the possibility of a substantial functional decline in cholinergic transmission with age, which may contribute to the impairment of radial maze performance. While nicergoline has only negligible affinity for muscarinic cholinergic binding sites [30], and chronic administration does not affect muscarinic binding site number [32], or basal ACh release [6] in young adult rats, a different profile is observed in aged rats. Chronic treatment with nicergoline is associated with restored ACh levels (present study), and basal ACh release in aged rats [6], as well as restoring choline acetyltransferase activity and muscarinic receptor density in the brain of aged rats [32]. These corresponding neurochemical changes in aged rats are consistent with other observations of concomitant changes in neurochemical indices of forebrain cholinergic neurone function and parameters of impaired learning and memory in aged rats [4,22,43]. The changes in both radial maze performance and ACh levels by nicergoline indicate that at least one of the mechanisms through which nicergoline improved performance is by modulation of the cholinergic system. However, recent studies have also indicated that nicergoline modulates transduction systems that could contribute to the improvements in radial maze performance observed in this study. Chronic administration of nicergoline increases cortical phosphoinositide turnover in young Wistar rats and potentiates noradrenaline- or carbachol-induced inositol triphosphate accumulation [7]. Di Luca and colleagues [10] have also shown that nicergoline enhances protein kinase C translocation to the membrane compartment in striatum and hippocampus. These changes in transduction systems are associated with synaptic plasticity, cognition, and are impaired in aged rats and patients with Alzheimer’s disease [8,23,33,45] and could be
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also be related to the behavioural changes in aged rats treated chronically with nicergoline in this study. In conclusion, chronic nicergoline treatment improved radial maze performance in aged rats and reversed age-associated changes in cortical and striatal ACh levels in these rats. The associated changes in learning and brain ACh levels suggests a cholinergic mechanism of action for nicergoline. However, nicergoline’s effects on transducer systems may also contribute to the overall changes in radial maze performance in aged rats. The improvement in radial maze performance was associated with enhanced maze exploration. It is unlikely that nicergoline directly stimulated locomotor activity in these rats, and that the changes in radial maze performance was secondary to increased activity. Nicergoline has been shown to improve vigilance and attention clinically. It is conceivable that the improved radial maze performance in aged rats by nicergoline could have been influenced by changes in attention in these rats. Finally, nicergoline’s ability to increase dopamine turnover in mesolimbic areas could alter motivated or incentive learning in these rats. It is clear that further studies would be required to determine the effects of nicergoline upon motivation and attention to characterise the cognitive-enhancing effect of nicergoline more fully. ACKNOWLEDGEMENTS
The authors wish to acknowledge the participation of all the members of the CNS preclinical research who contributed throughout this experiment, Dr. Maurizio Rocchetti for his excellent statistical advice, and to give special thanks to Professor Trevor Archer and Dr. Erik Wong for their comments and suggestions during the preparation of the manuscript. The authors affirm that these studies were carried out in accordance with the principles of the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health (NIH Publication No. 85-23, revised 1985).
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