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Effects of vasoactive intestinal peptide (VIP) on scopolamine-induced amnesia in the rat
Neuropeptides (1994) 26,153-l 58 0 Longman Group UK Ltd 1994
Effects of Vasoactive Intestinal Peptide (VIP) on Scopolamine-induced Amnesia in the Rat Y. YAMAGUCHI, and H. KOBAYASHI Research laboratory, Zenyaku Kogyo Co., Ltd, 2-33-7, Japan (Reprint requests to HK)
Ohizumi-machi,
Nerima-ku,
Tokyo 178,
Abstract-The effects of vasoactive intestinal peptide (VIP) on spatial cognitive deficits induced in the rat by injections of scopolamine were examined in a radial arm maze. A single intraperitoneal (Lp.), subcutaneous (s.c.) or intracerebroventricular (i.c.v.) injection of VIP inhibited the reduction in the number of initial correct responses in rats with scopolamineinduced amnesia. The inhibition was associated with a bell-shaped dose-response curve. Thus, VIP appears to have an ameliorating effect on spatial cognitive deficits induced by scopolamine in the rat.
Introduction VIP was first isolated from the pig small intestine,l and VIP was subsequently also found in the central nervous system of vertebrates.2 In the rat, VIP has been shown to exert a powerful excitatory effect on CA1 neurons in the hippocampus and to activate choline acetyltransferase (ChAT) in the dorsal and ventral regions of the hippocan-~pus.~ Since the hippocampus plays a critical role in memory modulation?-’ VIP might be expected to play an important role in memory modulation in conjunction with cholinergic neurons. It has been demonstrated that, in rats, i.c.v. injection of VIP inhibits memory retention in a stepDate received 8 July 1993 Date accepted 13 July 1993 Correspondence to: Dr H. Kobayashi, Research Laboratory, Zenyaku Kogyo Co., Ltd, 2-33-7 Ohizumi-machi, Nerima-ku, Tokyo 178, Japan.
through passive avoidance task.* Moreover, in mice, i.c.v. or intrahippocampal injection of VIP impaired memory retention in left-right footshock avoidance in a T-maze task,g while i.c.v. or intrahippocampal injection of a VIP-receptor antagonist, [4X1-DPhe6,Leu17]VIP, enhanced memory retention in a left-right footshock avoidance in a T-maze task.9 All these reports suggest that VIP is an amnestic neuropeptide. By contrast, Glowa et aI’* reported that i.c.v. injection of a VIP receptor antagonist, a hybrid molecule of VIP and neurotensin which consisted of 28 amino acids,” produced retardation of spatial discrimination in the Morris water maze in rats. This finding suggests that VIP acts not as an amnestic, but as an antiamnestic neuropeptide. Thus, the results concerning the effects of VIP on memory processes are contradictory. In the present study, the effects of VIP on performance in a radial arm maze were examined to determine whether VIP is an amnestic or an antiamnestic agent. 153
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Materials and methods Animals Male Sprague-Dawley rats, weighing from 250-300 g, were obtained from Charles River Inc. (Kanagawa, Japan). They were housed individually in a room maintained at around 22°C with a 12-h light/dark cycle. Apparatus Each animal was tested in a standard radial arm maze, which was a slightly modified version of the one originally used by Olton and Samuelson.’ The radial arm maze, situated 50 cm above the floor, consisted of an octagonal center platform (28 cm in diameter) with 8 equally spaced radial arms (50 cm long x 12 cm wide). A circular food well (1 cm deep and 2 cm in diameter) was located at the far end of each arm. Implantation of cannulas The animals were anesthetized by i.p. injection with 50 mg/kg pentobarbital sodium salt (Tokyo Kasei Co., Tokyo, Japan). A stainless-steel guide cammla (21 gauge) was implanted stereotaxically 0.8 mm posterior, 1.5 mm lateral to the bregma and 3.5 mm below the surface of the cranium, according to the atlas of Paxinos and Watson.13 The tip of the camnda was adjusted such that it was located in the lateral ventricle. Two stainless-screws were placed around the cannula as anchors and fixed firmly to the skull
with acrylic dental cement. To prevent blood coagulation within the cannula while it was not in use, a piece ofthin stainless-steel wire was inserted into the cannula. I.c.v. injections were given through an internal cannula (26 gauge), the tip of which was positioned 1 mm below the tip of the guide cannula. The other tip of the internal cannula was connected to a lo-p1 Hamilton syringe by polyethylene tubing. At the end of each experiment, the tip of the guide cannula was checked visually to ascertain whether it was located in the lateral ventricle. Experimental procedures The experimental protocol is summarized in Figure 1. In animals to be injected i.c.v., the pretraining started 8 d after implantation of the cannula. A restricted feeding schedule was started on the day 1 of pretraining in experiments with i.p., S.C.or i.c.v. injections. The body weights of the rats fell to approximately 80-85‘7~ of the starting body weights during pretraining. Subsequently, the body weights increased by 5 g per week per rat with the restricted feeding schedule. Pretraining On day 1 of the restricted feeding schedule, animals were placed in groups (4-6 animals) on the central hub of the maze for 10 min. On days 2,3 and 4, rats were placed on the maze in similar groups for 10 min with lumps of crystallized sugar scattered on the platform and arms. During this period each rat was han-
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Fig. 1 Experimental schedule for examina tion of the effects of administration of VIP on scopolamine-induced amnesia in rats in the radial arm maze. VIP (i.c.v., i.p. or s.c.) was injected once 30 min before rats were placed in the maze, and scopolamine (i.p.) was injected once 20 min before rats were placed in the maze.
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dled for approximately 5 min daily. For 2 days after pretraining, rats were not subjected to any treatment.
ante (ANOVA) followed by Dunnett’s multiple comparison test.
Training trials Beginning on day 7 of the restricted feeding schedule, training trials were begun. Each rat received 3 training trials (1-3 in Fig. l), with one trial per day for 5 days a week. Prior to each trial, the wells in all arms were baited with 3 lumps of crystallized sugar (approximately 50 mg in total) and then the rat was placed alone on the platform. Each trial continued until the bait in all 8 wells had been consumed, until 16 choices had been made, or until 10 min had elapsed, whichever occurred first. Test trials Test trials were carried out from the 4th week to the 10th or 11th week of training trials, and on every 4th day of each trial. Administration of drugs VIP (Peptide Institute Inc., Osaka, Japan) was dissolved in saline. Doses were 25,50,75,100 and 250 pikg for single i.p. injections; 1, 5, 10, 50 and 100 p/kg for single S.C.injections and 10,25,50 and 100 @-at for single i.c.v. injections. Injection volumes were 1 ml/kg for i.p. and S.C.injections, and 2 pl for i.c.v. injections. Injections were given 30 min before rats were put in the maze. Scopolamine hydrobromide (Merck, Darmstadt, Germany), dissolved in saline, was injected i.p. at a dose of 0.5 mg/kg in a volume of 1 ml/kg 20 min before rats were put in the maze. In the control experiments, rats received an equal volume of the vehicle. Assessment of performance The rats were required to learn how to obtain food from the well in each arm and to remember not to re-enter arms in which they had already found food. Re-entry into an arm that had previously been visited was scored as an error. Numbers of consecutive correct choices prior to re-entry into a previously visited arm (the number of initial correct responses) were recorded as an index of performance. Statistical analysis The statistical significance of differences between groups was calculated by one-way analysis of vari-
Results I.p. administration of scopolamine The number of initial correct responses was significantly decreased by injection of scopolamine, compared with control, saline-treated group (Figs 2,3 & 4). Thus, amnesia was induced by a single i.p. administration of scopolamine at a dose of 0.5 mgkg. 1.p. administration of VIP The number of initial correct responses was increased significantly by i.p. administration of VIP at doses of 50 and 75 pg/kg, given 10 min before injection of scopolamine. The effect was associated with a bell-shaped dose-response curve (Fig. 2). Thus, i.p. administration of VIP mitigated the amnesia induced by scopolamine. Xc. administration of V.P The number of initial correct responses was enhanced significantly by S.C.administration of VIP at doses of 5, 10 and 50 pg/kg, given 10 min before
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Fig. 2 Effects of i.p. administration of VIP on scopolamineinduced amnesia in rats in the radial arm maze. Each column represents the mean number of initial correct responses and vertical bars show SEM. The number in each column shows the number of animals used in the experiment. ##p < 0.01, compared with a saline control group. **p c 0.01, compared with a group treated with scopolamine only.
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between the earlier results and ours may be due to a difference in the nature ofthe reinforcement. In these tasks, animals were trained by a conditioned avoidance which was reinforced by aversive electric stimuli. Since such stimuli are unlikely to occur under normal life conditions, Ogawa et alI4pointed out that such anunnatural experimental procedure is far from ideal for the screening of antidementia drugs. Moreover, Watanabe et al” also pointed out recently that the memory mechanisms used in these tasks did not seem to be identical to those which are disrupted in amnesic patients, since these patients were reportedly able to make appropriate avoidance responses. I6 Therefore, it may be difficult to evaluate whether or not the results from a learning task reinforced with electric stimuli are due, in fact, to memory. However, it is considered that rats are not frightened by the radial arm maze task used in our experiments, in which animals are positively reinforced with the acquisition of food. Moreover, Watanabe et alI5 reported that results obtained from a radial maze task might be better suited for evaluation of memory performance in rats than tasks which are negatively reinforced with electric shock. Thus, our results obtained with the radial arm maze task demonstrate clearly that VIP has an ameliorating effect on memory processes.
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Fig. 3 Effects of S.C. administration of VIP on scopolamineinduced amnesia in rats in the radial arm maze. See Figure 2 for explanations. ###p< 0.01, compared with a saline control group. +p
injection of scopolamine. The responses were associated with a bell-shaped dose-response curve (Fig. 3). The results showed that S.C.injection of VIP mitigated the amnestic effect of scopolamine.
I. C.V.injection of VIP The number of initial correct responses was also increased significantly by i.c.v. injection of VIP at doses of 25 and 50 r&at, given 10 min before injection of scopolamine. The responses were associated with a bell-shaped dose-response curve (Fig. 4). Thus, i.c.v. administration of VIP also mitigated the amnestic effect of scopolamine.
r.
Discussion In the present study, single injections of VIP via three different routes (i.p., S.C.and i.c.v.) inhibited the reduction in numbers of initial correct responses caused by scopolamine, indicating that VIP has antiamnestic effects. However, amnestic effects of VIP have been reported by others: i.c.v. injection of VIP inhibited performance of a learning task which is negatively reinforced with electric shock, such as a step-through passive avoidance task and pole-jumping active avoidance task inrats* and a left-right footshock avoidance task in mice.g Thus, earlier reports are inconsistent with our results. The difference
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Fig. 4 Effects 0fi.c.v. injection ofVIP on scopolamine-induced amnesia in rats in the radial arm maze. See Figure 2 for explanations. ##p < 0.0 1, compared with a saline control group. lp < 0.05, **p < 0.01, compared with a group treated only with scopolamine.
VIP AND SCOPOLAMINE-INDUCED
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High doses of VIP (100 pg, i.p. and s.c.; 100 ng, i.c.v.) did not have any ameliorating effects under the present conditions. This observation is consistent with the observations by others that most cognition-improving drugs give bell-shaped doseresponse curves.7J7J* The minimum effective doses of VIP associated with reversal of memory deficits induced by scopolamine were 5 and 50 pg for S.C. and i.p. injections, respectively, suggesting that the S.C.route was more effective than the i.p. route. In the present study, i.c.v. injection of VIP caused the reversal of scopolamine-induced amnesia. VIP is considered to act through specific VIP receptors in the brain. Indeed, the highest density of binding sites for VIP has been found in the cerebral cortex and the dentate gyrus of the hippocampus in the rat Lg~20 and the highest concentrations of VIP in the brain have been found in the cerebral cortex, amygdala and hippocampus (see 21for review). It is well known that excitotoxic lesions of the nucleus basalis magnocellularis, the origin of cholinergic projections to the cortex and amygdala, produce impairment of the performance of learning tasks.22J3 Eckenstein and Baughman24 reported that cholinergic neurons in the cortex showed positive immunoreactivity for VIP. We found that scopolamine, a cholinergic receptor antagonist, induced memory deficits, as reported earlier by several groups of investigators .25-27 Together these findings suggest that VIP plays important roles in memory processes in cooperation with cholinergic mechanisms in the cortex and/or the hippocampus. It is known that lesions of the septo-hippocampal afferents5-’ and lesions of the hippocampusZs produce impairments in learning. Luine et al4 reported that VIP enhanced ChAT activity in the dorsal and ventral regions of the hippocampus. Therefore, it is possible that the ameliorating effects of VIP on memory processes might occur via enhancement of the ChAT activity in the hippocampus. 1.~. and S.C. administration of VIP resulted in reversal ofmemory deficits. It seems likely that VIP, injected i.p. and s.c., exerts its effects through cholinergic mechanisms.
Acknowledgements We are indebted to Mr K. Hashimoto, the Administrative Director, Research and Development, Zenyalcu Kogyo Co., Ltd, for his unwavering encouragement and support.
References 1. Said, S. I. and Mutt, B. Polypeptlde with broad biological activity: isolation from small intestine. Science 1970; 169: 1217-1218. 2. Magistretti, P. J., Dietl, M. M., Hof, P. R. et al. Vasoactive intestinal peptide as a mediator of intercellular communication in the cerebral cortex. Ann. NY Acad. Sci. 1988; 527: 110-129. 3. Dodd, J., Kelly, J. S. and Said, S. I. Excitation of CA1 neurones of the rat hippocampus by the octacompeptide, vasoactive intestmal polypeptde (VIP). Br. J. Phammcol. 1979; 66: 125. 4. Luine, V. N., Rostene, W., Rhodes, J. and McEwen, B. S. Activation of choline acetyltransferase by vasoactive intestinal peptide. J. Neurochem. 1984; 42: 113 l-l 134. 5. Hanan. J. J.. Salamone. J. D.. Simpson. J.. Iversen. S. D. and M&is; R. b. M. Place navigation in rats is impaired by lesions of medial septum and diagonal band but not nucleus basalis magnocelhtlarls. Behav. Brain Res. 1988; 27: 9-20. 6. Ho&a, A., Carino, M. A., Zabawska, J. and Lai, H. TRH analog MK-77 1 reverses neurochemical and learning deficits in medial septal-lesioned rats. Peptides 1989; 10: 121-124. 7. Matauoka, N., Maeda, N., Ohkubo, Y. and Yamaguchi, I. Differential effects of physostigmine and pilocarpine on the spatial memory deficits produced by two septo-hippocampal deafferentations in rats. Brain Res. 1991; 559: 233-240. 8. Cottrell, G. A., Veldhuis, H. D., Rostene, W. H. and de Kloet, E. R. Bohavioural actions of vasoactive intestinal peptide (VIP). Neuropeptides 1984; 4: 331-341. 9. Flood, J. F., Garland, J. S. and Morley, J. E. Vasoactive intestinal peptide (VIP): an amnestic neuropeptide. Peptides 1990; 11: 933-938. 10. Glowa, J. R., Panlilio, L. V., Bremreman, D. E., Gozes, I., Frldkin, M. and Hill, J. M. Learning impairment following intracerobral administration of the HIV envelope protein gpl20 or a VIP antagonist. Brain Ros. 1992; 570: 49-53. 11. Gozos, I., Moltzer, E., Rubinrout, S., Brenneman, D. E. and Fridkin, M. Vasoactive intestinal peptide potentiates sexual behavior: inhibition by novel antagonist. Endocrinology 1989; 125: 2945-2949. 12. Olton, D. S. and Samuelson, R J. Remembrance of places passed: spatialmemory inrats.J. Exp. Psychol. Anim. Behav. Processes 1976; 2: 97-l 16. 13. Paxinos, G. and Watson, C. The Rat Brain in Stereotaxic Coordinates. New York: Academic Press, 1982. 14. Ogawa, N., Haba, K., Sora, Y. H., Higashida, A., Sate, H. and Ogawa, S. Comparison of the effects of bifemelane hydrochloride and indeloxazine hydrochloride on scopolamine hydrobromide-induced impairment in radial maze performance. Clin. l-her. 1988; 10: 704-711. 15. Watanabe, H., Ni, J.-W., Ohta, H., Ni, X.-H. and Matsumoto, K. A kampo prescription, Shimotsu-to, improves scopolamino-induced spatial cognitive deficits in rats. Jap. J. Psychopharmacol. 1991; 11: 215-222. 16. Weiskrantz, L. and Wanington, E. K. Conditioning in amnesic patients. Neuropsychologia 1979; 17: 187-194. 17. Flood, J. F., Landry, D. W. and Jarvik, M. E. Cholinergic receptor interactions and their effects on long-term memory processing. Brain Res. 1981; 215: 177-185. 18. Cumin, R., Bandle, E. F., Gamzu, E. and Haefoly, W. E. Effects of the novel compound aniracotam (Ro 13-5057) upon impaired loaming and memory in rodents. Psychopharmacology 1982; 78: 104-l 11. 19. Besson, J., Sarrioau, A., Vial, M., Marie, J.-C. Rosselin, G. and Rostene, W. Characterization and autoradiographic dis-
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tribution of vasoactive intestinal peptide binding sites in the rat central nervous system. Brain Res. 1986; 398: 329-336. Dietl, M. M., Hof, P. R., Martin, J.-L., Magistretti, P. J. and Palacios, J. M. Autoradiographic analysis of the distribution of vasoactive intestinal peptide binding sites in the vertebrate central nervous system: a phylogenetic study. Brain Res. 1990; 520: 14-26. Rostene, W. H. Neurobiological and neuroendocrine fonctions of the vasoactive intestinal peptide (VIP). Prog. Neurobiol. 1984; 22: 103-129. Connor, D. J., Langlais, P. J. and Thal, L. J. Behavioral impairments after lesions of the nucleus basalis by ibotenic acid and auisaualic acid. Brain Res. 1991: 555: 84-90. Imaizumi: K.,.Kudo, Y., Shiosaka, S. et al. Specific cholinergic destruction in the basal magnocellular nucleus and impaired passive avoidance behavior of rodents. Brain Res. 1991; 551: 36-43.
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24. Eckenstein, F. and Baughman, R. W. Two types of cholinergic innervation in cortex, one co-localized with vasoactive intestinal polypeptide. Nature 1984; 309: 153-l 55. 25. Eckerman, D. A., Gordon, W. A., Edwards, J. D., MacPhail, R. C. and Gage, M. I. Effects of scopolamine, pentobarbital, and amphetamine on radial arm maze performance in the rat. Pharmacol. Biochem. Behav. 1979; 12: 595-602. 26. Wirsching, B. A., Beninger, R. J., Jhamandas, K., Boegman, R. J. and EL-Defrawy, S. R. Differential effects of scopolamine on working and reference memory of rats in the radial maze. Pharmacol. Biochem. Behav. 1984; 20: 659-662. 27. Flood, J. F. and Cherkin, A. Scopolamine effects on memory retention in mice: a model of dimentia? Behav. Neural Biol. 1986; 45: 169-184. 28. Morris, R. G. M., Garrud, P., Rawlins, J. N. P. and O’Keefe, J. Place navigation impaired in rats with hippocampal lesions. Nature 1982; 297: 681-683.
Effects of Vasoactive Intestinal Peptide (VIP) on Scopolamine-induced Amnesia in the Rat Y. YAMAGUCHI, and H. KOBAYASHI Research laboratory, Zenyaku Kogyo Co., Ltd, 2-33-7, Japan (Reprint requests to HK)
Ohizumi-machi,
Nerima-ku,
Tokyo 178,
Abstract-The effects of vasoactive intestinal peptide (VIP) on spatial cognitive deficits induced in the rat by injections of scopolamine were examined in a radial arm maze. A single intraperitoneal (Lp.), subcutaneous (s.c.) or intracerebroventricular (i.c.v.) injection of VIP inhibited the reduction in the number of initial correct responses in rats with scopolamineinduced amnesia. The inhibition was associated with a bell-shaped dose-response curve. Thus, VIP appears to have an ameliorating effect on spatial cognitive deficits induced by scopolamine in the rat.
Introduction VIP was first isolated from the pig small intestine,l and VIP was subsequently also found in the central nervous system of vertebrates.2 In the rat, VIP has been shown to exert a powerful excitatory effect on CA1 neurons in the hippocampus and to activate choline acetyltransferase (ChAT) in the dorsal and ventral regions of the hippocan-~pus.~ Since the hippocampus plays a critical role in memory modulation?-’ VIP might be expected to play an important role in memory modulation in conjunction with cholinergic neurons. It has been demonstrated that, in rats, i.c.v. injection of VIP inhibits memory retention in a stepDate received 8 July 1993 Date accepted 13 July 1993 Correspondence to: Dr H. Kobayashi, Research Laboratory, Zenyaku Kogyo Co., Ltd, 2-33-7 Ohizumi-machi, Nerima-ku, Tokyo 178, Japan.
through passive avoidance task.* Moreover, in mice, i.c.v. or intrahippocampal injection of VIP impaired memory retention in left-right footshock avoidance in a T-maze task,g while i.c.v. or intrahippocampal injection of a VIP-receptor antagonist, [4X1-DPhe6,Leu17]VIP, enhanced memory retention in a left-right footshock avoidance in a T-maze task.9 All these reports suggest that VIP is an amnestic neuropeptide. By contrast, Glowa et aI’* reported that i.c.v. injection of a VIP receptor antagonist, a hybrid molecule of VIP and neurotensin which consisted of 28 amino acids,” produced retardation of spatial discrimination in the Morris water maze in rats. This finding suggests that VIP acts not as an amnestic, but as an antiamnestic neuropeptide. Thus, the results concerning the effects of VIP on memory processes are contradictory. In the present study, the effects of VIP on performance in a radial arm maze were examined to determine whether VIP is an amnestic or an antiamnestic agent. 153
154
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Materials and methods Animals Male Sprague-Dawley rats, weighing from 250-300 g, were obtained from Charles River Inc. (Kanagawa, Japan). They were housed individually in a room maintained at around 22°C with a 12-h light/dark cycle. Apparatus Each animal was tested in a standard radial arm maze, which was a slightly modified version of the one originally used by Olton and Samuelson.’ The radial arm maze, situated 50 cm above the floor, consisted of an octagonal center platform (28 cm in diameter) with 8 equally spaced radial arms (50 cm long x 12 cm wide). A circular food well (1 cm deep and 2 cm in diameter) was located at the far end of each arm. Implantation of cannulas The animals were anesthetized by i.p. injection with 50 mg/kg pentobarbital sodium salt (Tokyo Kasei Co., Tokyo, Japan). A stainless-steel guide cammla (21 gauge) was implanted stereotaxically 0.8 mm posterior, 1.5 mm lateral to the bregma and 3.5 mm below the surface of the cranium, according to the atlas of Paxinos and Watson.13 The tip of the camnda was adjusted such that it was located in the lateral ventricle. Two stainless-screws were placed around the cannula as anchors and fixed firmly to the skull
with acrylic dental cement. To prevent blood coagulation within the cannula while it was not in use, a piece ofthin stainless-steel wire was inserted into the cannula. I.c.v. injections were given through an internal cannula (26 gauge), the tip of which was positioned 1 mm below the tip of the guide cannula. The other tip of the internal cannula was connected to a lo-p1 Hamilton syringe by polyethylene tubing. At the end of each experiment, the tip of the guide cannula was checked visually to ascertain whether it was located in the lateral ventricle. Experimental procedures The experimental protocol is summarized in Figure 1. In animals to be injected i.c.v., the pretraining started 8 d after implantation of the cannula. A restricted feeding schedule was started on the day 1 of pretraining in experiments with i.p., S.C.or i.c.v. injections. The body weights of the rats fell to approximately 80-85‘7~ of the starting body weights during pretraining. Subsequently, the body weights increased by 5 g per week per rat with the restricted feeding schedule. Pretraining On day 1 of the restricted feeding schedule, animals were placed in groups (4-6 animals) on the central hub of the maze for 10 min. On days 2,3 and 4, rats were placed on the maze in similar groups for 10 min with lumps of crystallized sugar scattered on the platform and arms. During this period each rat was han-
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Fig. 1 Experimental schedule for examina tion of the effects of administration of VIP on scopolamine-induced amnesia in rats in the radial arm maze. VIP (i.c.v., i.p. or s.c.) was injected once 30 min before rats were placed in the maze, and scopolamine (i.p.) was injected once 20 min before rats were placed in the maze.
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dled for approximately 5 min daily. For 2 days after pretraining, rats were not subjected to any treatment.
ante (ANOVA) followed by Dunnett’s multiple comparison test.
Training trials Beginning on day 7 of the restricted feeding schedule, training trials were begun. Each rat received 3 training trials (1-3 in Fig. l), with one trial per day for 5 days a week. Prior to each trial, the wells in all arms were baited with 3 lumps of crystallized sugar (approximately 50 mg in total) and then the rat was placed alone on the platform. Each trial continued until the bait in all 8 wells had been consumed, until 16 choices had been made, or until 10 min had elapsed, whichever occurred first. Test trials Test trials were carried out from the 4th week to the 10th or 11th week of training trials, and on every 4th day of each trial. Administration of drugs VIP (Peptide Institute Inc., Osaka, Japan) was dissolved in saline. Doses were 25,50,75,100 and 250 pikg for single i.p. injections; 1, 5, 10, 50 and 100 p/kg for single S.C.injections and 10,25,50 and 100 @-at for single i.c.v. injections. Injection volumes were 1 ml/kg for i.p. and S.C.injections, and 2 pl for i.c.v. injections. Injections were given 30 min before rats were put in the maze. Scopolamine hydrobromide (Merck, Darmstadt, Germany), dissolved in saline, was injected i.p. at a dose of 0.5 mg/kg in a volume of 1 ml/kg 20 min before rats were put in the maze. In the control experiments, rats received an equal volume of the vehicle. Assessment of performance The rats were required to learn how to obtain food from the well in each arm and to remember not to re-enter arms in which they had already found food. Re-entry into an arm that had previously been visited was scored as an error. Numbers of consecutive correct choices prior to re-entry into a previously visited arm (the number of initial correct responses) were recorded as an index of performance. Statistical analysis The statistical significance of differences between groups was calculated by one-way analysis of vari-
Results I.p. administration of scopolamine The number of initial correct responses was significantly decreased by injection of scopolamine, compared with control, saline-treated group (Figs 2,3 & 4). Thus, amnesia was induced by a single i.p. administration of scopolamine at a dose of 0.5 mgkg. 1.p. administration of VIP The number of initial correct responses was increased significantly by i.p. administration of VIP at doses of 50 and 75 pg/kg, given 10 min before injection of scopolamine. The effect was associated with a bell-shaped dose-response curve (Fig. 2). Thus, i.p. administration of VIP mitigated the amnesia induced by scopolamine. Xc. administration of V.P The number of initial correct responses was enhanced significantly by S.C.administration of VIP at doses of 5, 10 and 50 pg/kg, given 10 min before
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Fig. 2 Effects of i.p. administration of VIP on scopolamineinduced amnesia in rats in the radial arm maze. Each column represents the mean number of initial correct responses and vertical bars show SEM. The number in each column shows the number of animals used in the experiment. ##p < 0.01, compared with a saline control group. **p c 0.01, compared with a group treated with scopolamine only.
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between the earlier results and ours may be due to a difference in the nature ofthe reinforcement. In these tasks, animals were trained by a conditioned avoidance which was reinforced by aversive electric stimuli. Since such stimuli are unlikely to occur under normal life conditions, Ogawa et alI4pointed out that such anunnatural experimental procedure is far from ideal for the screening of antidementia drugs. Moreover, Watanabe et al” also pointed out recently that the memory mechanisms used in these tasks did not seem to be identical to those which are disrupted in amnesic patients, since these patients were reportedly able to make appropriate avoidance responses. I6 Therefore, it may be difficult to evaluate whether or not the results from a learning task reinforced with electric stimuli are due, in fact, to memory. However, it is considered that rats are not frightened by the radial arm maze task used in our experiments, in which animals are positively reinforced with the acquisition of food. Moreover, Watanabe et alI5 reported that results obtained from a radial maze task might be better suited for evaluation of memory performance in rats than tasks which are negatively reinforced with electric shock. Thus, our results obtained with the radial arm maze task demonstrate clearly that VIP has an ameliorating effect on memory processes.
15 0
8 16
i
5
1
14
16
15
10
50 100
VIP @g/kg; 8.c.) Scopolamine (Obmg/kg)
Fig. 3 Effects of S.C. administration of VIP on scopolamineinduced amnesia in rats in the radial arm maze. See Figure 2 for explanations. ###p< 0.01, compared with a saline control group. +p
injection of scopolamine. The responses were associated with a bell-shaped dose-response curve (Fig. 3). The results showed that S.C.injection of VIP mitigated the amnestic effect of scopolamine.
I. C.V.injection of VIP The number of initial correct responses was also increased significantly by i.c.v. injection of VIP at doses of 25 and 50 r&at, given 10 min before injection of scopolamine. The responses were associated with a bell-shaped dose-response curve (Fig. 4). Thus, i.c.v. administration of VIP also mitigated the amnestic effect of scopolamine.
r.
Discussion In the present study, single injections of VIP via three different routes (i.p., S.C.and i.c.v.) inhibited the reduction in numbers of initial correct responses caused by scopolamine, indicating that VIP has antiamnestic effects. However, amnestic effects of VIP have been reported by others: i.c.v. injection of VIP inhibited performance of a learning task which is negatively reinforced with electric shock, such as a step-through passive avoidance task and pole-jumping active avoidance task inrats* and a left-right footshock avoidance task in mice.g Thus, earlier reports are inconsistent with our results. The difference
.* t-l 3”
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1I1
Control
*+ D-f 1cl **
*
12 0
1’ 1 l( I
1c
:
25
[
10
10
50
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VIP (ng; i.c.v.1 Scopolamine (0.5mg/kg)
Fig. 4 Effects 0fi.c.v. injection ofVIP on scopolamine-induced amnesia in rats in the radial arm maze. See Figure 2 for explanations. ##p < 0.0 1, compared with a saline control group. lp < 0.05, **p < 0.01, compared with a group treated only with scopolamine.
VIP AND SCOPOLAMINE-INDUCED
157
AMNESIA
High doses of VIP (100 pg, i.p. and s.c.; 100 ng, i.c.v.) did not have any ameliorating effects under the present conditions. This observation is consistent with the observations by others that most cognition-improving drugs give bell-shaped doseresponse curves.7J7J* The minimum effective doses of VIP associated with reversal of memory deficits induced by scopolamine were 5 and 50 pg for S.C. and i.p. injections, respectively, suggesting that the S.C.route was more effective than the i.p. route. In the present study, i.c.v. injection of VIP caused the reversal of scopolamine-induced amnesia. VIP is considered to act through specific VIP receptors in the brain. Indeed, the highest density of binding sites for VIP has been found in the cerebral cortex and the dentate gyrus of the hippocampus in the rat Lg~20 and the highest concentrations of VIP in the brain have been found in the cerebral cortex, amygdala and hippocampus (see 21for review). It is well known that excitotoxic lesions of the nucleus basalis magnocellularis, the origin of cholinergic projections to the cortex and amygdala, produce impairment of the performance of learning tasks.22J3 Eckenstein and Baughman24 reported that cholinergic neurons in the cortex showed positive immunoreactivity for VIP. We found that scopolamine, a cholinergic receptor antagonist, induced memory deficits, as reported earlier by several groups of investigators .25-27 Together these findings suggest that VIP plays important roles in memory processes in cooperation with cholinergic mechanisms in the cortex and/or the hippocampus. It is known that lesions of the septo-hippocampal afferents5-’ and lesions of the hippocampusZs produce impairments in learning. Luine et al4 reported that VIP enhanced ChAT activity in the dorsal and ventral regions of the hippocampus. Therefore, it is possible that the ameliorating effects of VIP on memory processes might occur via enhancement of the ChAT activity in the hippocampus. 1.~. and S.C. administration of VIP resulted in reversal ofmemory deficits. It seems likely that VIP, injected i.p. and s.c., exerts its effects through cholinergic mechanisms.
Acknowledgements We are indebted to Mr K. Hashimoto, the Administrative Director, Research and Development, Zenyalcu Kogyo Co., Ltd, for his unwavering encouragement and support.
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