Effects of chronic intraventricular infusion of heparin glycosaminoglycan on learning and brain acetylcholine parameters in aged rats

Behavioural Brain Research 147 (2003) 115–123

Research report

Effects of chronic intraventricular infusion of heparin glycosaminoglycan on learning and brain acetylcholine parameters in aged rats Karel Jezek a , Daniela Schulz a , Maria Angelica De Souza Silva a , Hans-Werner Müller b,c , Joseph P. Huston a,c , Rüdiger U. Hasenöhrl d,∗ a

Institute of Physiological Psychology, University of Düsseldorf, 40225 Düsseldorf, Germany b Department of Neurology, University of Düsseldorf, 40225 Düsseldorf, Germany c Center for Biological and Medical Research, University of Düsseldorf, 40225 Düsseldorf, Germany d Department of Psychology, University of Hertfordshire, College Lane, Hatfield, Herts AL10 9AB, UK Received 12 April 2003; received in revised form 14 April 2003; accepted 14 April 2003

Abstract We reported previously that the glycosaminoglycan heparin (HP) has the facility to improve learning in adult rodents when administered into the nucleus basalis of the ventral pallidum. Here we gauged the effects of chronic intraventricular infusion of HP (20 ng per day over 28 days) in 26-month-old rats in terms of Morris water maze performance, habituation to a novel open field, retention of a step-through inhibitory avoidance task and changes in forebrain acetylcholine (ACh) levels. Control groups included vehicle-infused old and adult (3-month-old) rats. The chronic infusion of HP did not significantly influence the performance of the old animals in any of the learning and memory tasks employed. HP only slightly facilitated the retention of the inhibitory avoidance task and the rate of habituation in the open-field paradigm. In the water maze, the glycosaminoglycan did not counteract the navigation deficits observed for aged controls and even impaired performance during the initial place-learning trials. After behavioural testing, tissue levels of ACh were determined in frontal cortex, ventral striatum, neostriatum and hippocampus without detecting any obvious neurochemical differences between groups. The current results, together with our previous work, indicate that HP differentially affects learning and memory parameters in adult and aged rats. Thus, whereas the glycosaminoglycan proved effective in facilitating mnemonic functions in normal adult animals, no such a clear-cut beneficial effect was observed in behaviourally impaired old rats. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Ageing; Memory; Extracellular matrix; Proteoglycan; Heparan sulphate; Polysaccharides

1. Introduction Deficits in associative functions seen with senescence may reflect atrophy, neuronal loss, reduced number/efficacy of synapses and a decrease in transmitter release but also other, more subtle, structural changes of the brain [34]. These include astrogliosis and changes in the macromolecular composition of the extracellular matrix [33,37]. Neurons are typically surrounded by perineuronal nets comprised of glycosaminoglycans (GAGs) and other glycoproteins [3]. GAGs are a family of structurally distinct polysaccharide macromolecules. The major GAGs in the mammalian nervous system are chondroitin sulphate and heparan sulphate (HS), which are located in the extracellular space (ECS) and ∗ Corresponding author. Tel.: +44-1707-28-4618; fax: +44-1707-28-5073. E-mail address: [email protected] (R.U. Hasenöhrl).

on the surface of cells [30]. GAGs have been implicated in several forms of synaptic plasticity [2] and might even be operative in neural mechanisms related to learning and memory formation. We recently reported that chondroitin sulphate as well as biglycan, a protein-bound form of the GAG [20], can facilitate learning in adult rats when administered into the ventral pallidum [13,16]. This basal forebrain structure is known for its high concentration of cholinergic cells and is affected seriously by ageing and dementia [26]. Injected into the same area of the brain, both chondroitin sulphate as well as biglycan led to a long-lasting ventral pallidal-cortical cholinergic activation suggesting that the GAGs can modify the ‘functional state’ of basal forebrain cells [16]. A direct link between GAGs and functional brain ageing was provided recently by showing that the degree of impaired maze performance of old rats correlates, for one, with the reduction of hippocampal chondroitin sulphate and, secondly,

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with the decrease in ECS volume and the loss of diffusion coefficients for neuroactive substances in hippocampus and cortex [38]. Thus, cognitive deficits during ageing may be related to a decreased availability of GAGs, and, as a consequence, to dysfunctional changes of the extracellular matrix [36,37]. In line with this premise, the chronic intraventricular infusion of chondroitin sulphate and biglycan improved the maze performance of memory-impaired old rats [16]. HS and closely-related heparin (HP) comprise the other major group of GAGs in the brain. HP is more highly charged than HS, by virtue of its higher sulphation. Several physiological effects have been ascribed to HP, most of which are independent from its first-described and best-characterised activity as an anticoagulant. Heparinoids can function as co-receptors for growth factors and cytokines [39], play a role in cell adhesion/survival [9,21] and even can exert protective effects against excitotoxin- and amyloid ␤-induced neurotoxicity [7,19]. There are few studies implicating HP in mnestic functions. The chronic oral administration of the sulphomucopolysaccharide ateroid reduced learning deficits in old rats and reversed age-related disturbances in nucleus accumbens dopamine metabolism [24]; however, since ateroid is a preparation composed of various GAGs, it is not clear whether the observed beneficial effects were actually caused by HP. The repeated intraperitoneal injection of HP improved the performance of adult rats in complex maze learning tasks and increased brain monoamine levels; the behavioural and neurochemical effects were observed after drug treatment had been terminated, suggesting that HP can have long-lasting beneficial effects on brain function [22]. We recently found that HP is equipotent to chondroitin sulphate in facilitating inhibitory avoidance retention in adult rats after injection into the ventral pallidum; furthermore, similar to chondroitin sulphate, HP increased frontal cortical acetylcholine (ACh) strengthening the presumptive relationship between the promnestic effects of ventral pallidal GAG injection and cholinergic activity [6]. In reference to these findings, we asked in the present study whether HP could also counteract age-related performance deficits. Thus, we gauged the effect of chronic intraventricular infusion of HP on the performance of aged rats in a set of learning tasks, which differed in terms of complexity and reward contingencies (habituation, inhibitory avoidance, Morris water maze). Furthermore, in order to assess possible effects of HP on central cholinergic markers in these animals, we performed a postmortem analysis of ACh in a number of forebrain regions after termination of the behavioural tests.

2. Materials and methods 2.1. Animals The experiment was carried out in accordance with the German Law on the Protection of Animals and was approved by the state authority. Male Wistar rats 3 (250–280 g) and

26 (470–640 g) months of age (TVA; University of Düsseldorf) were kept under standard laboratory conditions with free access to food and water. A 12:12 h light:dark cycle was imposed (lights-on at 7:00 a.m.) and all behavioural experiments were performed during the lights-on period except for the open-field tests, which were carried out during the dark phase. 2.2. Surgery and heparin infusion Under anaesthesia (ketamine 100 mg, xylazine 20 mg/kg; i.p.), adult and aged rats were implanted unilaterally with a cannula into the lateral ventricle (see [14] for details). The cannula was connected to an Alzet model 2004 osmotic minipump placed subcutaneous in the dorsal neck area. In one group of old rats (n = 12), the pump was filled with PBS containing HP from porcine intestinal mucosa (Sigma, Germany). Intraventricular infusion was performed over 28 days with an estimated dose of 20 ng HP per day; control groups included old (n = 8) and adult rats (n = 5), which were implanted with pumps filled with PBS only. 2.3. Behavioural testing procedures 2.3.1. Water maze The water maze apparatus and the experimental design have been described in detail elsewhere [14,16]. After 1 week of chronic HP or PBS infusion, adult and aged animals were habituated to the maze by placing each subject into the apparatus for 90 s with no opportunity to escape. Commencing on the following day the rats were tested in the hidden-platform version of the maze (18 cm in diameter black escape platform fixed in the centre of one quadrant of the maze 2.0 cm below water level) on nine consecutive days (four trials per day). On day 10 (extinction trial), the hidden platform was removed and the animals were tested as described for the habituation trial. In the visible-platform version of the maze performed on day 11 the rats were tested as described for the hidden-platform task, with the exception that the escape platform was made visible by a contrast cue (25 cm high, white with black stripes) and placed in the centre of the quadrant opposite to its original location (four trials). After a delay of 10 days, the animals were re-exposed to the maze to assess long-term retention of the hidden-platform task (four trials). Behavioural parameters (time to platform; swimming speed) were tracked and analysed with EthoVision (Noldus, The Netherlands). 2.3.2. Open field Behavioural tests were performed during the rat’s night active period between 8:00 p.m. and 1:00 a.m. The apparatus used for habituation learning was a gray PVC open field (60 cm × 60 cm × 38 cm) with a smooth gray floor. On two consecutive days (day 1, baseline; day 2, test), exploration in the open field was measured for 10 min (see [11] for details). The following types of behaviour were recorded:

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locomotion (forward and backward movements using all limbs), rearing (standing on the hind legs with the forelegs in the air or against the wall), exploration without locomotion (sitting with movements of forepaws and/or head), grooming (licking, scratching, biting of the body surface) and quiet (motionless). Habituation was defined as a decrease in exploratory activity from baseline to test. To determine the rate of habituation the time spent during locomotion and exploration without locomotion as well as the number of rearings were each expressed as percentages of the corresponding baseline behaviour on day 1. Furthermore, the sojourn time in the peripheral (a sector of 10 cm along the walls) and in the central zone (50 cm × 50 cm) of the open field was measured to assess possible anxioactive properties of the treatment.

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2.5. Data analysis Behavioural and neurochemical data were analysed between-groups with the Mann–Whitney U-test and within-groups with the Wilcoxon test for matched samples. For the hidden-platform trials in the water maze the curve level (a0) as an index for the average performance and the linear trend component (a1) as an estimate for the acquisition rate were determined for each animal and evaluated for between-group differences with the Mann–Whitney U-test [12,23]. Exact P-values were used as a measure of effect.

3. Results 3.1. Water maze

2.3.3. Step-through inhibitory avoidance The step-through avoidance apparatus was an oblong PVC box (40 cm × 22 cm × 22 cm), which had a door separating a well-lit white start-compartment from a dimly-lit black shock-compartment. The shock-compartment contained an electrifiable grid floor through which a foot shock could be delivered. Each rat had to undergo three baseline trials in the step-through paradigm at 30 min intervals. During each trial, the animal was placed into the start-compartment with its back toward the door and 5 s later, the guillotine door was raised. After the animal had entered the shock-compartment, the door was closed and 5 s later the rat was returned to its home cage. Immediately after entering the black compartment during the third baseline (learning) trial, the rats received an inescapable foot-shock (0.7 mA/1 s). Retention of the step-through response was measured twice: at 1.5 h after training, in order to assess short-term memory (STM), and at 24 h after training, in order to assess long-term memory (LTM) of the task (see [18] for methodological details). In the test sessions, no foot shock was given and the step-through latency was cut off at 300 s. 2.4. Neurochemical analysis After behavioural testing, the rats were anaesthetised with sodium pentobarbital (60 mg/kg; i.p.) and decapitated [32]. Their brains were quickly removed and the following forebrain areas were dissected out bilaterally on ice: frontal cortex (cortical tissue anterior to the genus of the corpus callosum), ventral striatum (nucleus accumbens, olfactory tubercle, anterior parts of ventral pallidum), neostriatum (anterior parts of caudate-putamen with globus pallidus as posterior border), and hippocampus (anterior parts of the hippocampal formation with CA1, CA3 and dentate gyrus). The samples were weighed, homogenised in 0.05 M HClO4 with an ultrasonic homogeniser, centrifuged, filtered and kept at −70 ◦ C. The samples were analysed for ACh with HPLC-EC according to the method described by Damsma and Westerink [5], except for the internal standard, which was adopted from Potter et al. [28].

As depicted in Fig. 1A, vehicle-infused old animals showed an impairment in learning to navigate the maze compared with adult controls, as indicated by increased time to find the hidden platform during training (a0; P = 0.003) and retention trials (a0; P = 0.008). Old rats with chronic HP infusion required more time to find the hidden platform in the course of training than PBS-infused old animals (a0; P = 0.074); they did not differ from aged controls with regard to the slope of their learning curves (a1; P = 0.104). Furthermore, the two groups of old rats did not differ in long-term retention of the hidden-platform task (P = 0.529). During the extinction trial on day 10, vehicle-infused aged controls spent less time in the quadrant of the maze that had previously contained the escape platform (i.e. platform quadrant) compared with vehicle-injected adult controls (P = 0.013; Fig. 1B); the chronic HP infusion did not influence the quadrant time (P = 0.269). In the visible-platform task (Fig. 1A), the time it took the old vehicle-infused animals to escape onto the platform was significantly increased compared with adult controls (P = 0.003); the escape scores of the two aged groups were comparable (P = 0.529). The groups of adult and old rats did not differ from each other in swim speed registered during training (range: 23.52 ± 1.80 cm/s to 25.94 ± 0.98 cm/s), extinction (range: 23.66 ± 2.47 cm/s to 26.24 ± 1.02 cm/s), cued (range: 21.24 ± 2.22 cm/s to 23.72 ± 1.56 cm/s), and long-term retention trials (range: 22.39 ± 1.93 cm/s to 25.46 ± 1.60 cm/s; corresponding P-values >0.10). 3.2. Open-field behaviour During the first exposure to the novel open field (baseline), vehicle-infused old animals showed less locomotion (P = 0.003), rearing (P = 0.003), exploration without locomotion (P = 0.079) and more quiescence than adult controls (P = 0.003), while the two groups did not differ in the amount of grooming (P = 0.884). Furthermore, aged PBS-infused rats spent less time in the central zone

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Fig. 1. Maze performance of 26-month-old rats infused chronically with heparin (HPold). (A) Mean (+S.E.M.) time to find the platform during training and retention of the hidden-platform task and to escape onto the visible platform on cued trials. (B) Mean (+S.E.M.) percentage of time spent in the platform quadrant during the 90-s spatial probe ‘extinction’ trial. Control groups included vehicle-infused adult (PBSadult) and old (PBSold) rats.

of the open field than adult controls (P = 0.003), suggestive of increased fear/anxiety. The chronic infusion of HP did not influence the behavioural pattern of the old animals (P-values >0.10; data not shown). The effects of continuous HP infusion on between-session habituation are shown in Fig. 2. When comparing behaviour on day 2 to that on day 1, it was found that adult vehicle controls displayed a decrease in locomotion (P = 0.043) and number of rearing (P = 0.043), whereas the amount of exploration without locomotion did not change across trials (P = 0.500). HP-infused old rats but not aged vehicle-controls showed reduced exploration without locomotion (HPold, P = 0.050; PBSold, P = 0.674), while in both groups the amount of locomotion did not change across trials (HPold, P = 0.484; PBSold, P = 0.484). Furthermore, both groups of old rats

had less rearing during the test trial as compared to baseline (HPold, P = 0.034; PBSold, P = 0.035). However, comparing the different behaviour parameters in HP-treated animals with that of old vehicle controls during the test trial did not yield any differences (locomotion: HPold versus PBSold, P = 0.674; exploration without locomotion: HPold versus PBSold, P = 0.753; rearing: HPold versus PBSold, P = 0.833). 3.3. Step-through avoidance During the learning trial, PBS- and HP-infused old rats did not differ from each other in the latency to enter the black compartment (P = 0.268), while latency scores of both aged groups were increased relative to adult controls

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Fig. 2. Rate of habituation to a novel open field for 26-month-old rats infused chronically with heparin (HPold). The different parameters of exploratory activity during the second exposure to the open field are expressed as mean (−S.E.M.) percentage of corresponding baseline values (=100%). Control groups included vehicle-infused adult (PBSadult) and old (PBSold) rats. Baseline scores: Locomotion: PBSadult, 222.54 ± 29.94; PBSold, 61.10 ± 11.89; HPold, 66.15 ± 10.19. Exploration: PBSadult, 311.74 ± 31.87; PBSold, 231.29 ± 34.49; HPold, 242.24 ± 21.80. Rearing: PBSadult, 71.00 ± 7.67; PBSold, 16.25 ± 3.11; HPold, 17.75 ± 2.58.

(median ± 25/75 percentiles): PBSadult, 2.0 s (2.0/7.0); PBSold, 10.0 s (6.8/16.5), P = 0.015; HPold, 14.0 s (9.0/17.8), P = 0.008. Comparison between training and retention latencies revealed that all groups of animals had learned the task (both STM and LTM sessions: PBSadult, P = 0.043; PBSold, P = 0.025; HPold, P = 0.012). Furthermore, all treatment groups had similar step-through latencies during LTM as compared with STM trials (PBSadult,

P = 0.715; PBSold, P = 1.00; HPold, P = 0.893). For evaluation of between-group differences in the retention performance, step-through latencies revealed during STM and LTM sessions were adjusted to corresponding baseline values by subtracting the baseline avoidance scores from the respective STM and LTM latencies. As depicted in Fig. 3, the vehicle-infused old animals did not differ from adult controls in step-through latencies during the STM and LTM trials

Fig. 3. Inhibitory avoidance performance of 26-month-old rats infused chronically with heparin (HPold). Ordinate: median (±25/75 percentiles) test session step-through latencies in seconds. During the learning trial, the rats received a foot-shock contingent on the step-through response. The animals were tested for retention twice: at 1.5 h (STM) and 24 h after the learning trial (LTM). Control groups included vehicle-infused adult (PBSadult) and aged (PBSold) rats.

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Table 1 Mean (±S.E.M.) concentration (pmol/mg) of acetylcholine in respective forebrain areas of aged rats infused chronically with heparin (HPold)

HPold PBSold PBSadult

FC

VS

NS

HC

11.96 ± 0.77 11.85 ± 0.81 14.72 ± 2.03

26.21 ± 3.44 31.52 ± 3.29 50.11 ± 14.95

27.02 ± 3.03 22.67 ± 1.64 31.40 ± 4.39

20.24 ± 1.95 17.90 ± 1.57 24.75 ± 6.02

Control groups included vehicle-infused adult (PBSadult) and aged (PBSold) animals; FC: frontal cortex, VS: ventral striatum, NS: neostriatum, HC: hippocampus.

(both P-values = 0.464). The chronic infusion of HP facilitated learning of the step-through avoidance task in the old animals, as reflected by increased latencies to step-through during both the STM (HPold versus PBSold, P = 0.074) and the LTM (HPold versus PBSold, P = 0.059) sessions. 3.4. Neurochemistry As shown in Table 1, the groups of vehicle- and HP-infused aged rats did not differ from each other in cortical (P = 1.00), ventral- and neostriatal (each P = 0.317) as well as hippocampal ACh levels (P = 0.352); their respective ACh concentrations were somewhat decreased relative to adult controls but the differences were marginal with P-values ranging from 0.257 to 0.571.

4. Discussion Our results confirm earlier findings showing that old rats are deficient in a variety of behavioural tests [31] and also indicate that the degree of age-related impairment is not simply a function of age but depends on the specific tasks used to assess such deficits [25]. In the present study, old vehicle-infused rats evidenced a severe deficit in learning to navigate the water maze, a reduction in open-field activity and an increase in fear/anxiety. However, the aged animals did not differ from adult controls in terms of habituation and inhibitory avoidance learning, providing further evidence that age-related declines in different functional brain systems may progress independently [10]. Contrary to hypothesis, the long-term infusion of HP did not significantly improve the performance of the old rats in any of the learning and memory tasks employed. HP only slightly facilitated the short- and long-term retention of the inhibitory avoidance task and the rate of habituation in the open-field paradigm. In the water maze the glycosaminoglycan did not counteract the navigation deficits observed for aged controls and even impaired the performance during the initial place-learning trials. Furthermore, the neurochemical analysis yielded no obvious differences in measures of forebrain ACh tissue concentrations in HP-infused old rats as compared with vehicle-treated adult and aged controls. Based on our previous evidence for promnestic effects of HP in adult animals [6], we held it possible that the GAG might also improve the performance of behaviourally im-

paired aged rats in the water maze task. However, the chronic infusion of HP failed to counteract the navigation deficits observed for aged controls in the course of acquisition and long-term retention of the hidden-platform task. Actually, HP-infused animals even showed inferior learning during the initial observation period reaching control scores only within the last four days of training, when both aged groups attained an asymptotic level of performance. Furthermore, compared to aged controls the old rats of the HP group had a comparable swim speed across daily place trials and the time to escape onto the visible platform was similar in both groups of old rats. Thus, it is unlikely that potential promnestic effects of HP were confounded or masked by changes in motor performance and/or visually guided behaviour. The lack of effect observed for HP in the water maze task is also surprising in the light of previous studies showing that other structurally related GAGs and proteoglycans, such as chondroitin sulphate and biglycan, can diminish age-related maze learning deficits [16]. The following particulars of the present study may be of relevance for this discrepancy: For one, HP, unlike the other gluco- and galactosaminoglycans, exerts strong anticoagulant effects [1]. Thus, it is feasible that the GAG exacerbated haemorrhage caused by cannulation surgery and, by this way, interfered negatively with the performance in the water maze especially at the beginning of the behavioural assessment. However, histological inspection of the cannula sites revealed no apparent tissue damage, which we would expect to impair performance not only in the water maze but also in the other learning tasks. Furthermore, treatment with the HP analogue enoxaparin starting immediately after an experimental brain injury was reported to reduce brain tissue damage and to decrease, rather than increase, lesion-induced learning and motor deficits [40]. Secondly, the mentioned ‘cognition enhancing’ effects of chondroitin sulphate and biglycan were observed in aged hybrid Fischer 344 × Brown Norway rats [16]. These animals typically exhibit ‘hybrid vigor’ [35] and show, for example, much better performance on the different versions of the water maze, more exploration in the open field and less motor disturbances compared with age-matched Wistar rats [14]. Accordingly, the aged Wistar rats used in the present experiment performed worse even after 9 days of daily maze training and did not display a substantial acquisition rate across trials. Thus, HP might be effective in aged rats with residual maze learning capacities but not in animals with extensive disturbances in associative functions.

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Furthermore, with regard to the poor maze navigation of the old vehicle-infused rats at the end of the initial acquisition phase it is also feasible that the training period might have been too short to reveal such a facilitatory effect. Thus, HP might have developed its beneficial effect on learning about 3 weeks after start of infusion when the GAG slightly improved the performance of the old rats in the habituation paradigm and the inhibitory avoidance task. This suggests that potential beneficial effects of HP on age-related memory decline could be time-dependent and evident only after a critical period of continuous drug-infusion. In previous experiments using between-session habituation as an index of memory, aged rats had to be disqualified because their baseline activity was too low to determine possible effects of a (pharmacological) manipulation on habituation [8]. In the cited experiment, behavioural assessment was performed during the animals’ inactive light phase, which might have aggravated the known age-related decrease in general activity [42]. Therefore, in the present study, we tested the animals in their active night period. As expected, the aged animals showed increased activity scores, which, however, were still lower than those of the adult rats. The reduced reactivity of aged rats to a novel environment might simply result from disturbances in motor functions but could also encompass changes in emotional/motivational variables [29]. Actually, both groups of aged rats spent less time in the centre of the arena, indicative of enhanced fear/anxiety. This suggests, for one, that the low basal activity in the aged rats might be induced in part by an increase in anxiety-driven behaviours which are incompatible with active exploration (freezing/immobility, centre field avoidance) and, secondly, that long-term HP infusion did not influence the emotional status of the aged rats. These findings are interesting in the light of a recent study, demonstrating that repeated intraperitoneal injection of HP can reduce fear/anxiety in adult rats without changing their general exploration pattern [27]. With repeated open-field exposure, that is, with increasing familiarity to this environment, HP-infused old animals showed a decrease in the relative amount of exploration without locomotion. No such change was evident in adult and aged controls, suggesting that HP improved habituation of exploratory activity (i.e. habituation ‘learning’). A detailed analysis of individual exploration parameters revealed that rearing, which is frequently used to gauge the amount of between-exposure habituation in adult rodents [4], failed to differentiate between the groups of old and adult rats. Apparently, exploration without locomotion proved to be the most sensitive index for the assessment of habituation/deficits in aged rats possibly because this measure is less prone to the influence of motor deficits and/or motivational changes. However, it has to be pointed out that the effects of HP on habituation were weak and only evident when comparing the rate of habituation within- rather than between-groups. In addition to its potential beneficial effect on stimulusoriented learning chronic HP infusion also slightly enhanced

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the performance of the aged rats in a modified one-trial inhibitory avoidance task, which was recommended for the assessment of drug-effects on STM and LTM processes in rodentia [18]. Each animal was tested at different time points after training, namely at 1.5 h from training, in order to measure STM, and then again at 24 h, in order to evaluate long-term retention (LTM) of the task. The main finding was that chronic HP infusion proved effective in facilitating both short- and long-term retention of the conditioned avoidance response. A quite similar promnestic profile of HP was obtained in adult rats using this paradigm in combination with a single post-trial injection of the GAG into the ventral pallidum [6]. Thus, it is possible that the continuous infusion of HP improved the retention performance of the old animals by modulating early memory storage processes, rather than by acting on performance variables during acquisition and/or retrieval of the task. Interestingly, the different treatment groups did not show a significant across-session performance decrement suggesting the absence of extinction after the STM trials. This observation adds further evidence to the assumption that the measures of STM and LTM might represent separate and largely independent types of memory [17]. Brain cholinergic mechanisms are crucially involved in the control of behavioural processes related to exploration, fear/anxiety, learning and memory [41]. Furthermore, deficits in attention and mnemonic functions in the course of brain ageing or as a symptom of dementia are thought to be related, in part, to degenerative changes in basal forebrain cholinergic systems [26]. We recently found that injection of HP into the ventral pallidum in a dose, which was also effective in facilitating learning, increased cholinergic activity in frontal cortex of adult rats [6]. This indicates that the promnestic effects of the GAG might involve changes in central cholinergic neurotransmission. Based on these findings, we anticipated between-group differences in the concentrations of ACh in the forebrain of adult and aged rats with regard to the factors age and pharmacological treatment. For this study, we choose the decapitation rather than the microwave irradiation as a sacrifice method since it has been shown by Sethy and Francis [32] that there was no effect of sacrifice method (by decapitation or microwave irradiation) on ACh concentration increase after the treatment with the cholinomimetic oxotremorine. However, the neurochemical measures did not reveal any clear-cut between-group differences in ACh concentrations in the cortical, striatal and hippocampal areas under inspection. We cannot rule out that potential age- or drug-related differences in transmitter levels were masked, for example, by the large variance of the neurochemical data. Furthermore, tissue levels of a given transmitter do not reflect its activity; thus, potential differences in ACh activity (i.e. release) may not be detected by the present method. Recent studies provided evidence that HP can produce a psychostimulant-like activation of biogenic amines [22]. This raises the possibility that the behavioural effects of the GAG might be

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attributable to changes in reinforcement-related neurochemical systems rather than in encoding or retrieval systems per se, which both are known to have a strong cholinergic component [15]. Finally, another aspect should be pointed out. Several studies have demonstrated that heparinoids and other high-molecular GAGs could function as neurotrophic factors for differentiating neurons, but at high concentrations in mature neurons, as in Alzheimer’s disease, could cause neuronal degeneration by stimulating the aggregation of ␤-amyloid and/or increasing the formation of neurofibrillary tangles [33]. Thus, one might expect that treatment with exogenous HP and related GAGs could have negative effects on learning and memory parameters, especially in behaviourally impaired old rats whose brains are not healthy anymore. The current results, together with our previous work partly support this notion by showing that GAGs differentially affect associative processes depending on the age and behavioural status of the animals. Thus, in normal adult rats [6] and in old animals with minor cognitive decline [16], GAGs were found to improve mnestic functions. However, in old animals with more severe behavioural deficits, like those in the present study, the GAG treatment proved to be ‘mnemoneutral’ and even interfered negatively with certain learning and memory parameters.

Acknowledgements This work was supported by Grant Hu 306/11-3 from the Deutsche Forschungsgemeinschaft.

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