J Neural Transm [P-D Sect] (1992) 4:99-106
Journal of Neural Transmission 9 Spriilger-Verlag 1992 Printed in Austria
The effects of alpha-2 adrenoceptor antagonist, atipamezole, on spatial learning in scopolamine-treated and aged rats J. Sirviii I, P. Riekkinen Jr. 1, E. MacDonald 2, M. Airaksinen2, R. Lammintausta 3, and P. J. Riekkinen I Department of 1Neurology, and 2Pharmacology and Toxicology, University of Kuopio, Kuopio, and 3Orion Corporation, Farmos R & D Pharmaceuticals, Turku, Finland Accepted October 14, 1991
Summary. In order to study whether noradrenergic drugs improve age-related cognitive dysfunctions the present experiments investigated whether atipamezole, a selective and specific alpha-2 antagonist, improves spatial learning impairment due to cholinergic blockade (scopolamine 0.8 mg/kg) or aging in rats. Previously, it has been shown that atipamezole dose-dependently (0.03-3.0 mg/ kg) increases the turnover of noradrenaline in rat brain. According to the present results, atipamezole (0.1, 0.3, 0.6 mg/kg) did not affect spatial learning/memory when assessed in a free swim trial of the water maze task in control rats. Furthermore, atipamezole (0.1, 0.6 mg/kg) did not improve learning deficit in scopolamine treated young rats. Higher doses (~> 1.0mg/kg) of atipamezole could not be tested, because they induce floating behaviour in rats. In aged rats, which were screened to be impaired in the initial acquisition of the water maze task, 0.3 mg/kg atipamezole impaired further learning of this task. Because previous studies suggest that age-related learning impairment in the water maze may be, at least partly, due to a cholinergic deficit, the present results suggest that atipamezole which increases the release of noradrenaline in brain does not alleviate this learning deficit. Keywords: Aging, alpha-2 adrenoceptor antagonist, noradrenergic system, rat, spatial learning/memory. Introduction Aging can be associated with disorders in memory functions. Cholinergic and noradrenergic dysfunctions have received much attention as a neurobiological background of age-related memory impairment (Bartus etal., 1982; Arnsten and Goldman-Rakic, 1987). Interactions between cholinergic and noradrenergic systems may play an important role in memory dysfunctions; a partial norad-
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renergic lesion aggravated cholinergic blockade (scopolamine)-induced deficits of rats in the performance of radial arm maze and water maze tasks assessing spatial memory (Decker and Gallagher, 1987; Riekkinen Jr. etal., 1990a). Spatial memory is also worse in aged rats, and this deficit has been proposed to be a good model of memory impairment in aged subjects (Gallagher and Pelleymounter, 1988). Electrophysiological (Aghajanian and Rogawski, 1983) and neurochemical studies (Langer, 1981) have shown that the firing rate of the locus coeruleus and the release of noradrenaline is regulated by alpha-2 adrenergic autoreceptors. The blockade of those autoreceptors increases the release of noradrenaline in brain. Atipamezole which is a selective and specific alpha-2 adrenoceptor antagonist (Virtanen etal., 1989), increased dose-dependently (0.03-3 mg/kg) the turnover of central noradrenaline (Scheinin etal., 1988). The present experiments were undertaken to study whether pharmacological activation of the noradrenergic system would improve age-related impairment of spatial learning and memory. Thus, we examined whether atipamezole improves age-related learning deficit of rats in a water maze task. Since the acquisition deficit of this task may vary considerably between individuals (Gage et al., 1984), we screened a population of aged rats for impairment in the initial acquisition of the water maze task before testing the effects of atipamezole on further learning of this task. In order to investigate whether atipamezole could improve learning impairment following cholinergic dysfunction, we also studied the effects of atipamezole on water maze performance in rats treated with a muscarinic antagonist (scopolamine) which is a widely used model of amnesia (Smith, 1988). Materials and methods
Animals In experiment l, young (4 months old) male rats were used. In experiment 2, young (4 months old, n = 9) and aged (18 months old, n = 59) male Kuo :Wistar rats were used. The rats were housed in light period (0700-2100), temperature (21 ~ and humidity (5060%) controlled environment.
Behavioral training The water maze apparatus (the pool and video tracking system) used to assess spatial learning and memory has been described previously (Riekkinen etal., 1990b). The rats were trained to find a submerged platform, the position of which was kept constant in the pool during training. The free swim trial (probe trial) was used to measure the spatial bias (percentage of the total distance swum in the previous training quadrant). The spatial bias in the probe trial is an index of memory: the higher the spatial bias, the better memory is assessed as being. In experiment 1, the rats were trained for 10 days (two trials/day, maximum duration 90 seconds). The free swim trial was the second trial of the 10th training day. In experiment 2, young and aged rats were screened for the initial acquisition of water maze task for four days (2 trials/day, maximum 70 seconds). In this experiment, we used a shorter training time, because aged rats were used. The aged rats were considered to be impaired in the
Atipamezole and spatial learning/memory in rats
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acquisition of the task if the mean escape latency of two trials was more than 40 seconds (and in both trials more than 30 seconds) on the 4th day of training. Escape latency of young rats ranged from 6-36 seconds (mean 17 seconds). Young rats and impaired aged rats were trained (and treated with saline or atipamezole) further for four days (two trials/ day, maximum duration 70 seconds). The probe trial was the second trial of the last day of training.
Drug treatments Scopolamine-HBr (Sigma, U.S.A.) was dissolved in saline and injected intraperitoneally (ip) 35 minutes before the daily trainings in the water maze. Atipamezole (Farmos Group, Turku, Finland) was dissolved in saline, and injected subcutaneously (sc) 30 minutes before the training of rats. Previously, atipamezole has been shown to increase the release of noradrenaline in rat brain dose-dependently (0.03-3.0 mg/kg) after systemic administration. According to our preliminary studies, at the doses of/> 1.0 mg/kg atiapamezole induces floating behaviour when rats are put into a water pool. Thus, the doses of ~< 0.6 mg/kg were used in water maze experiments. In experiment 1, the groups of rats included: saline (ip)+ saline (sc); saline (ip) + atipamezole (0.1 mg/kg, sc); saline (ip) + atipamezole (0.3 mg/kg, sc); saline (ip) + atipamezole (0.6 mg/kg, sc); scopolamine (0.8 mg/kg, ip) + saline (sc); scopolamine (0.8 mg/kg, ip) + atipamezole (0.1mg/kg, sc) and scopolamine (0.8mg/kg, ip) + atipamezole (0.6mg/kg, sc). The number of rats was 7-8 in each group. In experiment 2, young rats were treated with saline, and aged impaired rats were treated with saline (n = 7) or atipamezole (0.3 mg/kg, n = 7) 30 minutes before the daily trainings of the last four days.
Statistical analysis Behavioral data were analyzed using SPSS/PC + program [analysis of variance (ANOVA) or ANOVA and post-hoc Mann-Whitney U-test].
Results
Experiment 1 Because escape latencies were confounded by the effects of drug treatments on the speed of swimming (scopolamine increased swimming speed and atipamezole decreased it), spatial learning/memory ability of rats is expressed as spatial bias (Fig. 1). The rats treated with scopolamine (0.8 mg/kg) had a lower spatial bias than saline treated rats (p < 0.05, Mann-Whitney U-test). The spatial bias did not differ between saline and atipamezole (0.1-0.6 mg/kg) treated rats (p > 0.1, Mann-Whitney U-test). Moreover, no significant difference was found between saline and atipamezole (0.1 and 0.6 mg/kg) treated rats which received scopolamine (0.8 mg/kg) (p > 0.05, Mann-Whitney U-test).
Experiment 2 The difference between saline treated young rats and aged rats was not significant in the probe trial assessed after training period (p > 0.05, Mann-Whitney
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~0
......
:::';;:"
9
~ii;iii:
m
c
~
ff~ S
A 0.1
A0a N~ SA 0.6
~
Fq SA 0.1
NSlS A0.6
Fig. 1. The spatial bias of young rats treated with scopolamine and/or atipamezole. Group
abbreviations: C controls; A atipamezole 0.1-0.6mg/kg; S scopolamine 0.8mg/kg. p < 0.05 vs. controls, | p < 0.01. The results are expressed as mean + SD
SPATIAL BIAS (%) 40 - - -
LATENCY (sec) 50
40
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30 20 20 10
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day 2
9-I-- young, saline aged-i, ati 0.3
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day 3 ~
j
day 4 aged-i, saline
da' ,5
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aged - i, young L%~] saline
aged-i, [--J ati 0.3
Fig. 2. The acquisition of water maze task [escape latency (mean + SEM), left panel; spatial bias (mean + SD), right panel] in saline treated young rats, saline or atipamezole (0.3 mg/ kg) treated aged rats which were considered to be impaired in the initial acquisition of water maze task. Iv p < 0.05 vs. saline treated aged-i rats
U-test), a l t h o u g h saline treated aged rats were impaired in their initial acquisition o f the task (Fig. 2). A g e d rats treated with atipamezole (0.3 mg/kg) h a d a lower spatial bias t h a n their saline treated c o u n t e r p a r t s (p < 0.05, M a n n W h i t n e y U-test) (Fig. 2). The a m o u n t o f swimming [distance expressed as arbitrary units (pixels), m e a n + SD] in the probe trial did not significantly differ
Atipamezole and spatial learning/memory in rats
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between saline treated young rats (2221 4-208), saline treated aged rats (2456 4-293) and atipamezole 0.3mg/kg treated aged rats (2111 4- 434) (p > 0.05 using ANOVA). Discussion
According to our pilot studies, aged rats were impaired in the acquisition of water maze task, but the long-term retention of this task (assessed using a free swim trial 7 or 10 days after the initial training) was not markedly affected when compared to young rats (unpublished data). Because previous studies suggest that the deficit in the acquisition of water maze task may be, at least partly, related to cholinergic dysfunction in aged rats (Fischer etal., 1989; Gallagher etal., 1990), we tested the effects of atipamezole on water maze learning/memory in scopolamine treated young rats. According to the present results, atipamezole did not improve spatial learning/memory in young control rats when assessed in the free swim trial. Furthermore, it did not prevent the acquisition deficit in scopolamine treated rats. Previous studies have shown that atipamezole did not improve scopolamine-induced deterioration of cortical and hippocampal EEG (Riekkinen Jr. et al., 1990 c; Valjakka et al., 1991). These results suggest that atipamezole treatment does not alleviate cholinergic dysfunction in rats. According to the present results, the acquisition of the water maze task was impaired in aged rats, at least in a subpopulation of those rats. This finding is in agreement with previous studies (Gage et al., 1984; Zanotti et al., 1989; Gallagher etal., 1990). In aged rats which were considered to be impaired in the initial acquisition of the water maze task, atipamezole (0.3 mg/kg) tended to aggravate their acquisition deficit as assessed by the lower spatial bias compared to their saline treated counterparts. Preliminary studies using a small number of unscreened aged rats suggested that the performance of aged rats is slightly impaired (slightly decreased swimming speed, significantly increased escape latency and slightly increased escape distance to a hidden platform) even at a lower dose (0.03 mg/kg) (our unpublished data). Higher doses ( > 0.6mg/kg) could not be tested, because they induce floating behaviour in rats (our unpublished observation). Furtheremore, atipamezole (0.1-2.7 mg/kg) did not improve spatial short-term memory in young and aged rats (Sirvi6 et al., 1991 a). In addition to the evident ineffectiveness of atipamezole to alleviate cholinergic dysfunction related spatial learning/memory impairment, one reason for these negative results in the effects of atipamezole on spatial learning/ memory could be that aged rats do not have a cortical and hippocampal noradrenergic hypofunction (Harik et al., 1986; McIntosh and Westfall, 1987; Santiago et al., 1988; Valjakka et al., 1990 a; Gallagher et al., 1990). Furthermore, it could be argued that noradrenergic hypofunction does not impair spatial learning/memory, because partial or extensive depletions of noradrenaline in the forebrain do not impair spatially cued navigation in water maze (as also assessed in the present study) in adult rats (Riekkinen et al., 1990; Selden
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et al., 1990; Valjakka et al., 1990 b). However, our recent data suggest that partial noradrenaline depletion impairs water maze acquisition in aged rats, but not in young rats (Sirvi6 etal., 1991 b). It remains to be determined whether atipamezole is effective in improving spatial learning/memory in aged rats with induced partial noradrenaline depletion. Previously, it has been shown that alpha-2 agonists, especially guanfacine at low doses (0.0001-0.001 mg/kg), improved spatial short-term memory in aged monkeys, and this improvement was proposed to be due to the activation of postjunctional alpha-2 adrenoceptors (Arnsten and Goldman-Rakic, 1985, 1990; Arnsten et al., 1988). It is not known how effectively atipamezole binds to the postjunctional alpha-2 adrenoceptors versus alpha-2 autoreceptors. Thus, it could be argued that atipamezole may impair spatial memory in aged rats due to a blockade of postsynaptic alpha-2 adrenoceptors despite the increased turnover of noradrenaline. However, a low dose (0.001 mg/kg) of guanfacine did not improve the acquisition deficit of water maze task in aged rats (Sirvi6 etal., 1991 c). In conclusion, the present results suggest that atipamezole, a selective and specific alpha-2 adrenoceptor antagonist, does not improve spatial learning/ memory deficit due to aging or blockade of muscarinic receptors in rats.
Acknowledgements We are grateful for Ms. S. Palviainen for her excellent secretarial help. This study was supported by the Academy of Finland.
References Aghajanian GK, Rogawski MA (t983) The physiological role of alpha-adrenoceptors in CNS: new concepts from single-cell studies. Trends Pharmacol Sci 4:315-317 Arnsten AFT, Go!dman-Rakic PS (1985) az-Adrenergic mechanisms in prefrontal cortex associated with cognitive decline in aged non-human primates. Science 230:1273-1275 Arnsten AFT, Goldman-Rakic PS (1987) Noradrenergic mechanisms in age-related cognitive decline. J Neural Transm 24 [Suppl]: 317-324 Arnsten AFT, Goldman-Rakic PS (1990) Analysis of alpha-2 adrenergic agonist effects on the delayed nonmatch-to-sample performance of aged rhesus monkeys. Neurobiol Aging 11:583-590 Arnsten AFT, Cai JX, Goldman-Rakic PS (1988) The alpha-2 adrenergic agonist guanfacine improves memory in aged monkeys without sedative or hypotensive side effects: evidence for alpha-2 receptor subtypes. J Neurosci 8:4287-4298 Bartus RT, Dean RL, Beer B, Lippa A (1982) The cholinergic hypothesis of geriatric memory dysfunction. Science 217:408-417 Decker MW, Gallagher M (1987) Scopolamine-disruption of radial arm maze performance: modification by noradrenergic depletion. Brain Res 417:59-69 Fischer W, Gage FH, Bj6rklund A (1989) Degenerative changes in forebrain cholinergic nuclei correlate with cognitive impairments in aged rats. Eur J Neurosci 1:34--45 Gage FH, Dunnett SB, Bj6rklund A (1984) Spatial learning and motor deficits in aged rats. Neurobiol Aging 5:43-48
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Gallagher M, Burwell RD, Kodsi MH, McKinney M, Southerland S, Vella-Rountree L, Lewis MH (1990) Markers for biogenic amines in the aged rat brain: relationship to decline in spatial learning ability. Neurobiol Aging 11:507-514 Gallagher M, Pelleymounter MA (1988) Spatial learning deficits in old rats: a model for memory decline in the aged. Neurobiol Aging 9:549-556 Harik JI, McCracken KA (1986) Age-related increase in pre-synaptic noradrenergic markers of the rat cerebral cortex. Brain Res 381:125-130 McIntosh HH, Westfall TC (1987) Influence of aging on catecholamine levels, accumulation and release in F-344 rats. Neurobiol Aging 8:233-239 Langer Z (1981) Presynaptic regulation of the release of catecholamines. Pharmacol Rev 32:337-362 Riekkinen P Jr, Sirvi6 J, Valjakka A, Pitk/inen A, Partanen J, Riekkinen P (1990 a) The effects of concurrent manipulations of cholinergic and noradrenergic systems on neocortical EEG and spatial learning. Behav Neural Biol 54:204-210 Riekkinen P Jr, Sirvi6 J, Riekkinen P (1990 b) Similar memory impairments found in medial septal-vertical diagonal band of Broca and nucleus basalis lesioned rats: are memory defects induced by nucleus basalis lesions related to the degree of non-specific subcortical cell loss? Behav Brain Res 37:81-88 Riekkinen P Jr, Sirvi6 J, Jfik/ilfi P, Lammintausta R, Riekkinen P (1990 c) Effect of alpha2 antagonists and an agonist on EEG slowing induced by scopolamine and lesion of the nucleus basalis. Neuropharmacology 29:993-999 Santiago M, Machado A, Reinoso-Suarez F, Cano J (1988) Changes in biogenic amines in rat hippocampus during development and aging. Life Sci 42:2503-2508 Selden NRW, Cole BJ, Everitt BJ, Robbins TW (1990) Damage to cerulo-cortical noradrenergic projections impairs locally cued but enhances spatially cued water maze acquisition. Behav Brain Res 39:29-51 Scheinin H, MacDonald E, Scheinin M (1988) Behavioural and neurochemical effects of atipamezole a novel a2-adrenoceptor antagonist. Eur J Pharmacol 151:35-42 Smith G (1988) Animal models of Alzheimer's disease: experimental cholinergic denervation. Brain Res Rev 13:103-118 Sirvi6 J, Lukkarinen K, Riekkinen Jr P, Koivisto E, Virtanen R, Pennanen A, Riekkinen P (1991 a) The effects of atipamezole, an alpha-2 antagonist, on the performance of young and aged rats in the delayed non-matching to position task. Pharmacol Biochem Behav 39:1015-1020 Sirvi6 J, Riekkinen Jr P, Valjakka A, Jolkkonen J, Riekkinen PJ (1991 b) The effects of noradrenergic neurotoxin DSP-4 on the performance of young and aged rats in spatial navigation task. Brain Res 563:297-302 Sirvi6 J, Riekkinen P Jr, Vajanto I, Koivisto E, Riekkinen PJ (1991 c) The effects of guanfacine, alpha-2 agonist, on the performance of young and aged rats in spatial navigation task. Behav Neural Biol 56:101-107 Valjakka A, Sirvi6 J, Pitk/inen A, Riekkinen PJ (1990a) Brain amines and neocortical EEG in young and aged rats. Comp Biochem Physiol 96 C: 299-304 Valjakka A, Riekkinen P Jr, Sirvi6 J, Nieminen S, Airaksinen M, Miettinen R, Riekkinen P (1990 b) The effects of dorsal noradrenergic bundle lesions on spatial learning, locomotor activity, and reaction to novelty. Behav Neural Biol 54:323-329 Valjakka A, Lukkarinen K, Koivisto E, Riekkinen Jr P, Miettinen R, Airaksinen MM, Lammintausta R, Riekkinen P (1991) Modulation of EEG rhythmicity and spike activity in the rat hippocampus by systemically administered tetrahydroaminoacridine, scopolamine and atipamezole. Brain Res Bull 26:739-745 Virtanen R, Savola J-M, Saano V (1989) Highly selective and specific antagonism of central and peripheral ~2-adrenoceptors by atipamezole. Arch Int Pharmacodyn Ther 297: 190-204
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Zanotti A, Valzelli L, Toffano G (1989) Chronic phosphatidyl serine treatment improves spatial memory and passive avoidance in aged rats. Psychopharmacology 99:316-321 Authors' address: J. SirviS, PhD, Department of Neurology, University of Kuopio, P.O. Box 1627, SF-70211 Kuopio, Finland. Received April 11, 1991
Journal of Neural Transmission 9 Spriilger-Verlag 1992 Printed in Austria
The effects of alpha-2 adrenoceptor antagonist, atipamezole, on spatial learning in scopolamine-treated and aged rats J. Sirviii I, P. Riekkinen Jr. 1, E. MacDonald 2, M. Airaksinen2, R. Lammintausta 3, and P. J. Riekkinen I Department of 1Neurology, and 2Pharmacology and Toxicology, University of Kuopio, Kuopio, and 3Orion Corporation, Farmos R & D Pharmaceuticals, Turku, Finland Accepted October 14, 1991
Summary. In order to study whether noradrenergic drugs improve age-related cognitive dysfunctions the present experiments investigated whether atipamezole, a selective and specific alpha-2 antagonist, improves spatial learning impairment due to cholinergic blockade (scopolamine 0.8 mg/kg) or aging in rats. Previously, it has been shown that atipamezole dose-dependently (0.03-3.0 mg/ kg) increases the turnover of noradrenaline in rat brain. According to the present results, atipamezole (0.1, 0.3, 0.6 mg/kg) did not affect spatial learning/memory when assessed in a free swim trial of the water maze task in control rats. Furthermore, atipamezole (0.1, 0.6 mg/kg) did not improve learning deficit in scopolamine treated young rats. Higher doses (~> 1.0mg/kg) of atipamezole could not be tested, because they induce floating behaviour in rats. In aged rats, which were screened to be impaired in the initial acquisition of the water maze task, 0.3 mg/kg atipamezole impaired further learning of this task. Because previous studies suggest that age-related learning impairment in the water maze may be, at least partly, due to a cholinergic deficit, the present results suggest that atipamezole which increases the release of noradrenaline in brain does not alleviate this learning deficit. Keywords: Aging, alpha-2 adrenoceptor antagonist, noradrenergic system, rat, spatial learning/memory. Introduction Aging can be associated with disorders in memory functions. Cholinergic and noradrenergic dysfunctions have received much attention as a neurobiological background of age-related memory impairment (Bartus etal., 1982; Arnsten and Goldman-Rakic, 1987). Interactions between cholinergic and noradrenergic systems may play an important role in memory dysfunctions; a partial norad-
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renergic lesion aggravated cholinergic blockade (scopolamine)-induced deficits of rats in the performance of radial arm maze and water maze tasks assessing spatial memory (Decker and Gallagher, 1987; Riekkinen Jr. etal., 1990a). Spatial memory is also worse in aged rats, and this deficit has been proposed to be a good model of memory impairment in aged subjects (Gallagher and Pelleymounter, 1988). Electrophysiological (Aghajanian and Rogawski, 1983) and neurochemical studies (Langer, 1981) have shown that the firing rate of the locus coeruleus and the release of noradrenaline is regulated by alpha-2 adrenergic autoreceptors. The blockade of those autoreceptors increases the release of noradrenaline in brain. Atipamezole which is a selective and specific alpha-2 adrenoceptor antagonist (Virtanen etal., 1989), increased dose-dependently (0.03-3 mg/kg) the turnover of central noradrenaline (Scheinin etal., 1988). The present experiments were undertaken to study whether pharmacological activation of the noradrenergic system would improve age-related impairment of spatial learning and memory. Thus, we examined whether atipamezole improves age-related learning deficit of rats in a water maze task. Since the acquisition deficit of this task may vary considerably between individuals (Gage et al., 1984), we screened a population of aged rats for impairment in the initial acquisition of the water maze task before testing the effects of atipamezole on further learning of this task. In order to investigate whether atipamezole could improve learning impairment following cholinergic dysfunction, we also studied the effects of atipamezole on water maze performance in rats treated with a muscarinic antagonist (scopolamine) which is a widely used model of amnesia (Smith, 1988). Materials and methods
Animals In experiment l, young (4 months old) male rats were used. In experiment 2, young (4 months old, n = 9) and aged (18 months old, n = 59) male Kuo :Wistar rats were used. The rats were housed in light period (0700-2100), temperature (21 ~ and humidity (5060%) controlled environment.
Behavioral training The water maze apparatus (the pool and video tracking system) used to assess spatial learning and memory has been described previously (Riekkinen etal., 1990b). The rats were trained to find a submerged platform, the position of which was kept constant in the pool during training. The free swim trial (probe trial) was used to measure the spatial bias (percentage of the total distance swum in the previous training quadrant). The spatial bias in the probe trial is an index of memory: the higher the spatial bias, the better memory is assessed as being. In experiment 1, the rats were trained for 10 days (two trials/day, maximum duration 90 seconds). The free swim trial was the second trial of the 10th training day. In experiment 2, young and aged rats were screened for the initial acquisition of water maze task for four days (2 trials/day, maximum 70 seconds). In this experiment, we used a shorter training time, because aged rats were used. The aged rats were considered to be impaired in the
Atipamezole and spatial learning/memory in rats
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acquisition of the task if the mean escape latency of two trials was more than 40 seconds (and in both trials more than 30 seconds) on the 4th day of training. Escape latency of young rats ranged from 6-36 seconds (mean 17 seconds). Young rats and impaired aged rats were trained (and treated with saline or atipamezole) further for four days (two trials/ day, maximum duration 70 seconds). The probe trial was the second trial of the last day of training.
Drug treatments Scopolamine-HBr (Sigma, U.S.A.) was dissolved in saline and injected intraperitoneally (ip) 35 minutes before the daily trainings in the water maze. Atipamezole (Farmos Group, Turku, Finland) was dissolved in saline, and injected subcutaneously (sc) 30 minutes before the training of rats. Previously, atipamezole has been shown to increase the release of noradrenaline in rat brain dose-dependently (0.03-3.0 mg/kg) after systemic administration. According to our preliminary studies, at the doses of/> 1.0 mg/kg atiapamezole induces floating behaviour when rats are put into a water pool. Thus, the doses of ~< 0.6 mg/kg were used in water maze experiments. In experiment 1, the groups of rats included: saline (ip)+ saline (sc); saline (ip) + atipamezole (0.1 mg/kg, sc); saline (ip) + atipamezole (0.3 mg/kg, sc); saline (ip) + atipamezole (0.6 mg/kg, sc); scopolamine (0.8 mg/kg, ip) + saline (sc); scopolamine (0.8 mg/kg, ip) + atipamezole (0.1mg/kg, sc) and scopolamine (0.8mg/kg, ip) + atipamezole (0.6mg/kg, sc). The number of rats was 7-8 in each group. In experiment 2, young rats were treated with saline, and aged impaired rats were treated with saline (n = 7) or atipamezole (0.3 mg/kg, n = 7) 30 minutes before the daily trainings of the last four days.
Statistical analysis Behavioral data were analyzed using SPSS/PC + program [analysis of variance (ANOVA) or ANOVA and post-hoc Mann-Whitney U-test].
Results
Experiment 1 Because escape latencies were confounded by the effects of drug treatments on the speed of swimming (scopolamine increased swimming speed and atipamezole decreased it), spatial learning/memory ability of rats is expressed as spatial bias (Fig. 1). The rats treated with scopolamine (0.8 mg/kg) had a lower spatial bias than saline treated rats (p < 0.05, Mann-Whitney U-test). The spatial bias did not differ between saline and atipamezole (0.1-0.6 mg/kg) treated rats (p > 0.1, Mann-Whitney U-test). Moreover, no significant difference was found between saline and atipamezole (0.1 and 0.6 mg/kg) treated rats which received scopolamine (0.8 mg/kg) (p > 0.05, Mann-Whitney U-test).
Experiment 2 The difference between saline treated young rats and aged rats was not significant in the probe trial assessed after training period (p > 0.05, Mann-Whitney
102
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~0
......
:::';;:"
9
~ii;iii:
m
c
~
ff~ S
A 0.1
A0a N~ SA 0.6
~
Fq SA 0.1
NSlS A0.6
Fig. 1. The spatial bias of young rats treated with scopolamine and/or atipamezole. Group
abbreviations: C controls; A atipamezole 0.1-0.6mg/kg; S scopolamine 0.8mg/kg. p < 0.05 vs. controls, | p < 0.01. The results are expressed as mean + SD
SPATIAL BIAS (%) 40 - - -
LATENCY (sec) 50
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30 20 20 10
10
r
Oay 1
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9-I-- young, saline aged-i, ati 0.3
i
day 3 ~
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day 4 aged-i, saline
da' ,5
0
II
aged - i, young L%~] saline
aged-i, [--J ati 0.3
Fig. 2. The acquisition of water maze task [escape latency (mean + SEM), left panel; spatial bias (mean + SD), right panel] in saline treated young rats, saline or atipamezole (0.3 mg/ kg) treated aged rats which were considered to be impaired in the initial acquisition of water maze task. Iv p < 0.05 vs. saline treated aged-i rats
U-test), a l t h o u g h saline treated aged rats were impaired in their initial acquisition o f the task (Fig. 2). A g e d rats treated with atipamezole (0.3 mg/kg) h a d a lower spatial bias t h a n their saline treated c o u n t e r p a r t s (p < 0.05, M a n n W h i t n e y U-test) (Fig. 2). The a m o u n t o f swimming [distance expressed as arbitrary units (pixels), m e a n + SD] in the probe trial did not significantly differ
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between saline treated young rats (2221 4-208), saline treated aged rats (2456 4-293) and atipamezole 0.3mg/kg treated aged rats (2111 4- 434) (p > 0.05 using ANOVA). Discussion
According to our pilot studies, aged rats were impaired in the acquisition of water maze task, but the long-term retention of this task (assessed using a free swim trial 7 or 10 days after the initial training) was not markedly affected when compared to young rats (unpublished data). Because previous studies suggest that the deficit in the acquisition of water maze task may be, at least partly, related to cholinergic dysfunction in aged rats (Fischer etal., 1989; Gallagher etal., 1990), we tested the effects of atipamezole on water maze learning/memory in scopolamine treated young rats. According to the present results, atipamezole did not improve spatial learning/memory in young control rats when assessed in the free swim trial. Furthermore, it did not prevent the acquisition deficit in scopolamine treated rats. Previous studies have shown that atipamezole did not improve scopolamine-induced deterioration of cortical and hippocampal EEG (Riekkinen Jr. et al., 1990 c; Valjakka et al., 1991). These results suggest that atipamezole treatment does not alleviate cholinergic dysfunction in rats. According to the present results, the acquisition of the water maze task was impaired in aged rats, at least in a subpopulation of those rats. This finding is in agreement with previous studies (Gage et al., 1984; Zanotti et al., 1989; Gallagher etal., 1990). In aged rats which were considered to be impaired in the initial acquisition of the water maze task, atipamezole (0.3 mg/kg) tended to aggravate their acquisition deficit as assessed by the lower spatial bias compared to their saline treated counterparts. Preliminary studies using a small number of unscreened aged rats suggested that the performance of aged rats is slightly impaired (slightly decreased swimming speed, significantly increased escape latency and slightly increased escape distance to a hidden platform) even at a lower dose (0.03 mg/kg) (our unpublished data). Higher doses ( > 0.6mg/kg) could not be tested, because they induce floating behaviour in rats (our unpublished observation). Furtheremore, atipamezole (0.1-2.7 mg/kg) did not improve spatial short-term memory in young and aged rats (Sirvi6 et al., 1991 a). In addition to the evident ineffectiveness of atipamezole to alleviate cholinergic dysfunction related spatial learning/memory impairment, one reason for these negative results in the effects of atipamezole on spatial learning/ memory could be that aged rats do not have a cortical and hippocampal noradrenergic hypofunction (Harik et al., 1986; McIntosh and Westfall, 1987; Santiago et al., 1988; Valjakka et al., 1990 a; Gallagher et al., 1990). Furthermore, it could be argued that noradrenergic hypofunction does not impair spatial learning/memory, because partial or extensive depletions of noradrenaline in the forebrain do not impair spatially cued navigation in water maze (as also assessed in the present study) in adult rats (Riekkinen et al., 1990; Selden
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et al., 1990; Valjakka et al., 1990 b). However, our recent data suggest that partial noradrenaline depletion impairs water maze acquisition in aged rats, but not in young rats (Sirvi6 etal., 1991 b). It remains to be determined whether atipamezole is effective in improving spatial learning/memory in aged rats with induced partial noradrenaline depletion. Previously, it has been shown that alpha-2 agonists, especially guanfacine at low doses (0.0001-0.001 mg/kg), improved spatial short-term memory in aged monkeys, and this improvement was proposed to be due to the activation of postjunctional alpha-2 adrenoceptors (Arnsten and Goldman-Rakic, 1985, 1990; Arnsten et al., 1988). It is not known how effectively atipamezole binds to the postjunctional alpha-2 adrenoceptors versus alpha-2 autoreceptors. Thus, it could be argued that atipamezole may impair spatial memory in aged rats due to a blockade of postsynaptic alpha-2 adrenoceptors despite the increased turnover of noradrenaline. However, a low dose (0.001 mg/kg) of guanfacine did not improve the acquisition deficit of water maze task in aged rats (Sirvi6 etal., 1991 c). In conclusion, the present results suggest that atipamezole, a selective and specific alpha-2 adrenoceptor antagonist, does not improve spatial learning/ memory deficit due to aging or blockade of muscarinic receptors in rats.
Acknowledgements We are grateful for Ms. S. Palviainen for her excellent secretarial help. This study was supported by the Academy of Finland.
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Zanotti A, Valzelli L, Toffano G (1989) Chronic phosphatidyl serine treatment improves spatial memory and passive avoidance in aged rats. Psychopharmacology 99:316-321 Authors' address: J. SirviS, PhD, Department of Neurology, University of Kuopio, P.O. Box 1627, SF-70211 Kuopio, Finland. Received April 11, 1991
