- Email: [email protected]
Effects of nitrendipine on reference and working memory of rats in three-panel runway
Pharmacological Research 50 (2004) 367–370
Effects of nitrendipine on reference and working memory of rats in three-panel runway Pınar Yamantürk Çelik∗ , Ya˘gız Üresin, Hüseyin Tonyalı Department of Pharmacology and Clinical Pharmacology, Istanbul Faculty of Medicine, Istanbul University, 34390 Capa, Istanbul, Turkey Accepted 19 January 2004
Abstract This study was designed to investigate whether calcium-channel blocker, nitrendipine affects memory of rats in three-panel runway test. Nitrendipine (2–4 mg kg−1 , intra peritoneally (i.p.)) neither enhanced nor impaired reference and working memory performances of young adult rats. However, it improved impairment in reference memory induced by anticholinergic drug scopolamine (3 mg kg−1 , i.p.) while it had no effects on impairment in working memory induced by the same drug. The results suggest that nitrendipine may be of benefit in the treatment of memory disturbances resulted from cholinergic deficit. © 2004 Elsevier Ltd. All rights reserved. Keywords: Calcium-channel blocker; Memory; Three-panel runway test; Scopolamine; Rats
1. Introduction Calcium-channel blockers have controversial effects on memory in animal models. These drugs were found to be able to enhance retention of passive avoidance learning and retention of appetitively motivated spatial discrimination learning in young adult mice [1]. They improve the retention deficits in animals exposed to electroconvulsive shock [2]. Yet, the long-term potentiation, experimental model for the synaptic changes that may underlie learning and memory has been found to be induced by different calcium sources [3,4]. Consistent with this, inhibition of calcium entry to the cell by calcium-channel blockers impairs memory performances of animals in some studies [5,6]. In fact, perturbations in calcium homeostasis frequently have been assumed as involved in the pathophysiology of vascular dementia and Alzheimer’s disease [7–9]. However, calcium hypothesis remains still obscure in Alzheimer’s disease [10]. There are stronger evidences that decreased central cholinergic transmission contributes significantly to the cognitive impairment associated with this disease [11]. On the other side, the relation between calcium and cholinergic system is conflicting. As known, cholinergic system [12] and hippocampus [13] are involved in memory formation. In hippocampal CA1 pyramidal neurons, nuclear cal∗ Corresponding
author. Tel.: +212-414-22-40; fax: +212-414-20-52. E-mail address: [email protected] (P. Yamantürk Çelik).
1043-6618/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2004.01.014
cium signalling is induced by cholinergic stimulation [14]. Yet, cholinergic agonist, muscarine causes calcium current decrement in the rat hippocampal pyramidal cells in vitro [15]. It is obvious that developing a better understanding of the interaction between calcium and cholinergic system is of critical importance to clarify more the role of calcium in memory disorders. Recently, a dihydropyridine calcium-channel blocker nitrendipine has been found to halve the rate of dementia in elderly hypertensive patients [9]. Thus, the role of cholinergic system in the effects of nitrendipine on memory is worth to investigate. At the present study, we assessed the effects of nitrendipine on reference and working memory performances of young adult rats with memory impairment induced by anticholinergic drug scopolamine in the three-panel runway test.
2. Methods 2.1. Subjects Male rats of Wistar strain (purchased from Institute for Experimental Medicine, Istanbul University) weighing 220–290 g at the beginning of the experiments were used. They were taken on a food deprivation schedule maintaining their weights at 80–85% of the free-feeding level. Rats were housed in groups of five per cage under constant
368
P. Yamantürk Çelik et al. / Pharmacological Research 50 (2004) 367–370
temperature (21 ± 2 ◦ C) and a 12 h light–dark cycle (light period: 7:00–19:00) with water ad libitum. All experiments were carried out according to the guidelines for the care of laboratory animals and in compliance with EEC Council Directive 86/609.
out three consecutive sessions, they were divided into groups that received injection of drugs as nitrendipine + saline, saline + scopolamine, nitrendipine + scopolamine, saline + saline, carboxymethyl cellulose + saline and carboxymethyl cellulose + scopolamine. Different animals were used to measure reference and working memory.
2.2. Drugs 2.4. Statistical analysis Nitrendipine (generous gift from Bayer, Turkey) at doses of 2 and 4 mg kg−1 , scopolamine hydrobromide (Sigma, St. Louis, MO) at the dose of 3 mg kg−1 were injected intraperitoneally (i.p.). Nitrendipine was dissolved in 0.5% solution of carboxymethyl cellulose mixing thoroughly before each injection and scopolamine was dissolved in saline and they were administered in volumes of 4 and 2 ml kg−1 , respectively. Rats were taken to the three-panel runway test 30 min after nitrendipine administration and 20 min after scopolamine administration. 2.3. Memory test Three-panel runway test was used to assess reference and working memory performances of rats according to the method previously described [16–18]. This test was chosen because its sensitivity is high and number of animals used is low to obtain statistical significance if there is significance. In brief, by means of a food deprivation schedule, rats were driven to find food pellet in the goal box of an apparatus (175 cm × 36 cm × 25 cm) composed of a start box, a goal box and consecutive choice points intervening between them. Before the goal box there were three choice points. Each choice point was composed a gate with three panels (12 cm × 25 cm). At the beginning of the test, a rat could pass through all the three-panel gates which were free of the front stoppers. Once the rats run the task repeatedly until the time elapsed to reach the goal box from the start box below 25 s the rats were given six consecutive trials (one session) per day. During the sessions, the front stopper of only one of the three-panel gates was removed at each choice point, so rats could pass through only one of the three-panel gates. For the reference memory, the locations of the correct (open) panel-gates (password) were kept constant within all sessions. For the working memory, the password was held constant only within a session changing in the next session. The number of times an animal tried to pass through incorrect panel-gate (error) and the time required for the animal to obtain food pellets (latency) were recorded for each rat along six trials of a session. The criterion of learning was less than six and less than 12 errors summed across the six trials of a session in reference and working memory tasks, respectively. The criterion was defined according to the study reported by Ohno et al. [18]. It seems that the average number of error is not decreasing more doing further trial under these conditions. Rats which were not reached the learning criterion were discarded from the study. After the rats achieved this criterion through-
The memory performances of rats in three-panel runway were analyzed by the same way with the previous report [18]. Namely, for the reference memory task, the number of errors and the latency were summed across all six trials of a session. Yet, they were summed from the second trial to the sixth trial of a session for the working memory since the first trial was given to present the correct panel-gate location in each session and did not reflect any memory function. All results were expressed as the mean±S.E. and analyzed using one-way analysis of variance. Further statistical analysis for individual groups was carried out by Dunnett’s test when F ratios reached significance. The criterion for statistical significance was P < 0.05.
3. Results Nitrendipine neither decreased nor increased number of errors and neither shortened nor prolonged latency values in both reference and working memory trials of young adult rats (Tables 1 and 2). Four milligrams per kg dose of it reversed scopolamine-induced increase in errors and prolongation in latency for reference memory but not for working memory trial (Tables 1 and 2). Carboxymethyl cellulose as vehicle of nitrendipine did not have any effects on these reTable 1 Effects of nitrendipine on reference memory performances of rats in three-panel runway test Drug
Trials 1–6 Number of errors
Saline + saline (control) Carboxymethyl cellulose + saline Nitrendipine(2) + saline Nitrendipine(4) + saline Saline + scopolamine(3) Carboxymethyl cellulose + scopolamine(3) Nitrendipine(4) + scopolamine(3)
1.7 1.6 2.0 1.8 20.5 20.8
± ± ± ± ± ±
0.4 0.3 0.2 0.3 1.1∗ 0.8∗
2.7 ± 0.2∗∗ F = 213.58 P < 0.0001
Latency (s) 34.4 36.3 38.3 36.5 161.4 152.1
± ± ± ± ± ±
3.2 3.2 2.7 3.1 4.9∗ 3.6∗
38.2 ± 2.8∗∗ F = 283.57 P < 0.0001
Each value represents the mean ± S.E. of the parameters summed across all six trials of a session and number in parenthesis is dose administered i.p. (mg kg−1 ). Number of animals in groups was six or seven, totally 47. ∗ P < 0.01 comparing to control or vehicle group according to Dunnett’s test. ∗∗ P < 0.01 comparing to saline + scopolamine group according to Dunnett’s test.
P. Yamantürk Çelik et al. / Pharmacological Research 50 (2004) 367–370
369
Table 2 Effects of nitrendipine on working memory performances of rats in three-panel runway test Drug
Number of errors
Saline + saline (control) Carboxymethyl cellulose + saline Nitrendipine(2) + saline Nitrendipine(4) + Saline Saline + scopolamine(3) Carboxymethyl cellulose + scopolamine(3) Nitrendipine(4) + scopolamine(3)
4.7 ± 0.6 5.0 ± 0.7 5.1 ± 0.7 6.3 ± 0.6 6.2 ± 0.5 5.4 ± 0.5 6.4 ± 0.6 F = 1.338 P < 0.2639
Trial 1
Latency (s) Trials 2–6 3.5 ± 0.3 3.3 ± 0.4 3.6 ± 0.2 3.5 ± 0.2 23.4 ± 1.3∗ 24.4 ± 1.0∗ 23.8 ± 0.7 F = 197.99 P < 0.0001
Trial 1
Trial 2–6
12.2 ± 0.9 12.0 ± 0.9 11.1 ± 1.0 12.0 ± 1.1 22.4 ± 1.6∗ 22.8 ± 1.3∗ 24.2 ± 1.4 F = 23.39 P < 0.0001
33.8 ± 1.3 33.3 ± 1.7 34.5 ± 2.6 35.7 ± 1.5 147.1 ± 3.2∗ 151.1 ± 2.7∗ 144.5 ± 2.5 F = 662.55 P < 0.0001
Each value represents the mean ± S.E. of the parameters recorded in the first trial and those summed from the second to the sixth trials of a session. First trial which does not reflect any memory function was given to present the correct panel gate location. Number in parenthesis is dose administered i.p. (mg kg−1 ). Number of animals in groups was six or seven, totally 46. ∗ P < 0.01 comparing to control or vehicle group according to Dunnett’s test.
sults. In order to minimize the number of animals, low dose of nitrendipine was not examined in scopolamine-induced change in memory since the effect was obtained with its high dose.
4. Discussion In the present study, nitrendipine was administered at the dose which is found to reverse long-term memory loss induced by phenytoin and valproate in rats [19]. At this dose, nitrendipine did not alter memory performances of young adult rats and ameliorated reference but not working memory performances of rats received scopolamine in three-panel runway task. These results are consistent with the reports that suggest calcium-channel blockers improve memory impairments [2,20,21] but contradictory with the data indicate that they can enhance memory [1,22,23] or can impair memory [5,6]. In some studies, the mode of administration has been claimed to determine the effects of calcium-channel blockers on memory [24]. Namely, their central administration impaires [5,24] while systemic administration enhances memory [1,23,24]. Nevertheless, the influence of the mode of administration in the effects of calcium-channel blockers on memory is not proven. Because in other studies, it has been found opposite that they enhance memory retention when given centrally [1,22] or cause memory deficit when given systemically [6]. Shortly, in all these studies, calcium-channel blockers using various administration ways within various dose ranges were investigated in a wide animal model spectrum. Therefore, such contraversions may be expected. However, it seems that calcium may well have a physiological role in memory function but the exact nature of that role is controversial for both normal and abnormal memory at present. In the study of Norman and colleagues, the improving effect of another calcium-channel blocker, nimodipine on the object recognition memory of rats was attributed
to the prevention of possible excessive calcium current induced by an anticholinergic, scopolamine [21]. At the present study, nitrendipine may cause the alleviating effect on scopolamine-induced memory impairment by the same way. On the other side, in cholinergic synapse of the Aplysia buccal ganglion, the presynaptic calcium current is due to calcium influxes through only N- and P-type channels trigger acetylcholine release but not L-type [25]. Similar characteristic of neurotransmitter release has also been shown in mammalian brain, in hippocampal Schaffer collateral-CA1 synapses [26]. This irrelevance between acetylcholine release and calcium influxes through L-type channel may account for the absence of memory impairing effect of L-type channel blockers like nitrendipine in memory intact animals. Our data are insufficient to explain why scopolamine-induced reference memory impairment is reversed by nitrendipine while working memory impairment not. The question of how and what cellular changes lead to the calcium imbalances within the neuron in the aging process that cause memory disturbances has been found important [27]. In fact, many changes occur in calcium regulation with aging including a chronic elevation of calcium influx through increased numbers of calcium channels [28]. Ameliorating effects of calcium-channel blockers on memory disorders in aging [9] can be attributed to this finding. Besides, calcium-channel blockers as neurotransmitter modulators and/or via calcium’s theoretical role in neurofibrillary tangles, proteolysis, or neurofilament formation may represent a therapeutic opportunity for the patients with Alzheimer’s disease [29]. It has also been shown that among event-related potential recordings of hypertensive patients with complaints suggesting initial stages of cerebral ischaemia that is one of important causes of memory deficits in aging, the left frontal lead N1 amplitude is increased by calcium-channel blocker nimodipine suggesting augmentation in attention and vigilance [30]. Taking together, perturbations in calcium homeostasis may have an
370
P. Yamantürk Çelik et al. / Pharmacological Research 50 (2004) 367–370
important role in different pathophysiologies associated with memory deficit in aging and calcium-channel blockers seem to be effective in these pathologies. Nevertheless, it is not clear whether hemodinamic or direct neuronal effects are involved in the mechanisms behind these therapeutic effects of calcium-channel blockers. The results of the present study suggest that calciumchannel blockers may not produce memory loss and furthermore they may ameliorate memory impairments not only the via hemodinamic but also improving the effects of cholinergic deficit. Many pharmacologic agents that have different target molecules improve animal memory impaired by anticholinergic drugs. Calcium seems to be another candidate molecule in the mechanisms underlying anticholinergic drug-induced memory impairment. Possible involvement of other mechanisms in memory processes should also be considered to explain the effects of calcium-channel blockers on memory. The limitations of their useful effects on memory should be defined more since working memory impairment induced by scopolamine was not improved by nitrendipine in this study.
Acknowledgements We are grateful to Kadir Öztürk for his help in conducting the experiments.
References [1] Quartermain D, deSoria VG, Kwan A. Calcium channel antagonists enhance retention of passive avoidance and maze learning in mice. Neurobiol Learn Mem 2001;75:77–90. [2] Zupan G, Vitezic D, Mrsic J, Matesic D, Simonic A. Effects of nimodipine, felodipine and amlodipine on electroconvulsive shock-induced amnesia in the rat. Eur J Pharmacol 1996;310:103–6. [3] Lynch G, Larson J, Kelso S, Barrionuevo G, Schottler F. Intracellular injections of EGTA block induction of long-term potentiation. Nature 1983;321:519–22. [4] Raymond CR, Redman SJ. Different calcium sources are narrowly tuned to the induction of different forms of LTP. J Neurophysiol 2002;88:249–55. [5] Deyo RA, Nix DA, Parker TW. Nifedipine blocks retention of a visual discrimination task in chicks. Behav Neural Biol 1992;57:260–2. [6] Maurice T, Bayle J, Privat A. Learning impairment following acute administration of the calcium channel antagonist nimodipine in mice. Behav Pharmacol 1995;6:167–75. [7] Gibson GE, Peterson C. Calcium and the aging nervous system. Neurobiol Aging 1987;8:329–43. [8] Mattson MP. Calcium as sculptor and destroyer of neural circuitry. Exp Gerontol 1992;27:29–49. [9] Forette F, Seux ML, Staessen JA, Thijs L, Birkenhager WH, Babarskiene MR, et al. Prevention of dementia in randomised double-blind placebo-controlled systolic hypertension in Europe (Syst–Eur) trial. Lancet 1998;352:1347–51. [10] Fritze J, Walden J. Clinical findings with nimodipine in dementia: test of the calcium hypothesis. J Neural Transm Suppl 1995;46:439–53.
[11] Collerton D. Cholinergic function and intellectual decline in Alzheimer disease. Neurosci 1986;19:1–28. [12] Bartus RT, Dean RL, Beer B, Lippa AS. The cholinergic hypothesis of geriatric memory dysfunction. Science 1982;217:408–17. [13] Sakurai Y. Hippocampal and neocortical cell assemblies encode memory processes for different types of stimuli in the rat. J Neurosci 1996;16:2809–19. [14] Power JM, Sah P. Nuclear calcium signaling evoked by cholinergic stimulation in hippocampal CA1 pyramidal neurons. J Neurosci 2002;22:3454–62. [15] Gahwiler BH, Brown DA. Muscarine affects calcium-currents in rat hippocampal pyramidal cells in vitro. Neurosci Lett 1987;76:301–6. [16] Furuya Y, Yamamoto T, Yatsugi S, Ueki S. A new method for studying working memory by using the three-panel runway apparatus in rats. Jpn J Pharmacol 1988;46:183–8. [17] Ohno M, Yamamoto T, Ueki S. Effect of the -receptor agonist, U-50, 488H, on cerebral ischemia-induced impairment of working memory assessed in rats by a three-panel runway task. Eur J Pharmacol 1991;193:357–61. [18] Ohno M, Yamamoto T, Watanabe S. Effects of intrahippocampal injections of N-methyl-d-aspartate receptor antagonists and scopolamine on working and reference memory assessed in rats by a three-panel runway task. J Pharmacol Exp Ther 1992;263:943–50. [19] Balakrishnan S, Bhargava VK, Pandhi P. Anticonvulsant and psychomotor activity of nitrendipine alone and in combination with phenytoin and valproate in rats. Methods Find Exp Clin Pharmacol 1999;21:441–7. [20] Batuecas A, Pereira R, Centeno C, Pulido JA, Hernandez M, Bollati A, et al. Effects of chronic nimodipine on working memory of old rats in relation to defects in synaptosomal calcium homeostasis. Eur J Pharmacol 1998;350:141–50. [21] Norman G, Brooks SP, Hennebry GM, Eacott MJ, Little HJ. Nimodipine prevents scopolamine-induced impairments in object recognition. J Psychopharmacol 2002;16:153–61. [22] Quevedo J, Vianna M, Daroit D, Born AG, Kuyven CR, Roesler R, et al. L-type voltage-dependent calcium channel blocker nifedipine enhances memory retention when infused into the hippocampus. Neurobiol Learn Mem 1998;69:320–5. [23] Isaacson RL, Johnston JE, Vargas DM. The effect of a calcium antagonist on the retention of simple associational learning. Physiol Behav 1988;42:447–52. [24] Deyo RA, Hittner JM. Effects of the Ca2+ channel antagonist flunarizine on visual discrimination learning. Neurobiol Learn Mem 1995;64:10–6. [25] Fossier P, Baux G, Tauc L. N- and P-Type Ca2+ channels are involved in acetylcholine release at a neuroneuronal synapse. Only the N-type channel is the target of neuromodulators. Proc Natl Acad Sci USA 1994;91:4771–5. [26] Wu LG, Saggau P. Pharmacological identification of two types of presynaptic voltage-dependent calcium channels at CA3-CA1 synapses of the hippocampus. J Neurosci 1994;14:5613–22. [27] Khachaturian ZS. Hypothesis on the regulation of cytosol calcium concentration and the aging brain. Neurobiol Aging 1987;8:345–6. [28] Landfield PW. Aging-related increase in hippocampal calcium channels. Life Sci 1996;59:399–404. [29] Tollefson GD. Short-term effects of the calcium channel blocker nimodipine (Bay-e-9736) in the management of primary degenerative dementia. Biol Psychiatry 1990;27:1133–42. [30] Uresin Y, Ermutlu N, Özek M, Karamürsel S. Effects of nimodipine on ischaemic neurological complaints and ERP (event related potential) in hypertensive patients. EPHAR, Federation of European Pharmacological Societies Affiliate Member of IUPHAR. In: Proceedings of the International Symposium on Drug Targets in Heart and Brain Ischaemia. Florence, Italy: Abstract Book; 1997. p. 69.
Effects of nitrendipine on reference and working memory of rats in three-panel runway Pınar Yamantürk Çelik∗ , Ya˘gız Üresin, Hüseyin Tonyalı Department of Pharmacology and Clinical Pharmacology, Istanbul Faculty of Medicine, Istanbul University, 34390 Capa, Istanbul, Turkey Accepted 19 January 2004
Abstract This study was designed to investigate whether calcium-channel blocker, nitrendipine affects memory of rats in three-panel runway test. Nitrendipine (2–4 mg kg−1 , intra peritoneally (i.p.)) neither enhanced nor impaired reference and working memory performances of young adult rats. However, it improved impairment in reference memory induced by anticholinergic drug scopolamine (3 mg kg−1 , i.p.) while it had no effects on impairment in working memory induced by the same drug. The results suggest that nitrendipine may be of benefit in the treatment of memory disturbances resulted from cholinergic deficit. © 2004 Elsevier Ltd. All rights reserved. Keywords: Calcium-channel blocker; Memory; Three-panel runway test; Scopolamine; Rats
1. Introduction Calcium-channel blockers have controversial effects on memory in animal models. These drugs were found to be able to enhance retention of passive avoidance learning and retention of appetitively motivated spatial discrimination learning in young adult mice [1]. They improve the retention deficits in animals exposed to electroconvulsive shock [2]. Yet, the long-term potentiation, experimental model for the synaptic changes that may underlie learning and memory has been found to be induced by different calcium sources [3,4]. Consistent with this, inhibition of calcium entry to the cell by calcium-channel blockers impairs memory performances of animals in some studies [5,6]. In fact, perturbations in calcium homeostasis frequently have been assumed as involved in the pathophysiology of vascular dementia and Alzheimer’s disease [7–9]. However, calcium hypothesis remains still obscure in Alzheimer’s disease [10]. There are stronger evidences that decreased central cholinergic transmission contributes significantly to the cognitive impairment associated with this disease [11]. On the other side, the relation between calcium and cholinergic system is conflicting. As known, cholinergic system [12] and hippocampus [13] are involved in memory formation. In hippocampal CA1 pyramidal neurons, nuclear cal∗ Corresponding
author. Tel.: +212-414-22-40; fax: +212-414-20-52. E-mail address: [email protected] (P. Yamantürk Çelik).
1043-6618/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2004.01.014
cium signalling is induced by cholinergic stimulation [14]. Yet, cholinergic agonist, muscarine causes calcium current decrement in the rat hippocampal pyramidal cells in vitro [15]. It is obvious that developing a better understanding of the interaction between calcium and cholinergic system is of critical importance to clarify more the role of calcium in memory disorders. Recently, a dihydropyridine calcium-channel blocker nitrendipine has been found to halve the rate of dementia in elderly hypertensive patients [9]. Thus, the role of cholinergic system in the effects of nitrendipine on memory is worth to investigate. At the present study, we assessed the effects of nitrendipine on reference and working memory performances of young adult rats with memory impairment induced by anticholinergic drug scopolamine in the three-panel runway test.
2. Methods 2.1. Subjects Male rats of Wistar strain (purchased from Institute for Experimental Medicine, Istanbul University) weighing 220–290 g at the beginning of the experiments were used. They were taken on a food deprivation schedule maintaining their weights at 80–85% of the free-feeding level. Rats were housed in groups of five per cage under constant
368
P. Yamantürk Çelik et al. / Pharmacological Research 50 (2004) 367–370
temperature (21 ± 2 ◦ C) and a 12 h light–dark cycle (light period: 7:00–19:00) with water ad libitum. All experiments were carried out according to the guidelines for the care of laboratory animals and in compliance with EEC Council Directive 86/609.
out three consecutive sessions, they were divided into groups that received injection of drugs as nitrendipine + saline, saline + scopolamine, nitrendipine + scopolamine, saline + saline, carboxymethyl cellulose + saline and carboxymethyl cellulose + scopolamine. Different animals were used to measure reference and working memory.
2.2. Drugs 2.4. Statistical analysis Nitrendipine (generous gift from Bayer, Turkey) at doses of 2 and 4 mg kg−1 , scopolamine hydrobromide (Sigma, St. Louis, MO) at the dose of 3 mg kg−1 were injected intraperitoneally (i.p.). Nitrendipine was dissolved in 0.5% solution of carboxymethyl cellulose mixing thoroughly before each injection and scopolamine was dissolved in saline and they were administered in volumes of 4 and 2 ml kg−1 , respectively. Rats were taken to the three-panel runway test 30 min after nitrendipine administration and 20 min after scopolamine administration. 2.3. Memory test Three-panel runway test was used to assess reference and working memory performances of rats according to the method previously described [16–18]. This test was chosen because its sensitivity is high and number of animals used is low to obtain statistical significance if there is significance. In brief, by means of a food deprivation schedule, rats were driven to find food pellet in the goal box of an apparatus (175 cm × 36 cm × 25 cm) composed of a start box, a goal box and consecutive choice points intervening between them. Before the goal box there were three choice points. Each choice point was composed a gate with three panels (12 cm × 25 cm). At the beginning of the test, a rat could pass through all the three-panel gates which were free of the front stoppers. Once the rats run the task repeatedly until the time elapsed to reach the goal box from the start box below 25 s the rats were given six consecutive trials (one session) per day. During the sessions, the front stopper of only one of the three-panel gates was removed at each choice point, so rats could pass through only one of the three-panel gates. For the reference memory, the locations of the correct (open) panel-gates (password) were kept constant within all sessions. For the working memory, the password was held constant only within a session changing in the next session. The number of times an animal tried to pass through incorrect panel-gate (error) and the time required for the animal to obtain food pellets (latency) were recorded for each rat along six trials of a session. The criterion of learning was less than six and less than 12 errors summed across the six trials of a session in reference and working memory tasks, respectively. The criterion was defined according to the study reported by Ohno et al. [18]. It seems that the average number of error is not decreasing more doing further trial under these conditions. Rats which were not reached the learning criterion were discarded from the study. After the rats achieved this criterion through-
The memory performances of rats in three-panel runway were analyzed by the same way with the previous report [18]. Namely, for the reference memory task, the number of errors and the latency were summed across all six trials of a session. Yet, they were summed from the second trial to the sixth trial of a session for the working memory since the first trial was given to present the correct panel-gate location in each session and did not reflect any memory function. All results were expressed as the mean±S.E. and analyzed using one-way analysis of variance. Further statistical analysis for individual groups was carried out by Dunnett’s test when F ratios reached significance. The criterion for statistical significance was P < 0.05.
3. Results Nitrendipine neither decreased nor increased number of errors and neither shortened nor prolonged latency values in both reference and working memory trials of young adult rats (Tables 1 and 2). Four milligrams per kg dose of it reversed scopolamine-induced increase in errors and prolongation in latency for reference memory but not for working memory trial (Tables 1 and 2). Carboxymethyl cellulose as vehicle of nitrendipine did not have any effects on these reTable 1 Effects of nitrendipine on reference memory performances of rats in three-panel runway test Drug
Trials 1–6 Number of errors
Saline + saline (control) Carboxymethyl cellulose + saline Nitrendipine(2) + saline Nitrendipine(4) + saline Saline + scopolamine(3) Carboxymethyl cellulose + scopolamine(3) Nitrendipine(4) + scopolamine(3)
1.7 1.6 2.0 1.8 20.5 20.8
± ± ± ± ± ±
0.4 0.3 0.2 0.3 1.1∗ 0.8∗
2.7 ± 0.2∗∗ F = 213.58 P < 0.0001
Latency (s) 34.4 36.3 38.3 36.5 161.4 152.1
± ± ± ± ± ±
3.2 3.2 2.7 3.1 4.9∗ 3.6∗
38.2 ± 2.8∗∗ F = 283.57 P < 0.0001
Each value represents the mean ± S.E. of the parameters summed across all six trials of a session and number in parenthesis is dose administered i.p. (mg kg−1 ). Number of animals in groups was six or seven, totally 47. ∗ P < 0.01 comparing to control or vehicle group according to Dunnett’s test. ∗∗ P < 0.01 comparing to saline + scopolamine group according to Dunnett’s test.
P. Yamantürk Çelik et al. / Pharmacological Research 50 (2004) 367–370
369
Table 2 Effects of nitrendipine on working memory performances of rats in three-panel runway test Drug
Number of errors
Saline + saline (control) Carboxymethyl cellulose + saline Nitrendipine(2) + saline Nitrendipine(4) + Saline Saline + scopolamine(3) Carboxymethyl cellulose + scopolamine(3) Nitrendipine(4) + scopolamine(3)
4.7 ± 0.6 5.0 ± 0.7 5.1 ± 0.7 6.3 ± 0.6 6.2 ± 0.5 5.4 ± 0.5 6.4 ± 0.6 F = 1.338 P < 0.2639
Trial 1
Latency (s) Trials 2–6 3.5 ± 0.3 3.3 ± 0.4 3.6 ± 0.2 3.5 ± 0.2 23.4 ± 1.3∗ 24.4 ± 1.0∗ 23.8 ± 0.7 F = 197.99 P < 0.0001
Trial 1
Trial 2–6
12.2 ± 0.9 12.0 ± 0.9 11.1 ± 1.0 12.0 ± 1.1 22.4 ± 1.6∗ 22.8 ± 1.3∗ 24.2 ± 1.4 F = 23.39 P < 0.0001
33.8 ± 1.3 33.3 ± 1.7 34.5 ± 2.6 35.7 ± 1.5 147.1 ± 3.2∗ 151.1 ± 2.7∗ 144.5 ± 2.5 F = 662.55 P < 0.0001
Each value represents the mean ± S.E. of the parameters recorded in the first trial and those summed from the second to the sixth trials of a session. First trial which does not reflect any memory function was given to present the correct panel gate location. Number in parenthesis is dose administered i.p. (mg kg−1 ). Number of animals in groups was six or seven, totally 46. ∗ P < 0.01 comparing to control or vehicle group according to Dunnett’s test.
sults. In order to minimize the number of animals, low dose of nitrendipine was not examined in scopolamine-induced change in memory since the effect was obtained with its high dose.
4. Discussion In the present study, nitrendipine was administered at the dose which is found to reverse long-term memory loss induced by phenytoin and valproate in rats [19]. At this dose, nitrendipine did not alter memory performances of young adult rats and ameliorated reference but not working memory performances of rats received scopolamine in three-panel runway task. These results are consistent with the reports that suggest calcium-channel blockers improve memory impairments [2,20,21] but contradictory with the data indicate that they can enhance memory [1,22,23] or can impair memory [5,6]. In some studies, the mode of administration has been claimed to determine the effects of calcium-channel blockers on memory [24]. Namely, their central administration impaires [5,24] while systemic administration enhances memory [1,23,24]. Nevertheless, the influence of the mode of administration in the effects of calcium-channel blockers on memory is not proven. Because in other studies, it has been found opposite that they enhance memory retention when given centrally [1,22] or cause memory deficit when given systemically [6]. Shortly, in all these studies, calcium-channel blockers using various administration ways within various dose ranges were investigated in a wide animal model spectrum. Therefore, such contraversions may be expected. However, it seems that calcium may well have a physiological role in memory function but the exact nature of that role is controversial for both normal and abnormal memory at present. In the study of Norman and colleagues, the improving effect of another calcium-channel blocker, nimodipine on the object recognition memory of rats was attributed
to the prevention of possible excessive calcium current induced by an anticholinergic, scopolamine [21]. At the present study, nitrendipine may cause the alleviating effect on scopolamine-induced memory impairment by the same way. On the other side, in cholinergic synapse of the Aplysia buccal ganglion, the presynaptic calcium current is due to calcium influxes through only N- and P-type channels trigger acetylcholine release but not L-type [25]. Similar characteristic of neurotransmitter release has also been shown in mammalian brain, in hippocampal Schaffer collateral-CA1 synapses [26]. This irrelevance between acetylcholine release and calcium influxes through L-type channel may account for the absence of memory impairing effect of L-type channel blockers like nitrendipine in memory intact animals. Our data are insufficient to explain why scopolamine-induced reference memory impairment is reversed by nitrendipine while working memory impairment not. The question of how and what cellular changes lead to the calcium imbalances within the neuron in the aging process that cause memory disturbances has been found important [27]. In fact, many changes occur in calcium regulation with aging including a chronic elevation of calcium influx through increased numbers of calcium channels [28]. Ameliorating effects of calcium-channel blockers on memory disorders in aging [9] can be attributed to this finding. Besides, calcium-channel blockers as neurotransmitter modulators and/or via calcium’s theoretical role in neurofibrillary tangles, proteolysis, or neurofilament formation may represent a therapeutic opportunity for the patients with Alzheimer’s disease [29]. It has also been shown that among event-related potential recordings of hypertensive patients with complaints suggesting initial stages of cerebral ischaemia that is one of important causes of memory deficits in aging, the left frontal lead N1 amplitude is increased by calcium-channel blocker nimodipine suggesting augmentation in attention and vigilance [30]. Taking together, perturbations in calcium homeostasis may have an
370
P. Yamantürk Çelik et al. / Pharmacological Research 50 (2004) 367–370
important role in different pathophysiologies associated with memory deficit in aging and calcium-channel blockers seem to be effective in these pathologies. Nevertheless, it is not clear whether hemodinamic or direct neuronal effects are involved in the mechanisms behind these therapeutic effects of calcium-channel blockers. The results of the present study suggest that calciumchannel blockers may not produce memory loss and furthermore they may ameliorate memory impairments not only the via hemodinamic but also improving the effects of cholinergic deficit. Many pharmacologic agents that have different target molecules improve animal memory impaired by anticholinergic drugs. Calcium seems to be another candidate molecule in the mechanisms underlying anticholinergic drug-induced memory impairment. Possible involvement of other mechanisms in memory processes should also be considered to explain the effects of calcium-channel blockers on memory. The limitations of their useful effects on memory should be defined more since working memory impairment induced by scopolamine was not improved by nitrendipine in this study.
Acknowledgements We are grateful to Kadir Öztürk for his help in conducting the experiments.
References [1] Quartermain D, deSoria VG, Kwan A. Calcium channel antagonists enhance retention of passive avoidance and maze learning in mice. Neurobiol Learn Mem 2001;75:77–90. [2] Zupan G, Vitezic D, Mrsic J, Matesic D, Simonic A. Effects of nimodipine, felodipine and amlodipine on electroconvulsive shock-induced amnesia in the rat. Eur J Pharmacol 1996;310:103–6. [3] Lynch G, Larson J, Kelso S, Barrionuevo G, Schottler F. Intracellular injections of EGTA block induction of long-term potentiation. Nature 1983;321:519–22. [4] Raymond CR, Redman SJ. Different calcium sources are narrowly tuned to the induction of different forms of LTP. J Neurophysiol 2002;88:249–55. [5] Deyo RA, Nix DA, Parker TW. Nifedipine blocks retention of a visual discrimination task in chicks. Behav Neural Biol 1992;57:260–2. [6] Maurice T, Bayle J, Privat A. Learning impairment following acute administration of the calcium channel antagonist nimodipine in mice. Behav Pharmacol 1995;6:167–75. [7] Gibson GE, Peterson C. Calcium and the aging nervous system. Neurobiol Aging 1987;8:329–43. [8] Mattson MP. Calcium as sculptor and destroyer of neural circuitry. Exp Gerontol 1992;27:29–49. [9] Forette F, Seux ML, Staessen JA, Thijs L, Birkenhager WH, Babarskiene MR, et al. Prevention of dementia in randomised double-blind placebo-controlled systolic hypertension in Europe (Syst–Eur) trial. Lancet 1998;352:1347–51. [10] Fritze J, Walden J. Clinical findings with nimodipine in dementia: test of the calcium hypothesis. J Neural Transm Suppl 1995;46:439–53.
[11] Collerton D. Cholinergic function and intellectual decline in Alzheimer disease. Neurosci 1986;19:1–28. [12] Bartus RT, Dean RL, Beer B, Lippa AS. The cholinergic hypothesis of geriatric memory dysfunction. Science 1982;217:408–17. [13] Sakurai Y. Hippocampal and neocortical cell assemblies encode memory processes for different types of stimuli in the rat. J Neurosci 1996;16:2809–19. [14] Power JM, Sah P. Nuclear calcium signaling evoked by cholinergic stimulation in hippocampal CA1 pyramidal neurons. J Neurosci 2002;22:3454–62. [15] Gahwiler BH, Brown DA. Muscarine affects calcium-currents in rat hippocampal pyramidal cells in vitro. Neurosci Lett 1987;76:301–6. [16] Furuya Y, Yamamoto T, Yatsugi S, Ueki S. A new method for studying working memory by using the three-panel runway apparatus in rats. Jpn J Pharmacol 1988;46:183–8. [17] Ohno M, Yamamoto T, Ueki S. Effect of the -receptor agonist, U-50, 488H, on cerebral ischemia-induced impairment of working memory assessed in rats by a three-panel runway task. Eur J Pharmacol 1991;193:357–61. [18] Ohno M, Yamamoto T, Watanabe S. Effects of intrahippocampal injections of N-methyl-d-aspartate receptor antagonists and scopolamine on working and reference memory assessed in rats by a three-panel runway task. J Pharmacol Exp Ther 1992;263:943–50. [19] Balakrishnan S, Bhargava VK, Pandhi P. Anticonvulsant and psychomotor activity of nitrendipine alone and in combination with phenytoin and valproate in rats. Methods Find Exp Clin Pharmacol 1999;21:441–7. [20] Batuecas A, Pereira R, Centeno C, Pulido JA, Hernandez M, Bollati A, et al. Effects of chronic nimodipine on working memory of old rats in relation to defects in synaptosomal calcium homeostasis. Eur J Pharmacol 1998;350:141–50. [21] Norman G, Brooks SP, Hennebry GM, Eacott MJ, Little HJ. Nimodipine prevents scopolamine-induced impairments in object recognition. J Psychopharmacol 2002;16:153–61. [22] Quevedo J, Vianna M, Daroit D, Born AG, Kuyven CR, Roesler R, et al. L-type voltage-dependent calcium channel blocker nifedipine enhances memory retention when infused into the hippocampus. Neurobiol Learn Mem 1998;69:320–5. [23] Isaacson RL, Johnston JE, Vargas DM. The effect of a calcium antagonist on the retention of simple associational learning. Physiol Behav 1988;42:447–52. [24] Deyo RA, Hittner JM. Effects of the Ca2+ channel antagonist flunarizine on visual discrimination learning. Neurobiol Learn Mem 1995;64:10–6. [25] Fossier P, Baux G, Tauc L. N- and P-Type Ca2+ channels are involved in acetylcholine release at a neuroneuronal synapse. Only the N-type channel is the target of neuromodulators. Proc Natl Acad Sci USA 1994;91:4771–5. [26] Wu LG, Saggau P. Pharmacological identification of two types of presynaptic voltage-dependent calcium channels at CA3-CA1 synapses of the hippocampus. J Neurosci 1994;14:5613–22. [27] Khachaturian ZS. Hypothesis on the regulation of cytosol calcium concentration and the aging brain. Neurobiol Aging 1987;8:345–6. [28] Landfield PW. Aging-related increase in hippocampal calcium channels. Life Sci 1996;59:399–404. [29] Tollefson GD. Short-term effects of the calcium channel blocker nimodipine (Bay-e-9736) in the management of primary degenerative dementia. Biol Psychiatry 1990;27:1133–42. [30] Uresin Y, Ermutlu N, Özek M, Karamürsel S. Effects of nimodipine on ischaemic neurological complaints and ERP (event related potential) in hypertensive patients. EPHAR, Federation of European Pharmacological Societies Affiliate Member of IUPHAR. In: Proceedings of the International Symposium on Drug Targets in Heart and Brain Ischaemia. Florence, Italy: Abstract Book; 1997. p. 69.