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Effects of nicotine on spatial memory deficits in rats with septal lesions
Brain Research, 572 (1992) 281-285 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
281
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Effects of nicotine on spatial memory deficits in rats with septal lesions Michael W. Decker, Mark J. Majchrzak and David J. Anderson Neuroscience Research, Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064 (U.S.A.)
(Accepted 5 November 1991) Key words: Nicotine; Nicotinic cholinergic agonist; Medial septal area; Spatial memory; Rat
Impaired septohippocampal function has been implicated in the memory deficits associated with Alzheimer's disease (AD), and septal lesions have been used to model the cognitive deficits associated with AD. In this study, we assessed the effects of systemic administration of nicotine on lesion-induced deficits in the acquisition of a spatial discrimination version of the Morris water maze. Rats with radiofrequency lesions of the medial septum were required to learn which of two visible platforms in a pool of water provided a means of escape. On each of the first 4 days of training, the rats received an injection of (-)nicotine (0, 0.1 or 0.3 mg/kg, i.p.) before training. Nicotine markedly improved the performance of septal rats. This enhanced performance was maintained in rats subsequently tested 1 and 15 days later without additional drug treatment. Septal rats initially trained under nicotine were impaired, however, when the platform locations were reversed and training was conducted under saline. Our findings suggest that nicotinic receptor stimulation might be useful in the treatment of cognitive deficits. Cholinergic neurons in the nucleus basalis of Meynert (NBM) and the medial septum/diagonal band, which provide extensive cholinergic input to the cortex, amygdala and hippocampus, degenerate in Alzheimer's disease (AD)2; and experimental lesions of the rat homologues of these basal forebrain nuclei impair performance on a variety of m e m o r y tasks 3'8. These observations suggest that cholinergic dysfunction may underlie some of the cognitive deficits associated with A D . In part because muscarinic antagonists, such as scopolamine, also impair memory, much of the current research has emphasized the role of muscarinic cholinergic neurotransmission in learning and m e m o r y deficits associated with A D . Accordingly, preclinical exploration of treatment approaches has tended to focus on pharmacological enhancement of muscarinic tone. For example, several studies have investigated the effects of muscarinic agonists on the performance of NBM-lesioned rats 1°'16'22. However, evidence that the nicotinic receptor stimulation enhances 4'5'9'15'17 and nicotinic blockade impairs 12A4,IT'1s memory in experimental animals suggests that nicotinic cholinergic neurotransmission might also play an important role in learning and memory. Recent findings that nicotinic but not muscarinic receptors are decreased in the brains of A D victims also suggest that nicotinic cholinergic dysfunction might be particularly important in the m e m o r y deficits associated with AD 21'23. Thus, nicotinic receptor stimulation might be effective in attenuating some of the m e m o r y deficits as-
sociated with A D . Consistent with this suggestion are findings that nicotine appears to improve matching to sample performance in aged monkeys 1 and to enhance radial maze performance in rats with N B M lesions 22. Relatively little additional data regarding the effects of nicotine in animal models of A D is available. Therefore, in the current study, we assessed the effects of nicotine on spatial m e m o r y performance in a water maze in rats with lesions of the medial septum. Radiofrequency lesions of the medial septal nucleus were produced in male, L o n g - E v a n s rats under pentobarbital anesthesia using the coordinates: 0.5 m m anterior to bregma, 0.0 m m lateral to the midline, and 6.5 m m ventral to the skull surface. Radiofrequency current sufficient to maintain a temperature of 61°C at the electrode tip was passed for 25 s. Sham surgery consisted of lowering an electrode to a point 1.0 m m above the target location for the lesions, but passing no current. At the end of the behavioral experiments, lesions were evaluated by measuring choline acetyltransferase (CHAT) activity in the hippocampus using an adaptation of the procedure of Fonnum 6. Behavioral testing was conducted using a round pool (180 cm in diameter and 60 cm high) filled to a depth of 37 cm with water (26 + I°C) rendered opaque by the addition of powdered milk. Two weeks after surgery, rats were habituated to the water maze by training them to swim to a visible escape platform (13 cm in diameter). Four trials per day were conducted for two days.
Correspondence: M.W. Decker, Department 47W, AP-10, Abbott Laboratories, Abbott Park, IL 60064, U.S.A. Fax: (1) (708) 937-9195.
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Fig. 1. Errors during initial training and retention test sessions on the spatial discrimination water maze. A: mean errors (_+S.E.M.) during the first 4 days of training in septal-lesioned and sham rats,
Each session was preceded by an injection of saline or nicotine, Rats with septal lesions made more errors than sham rats, an effect that was attenuated by nicotine treatment (see text for statistical analysis; n = 9-10 per group). B: mean errors (+S.E.M.) for these same animals during a retention test session conducted one day after initial training. No injections were made before this session. Rats with septal lesions who had been trained under saline were impaired, but rats with septal lesions who had been trained under nicotine (0.3 mg/kg) were not (see text for statistical analysis). C: Mean errors (+S.E.M.) for a subset of these animals during a retention test session conducted two weeks later. No injections were made before this session. Enhanced performance in rats with septal lesions who had been trained under nicotine (0.3 mg/ kg) was largely maintained during this retention test (see text for statistical analysis; n = 5 per group).
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E a c h trial was initiated by placing the animal in the pool at one of four start positions that were located equidistantly a r o u n d the p e r i m e t e r of the pool. The escape platform was located in the center of one of the four quadrants of the pool defined by the start locations. Different platform positions and start positions were used for each trial administered on these habituation days. T h r e e days
later, the rats were trained on the spatial discrimination water maze. F o r the spatial discrimination water maze, two visible platforms similar in a p p e a r a n c e to the platform used in cue training were provided. H o w e v e r , only one of the platforms provided a means of escape from the water; the other was m a d e of e x p a n d e d polystyrene and thus was unstable. The platforms could be distinguished by their spatial locations, since the position of each platform r e m a i n e d constant in relation to extramaze cues available in the r o o m throughout training. Rats were given 6 trials p e r day, with the start location being changed from trial to trial. Contacts with the incorrect platform were scored as errors. On each of the first 4 days of training, ( - ) n i c o t i n e (0.1 or 0.3 mg/kg, i.p.) or saline was administered 15 rain before training. A retention 6-trial test session was conducted on the fifth day, and half of the animals who had received either saline or the higher dose of nicotine during initial training received an additional retention test session two weeks later. No additional drug was injected before these retention test sessions. The rats who had received either saline or the higher dose of nicotine but who did not receive the second retention test were retrained with the platform locations reversed beginning 10 days after the original training. During reversal training, the rats received the drug t r e a t m e n t opposite to that received during initial training (i.e. rats originally trained under nicotine were retrained under saline during the reversal phase). The general p r o c e d u r e s used during reversal training were the same as those used for initial training. Performance during drug t r e a t m e n t was analyzed using a 3-way r e p e a t e d measures A N O V A , with session as the r e p e a t e d measure and drug dose and lesion as between group measures. Performance during retention sessions (sessions not immediately p r e c e d e d by drug in-
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Fig. 2. Mean errors (+S.E.M.) during the test day following reversal training on the spatial discrimination water maze. These rats were trained in the spatial discrimination task with the platform locations reversed from those used during initial training. Drug treatment was also reversed from that during original training. Nicotine or saline injections preceded training on the first 4 days of training, but no injections were made before the retention test session illustrated in this figure. Rats with septal lesions were impaired but nicotine improved performance (see text for statistical analysis; n = 4-5 per group).
jection) were analyzed separately using a 2-way ANOVA. (-)Nicotine (hydrogen tartrate salt; Sigma) was dissolved in sterile 0.9% saline. Doses were calculated based on the weight of the base. Measurement of ChAT activity in the hippocampus revealed no overlap in the ChAT activity measured in the hippocampi of sham and septal rats. Behavioral data from one septal animal were discarded, however, because the hippocampal ChAT activity measured in its hippocampus was within 2 standard deviations of the control mean. Comparison of the 28 remaining septal animals with the 29 sham rats revealed an average decrease in ChAT activity of 65.1% in septal animals (33.84 + 2.45 nmol acetylcholine (ACh)/mg protein/40 min) relative to sham rats (98.03 + 1.40 nmol ACh/mg protein/40 min) (t55 = 22.89, P < 0.001). Although substantial decreases in ChAT activity were noted in the septal animals, radiofrequency lesions such as ours are likely to disrupt other systems projecting to the hippocampus arising from or coursing through the septal area. The most notable of these non-cholinergic systems is probably the rather extensive GABAergic input to the hippocampus originating in the medial septum 13. Unfortunately, despite the early promise of the neurotoxin AF64A, no method for producing specific cholinergic lesions is yet available. During initial training under nicotine (Fig. 1A), rats with medial septal lesions made significantly more errors than rats with sham lesions (F~,51 = 28.44, P < 0.0001).
Nicotine administration prior to each of these training sessions, however, produced a dose-dependent improvement in the performance of the septal rats (F2,51 = 4.61, P < 0.02 for the main effect of nicotine and F2,51 = 5.77, P < 0.01 for the nicotine by lesion interaction). Furthermore, this improvement was maintained during the oneday retention test, which was not preceded by the administration of nicotine (F1,51 = 31.70, P < 0.0001 for the lesion effect; F2,51 = 5.05, P < 0.01 for the nicotine effect; F2,51 = 7.12, P < 0.002 for the nicotine by lesion interaction; Fig. 1B). Long-term effects of training under nicotine (P < 0.05) were also apparent when half of the animals who had received either saline or 0.3 mg/kg of nicotine were retested after a 2 week delay without further drug administration (Fig. 1C). Thus, nicotine treatment improved acquisition of this spatial memory task in septal rats; and once the septal rats learned the task, they retained the information without additional administration of nicotine. Nicotine effects were also observed during reversal training. Fig. 2 illustrates the performance of these rats on the one-day reversal retention test (during which no drugs were administered). During this retention test session, a significant lesion effect was again noted (F1,15 = 9.98, P < 0.01), but animals who had been trained on this reversal task under nicotine during the previous 4 days performed significantly better than rats trained under saline (F1,15 = 5.49, P < 0.05). Septal animals who had n o t been impaired when initially trained under nicotine were impaired when trained under saline in this reversal task. Conversely, septal animals who had originally been impaired when trained under saline were not impaired when given nicotine during reversal training. Thus, although septal rats who learned the initial task under nicotine continued to perform this discrimination correctly without additional drug treatment, rats initially trained under nicotine did not perform normally on a new spatial discrimination in the absence of additional nicotine treatment. While these experiments strongly suggest that nicotine administration may ameliorate spatial memory deficits in medial septal animals, the mechanisms and site(s) of action are unclear. Nicotine could act directly on postsynaptic nicotinic cholinergic receptors in the hippocampus or other structures involved in learning and memory processing. Alternatively, the effects we observed with nicotine could be mediated presynaptically. Nicotine administration affects the release of several neurotransmitters believed to play a role in memory modulation, including dopamine, norepinephrine and serotonin 7, effects that might underlie the memory enhancement we observed. In addition, nicotine enhances the release of acetylcholine7, which might then mediate memory en-
284 h a n c e m e n t via muscarinic or nicotinic mechanisms. It is perhaps unlikely, however, that enhanced release of A C h in the hippocampus underlies our effects given the rather substantial reduction of cholinergic input to hippocampus produced by our lesions. Interestingly, tetrahydroaminoacridine ( T H A ) , a cholinesterase inhibitor, improves but does not completely reverse the reference m e m o r y deficits exhibited by rats with medial septal lesions in the hidden platform version of the Morris water maze 19'2°. The partial attenuation of reference m e m o r y deficits found with T H A in these studies contrasts with a more nearly complete reversal of deficits by nicotine in the current study. However, it is not possible to determine the relative efficacy of these two drugs by comparing across studies. The extent and placement of the lesions likely differed in these studies. In addition, we used a spatial discrimination version of the water maze which differs significantly from the hidden platform task used in the evaluation of T H A . Only a direct comparison of the effects of nicotine with those of T H A will be able to determine if there are substantial differences in the efficacy of these two treatment approaches.
1 Buccafusco, J.J. and Jackson, W.J., Beneficial effects of nicotine administered prior to a delayed matching-to-sample task in young and aged monkeys, Neurobiol. Aging, 12 (1991) 233-238. 2 Coyle, J.T., Price, D.L. and DeLong, M.R., Alzheimer's disease: a disorder of cortical cholinergie innervation, Science, 219 (1983) 1184-1190. 3 Dekker, A.J.A.M., Connor, D.J. and Thai, L.J., The role of cholinergic projections from the nucleus basalis in memory, Neurosci, Biobehav. Rev., 15 (1991) 299-317. 4 Dilts, S.L. and Berry, C.A., Effect of cholinergic drugs on passive avoidance in the mouse, J. Pharmacol. Exp. Ther., 158 (1967) 279-285. 5 Elrod, K., Buccafusco, J.J. and Jackson, W.J., Nicotine enhances delayed matching-to-sample performance in primates, Life Sci., 43 (1988) 277-287. 6 Fonnum, E, A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407409. 7 Fuxe, K., Andersson, K., H~irfstrand, A., Eneroth, P., Perez della Mora, M. and Agnati, L.E, Effects of nicotine on synaptic transmission in the brain. In S. Wonnacott, M.A.H. Russel and I.P. Stolerman (Eds.), Nicotinic Psychopharmacology: Molecular, Cellular, and Behavioural Aspects, Oxford University Press, Oxford, 1990, pp. 194-225. 8 Gray, J.A. and McNaughton, N., Comparison between the behavioural effects of septal and hippocampal lesions: a review, Neurosci. Biobehav. Rev., 7 (1982) 119-188. 9 Haroutunian, V., Barnes, E. and Davis, K.L., Cholinergic modulation of memory in rats, Psychopharmacology, 87 (1985) 266271. 10 Haroutunian, V., Kanof, P.D., Tsuboyama, G. and Davis, K.L., Restoration of cholinomimetic activity by clonidine in cholinergic plus noradrenergic lesioned rats, Brain Research, 507 (1990) 261-266. 11 Hodges, H., Allen, Y., Sinden, J. Mitchell, S.N., Arendt, T., Lantos, P.L. and Gray, J.A., The effects of cholinergic drugs
The current study demonstrates a nicotine-induced improvement in the acquisition of spatial information in the water maze in rats with septal lesions. These findings are consistent with previous reports of memory-enhancement with nicotine in aged primates ~ and NBM-lesioned rats 22 and extend the observation of memoryenhancing effects of nicotine to an additional model of impaired cognition. Our findings are also consistent with the recent finding that nicotine improves the radial maze performance of rats with alcohol-induced degeneration of the basal forebrain n . Further work will be necessary to investigate these potential mechanisms as well as to determine whether nicotine effects are due to effects on memory processing itself or effects on factors such as attention and distractibility. Nevertheless, the potential of pharmacological manipulation of nicotinic neurotransmission as a palliative treatment for A D appears to be worthy of further consideration.
This work was supported by Abbott Laboratories research funds. The authors would like to thank Dr. Stephen P. Arneric and Dr. Jorge D. Brioni for their comments on an earlier draft of this paper.
and cholinergic-rich foetal neural transplants on alcohol-induced deficits in radial maze performance in rats, Behaw Brain Res., 43 (1991) 7-28. 12 Jackson, W.J., Elrod, K. and Buccafusco, J.J., Delayed matching-to-sample in monkeys as a model for learning and memory deficits: role of brain nicotinic receptors. In E.M. Meyer, J.W. Simpkins and J. Yamamoto (Eds.), Novel Approaches to the Treatment of Alzheimer's Disease, Plenum, New York, 1989, pp. 39-52. 13 Kohler, C., Chan-Palay, V. and Wu, J., Septal neurons contalning glutamic acid decarboxylase immunoreactivity project to the hippocampal region in the rat brain, Anat. Embryol., 169
(1984) 41-44. 14 Levin, E.D., Castonguay, M. and Ellison, G:D., Effects of the nicotinic receptor blocker, mecamylamine, on radial-arm maze performance in rats, Behav. Neural Biol., 48 (1987) 206-212. 15 Levin, E.D., Lee, C., Rose, J.E., Reyes, A., EUison, G., Jarvik, M. and Gritz, E., Chronic nicotine and withdrawal effects on radial-arm maze performance in rats, Behav. Neural Biol., 53 (1990) 269-276. 16 Murray, C.L. and Fibiger, H.C., Piloearpine and physostigmine attenuate spatial memory impairments produced by lesions of the nucleus basalis magnoceltularis, Behav. Neurosci., 100 (1986) 23-32. 17 Oliverio, A., Effects of mecamylamine on avoidance conditioning and maze learning of mice, J. Pharmacol. Exp. Ther., 154 (1966) 350-356. 18 Riekkinen Jr., P., Sirvi6, J., Aaltonen, M. and Riekkinen, P., The effects of concurrent manipulations of nicotinic and muscarinic receptors on spatial and passive avoidance learning, Pharmacol. Biochem. Behav., 37 (1990) 405-4t0. 19 Riekkinen Jr., P., Sirvi6, J., Aaltonen, M. and Riekkinen, P., Tetrahydroaminoacridine alleviates medial septat lesion-induced and age-related spatial reference but not working memory deficits, Physiol. Behav., 49 (1991) 1147-1152. 20 Riekkinen Jr., P., Sirvi6, J. and Riekkinen, P., The effects of
285 21 Schr0der, H., Giacobini, E., Struble, R.G., Zilles, K. and Maelicke, A., Nicotinic cholinoceptive neurons of the frontal cortex are reduced in Alzheimer's disease, Neurobiol. Aging, 12 (1991) 259-262. 22 Tiison, H.A., McLamb, R.L., Shaw, S., Rodgers, B.C., Pediaditakis, P. and Cook, L., Radial-arm maze deficits produced by colchicine administered into the area of the nucleus basalis are
ameliorated by cholinergic agents, Brain Research, 438 (1988) 83-94. 23 Whitehouse, P.J., Martino, A.M., Antuono, P.G., Lowenstein, P.R., Coyle, J.T., Price, D.L. and Kellar, K.J., Nicotinic acetylcholine binding sites in Alzheimer's disease, Brain Research, 371 (1986) 146-151.
281
BRES 25037
Effects of nicotine on spatial memory deficits in rats with septal lesions Michael W. Decker, Mark J. Majchrzak and David J. Anderson Neuroscience Research, Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064 (U.S.A.)
(Accepted 5 November 1991) Key words: Nicotine; Nicotinic cholinergic agonist; Medial septal area; Spatial memory; Rat
Impaired septohippocampal function has been implicated in the memory deficits associated with Alzheimer's disease (AD), and septal lesions have been used to model the cognitive deficits associated with AD. In this study, we assessed the effects of systemic administration of nicotine on lesion-induced deficits in the acquisition of a spatial discrimination version of the Morris water maze. Rats with radiofrequency lesions of the medial septum were required to learn which of two visible platforms in a pool of water provided a means of escape. On each of the first 4 days of training, the rats received an injection of (-)nicotine (0, 0.1 or 0.3 mg/kg, i.p.) before training. Nicotine markedly improved the performance of septal rats. This enhanced performance was maintained in rats subsequently tested 1 and 15 days later without additional drug treatment. Septal rats initially trained under nicotine were impaired, however, when the platform locations were reversed and training was conducted under saline. Our findings suggest that nicotinic receptor stimulation might be useful in the treatment of cognitive deficits. Cholinergic neurons in the nucleus basalis of Meynert (NBM) and the medial septum/diagonal band, which provide extensive cholinergic input to the cortex, amygdala and hippocampus, degenerate in Alzheimer's disease (AD)2; and experimental lesions of the rat homologues of these basal forebrain nuclei impair performance on a variety of m e m o r y tasks 3'8. These observations suggest that cholinergic dysfunction may underlie some of the cognitive deficits associated with A D . In part because muscarinic antagonists, such as scopolamine, also impair memory, much of the current research has emphasized the role of muscarinic cholinergic neurotransmission in learning and m e m o r y deficits associated with A D . Accordingly, preclinical exploration of treatment approaches has tended to focus on pharmacological enhancement of muscarinic tone. For example, several studies have investigated the effects of muscarinic agonists on the performance of NBM-lesioned rats 1°'16'22. However, evidence that the nicotinic receptor stimulation enhances 4'5'9'15'17 and nicotinic blockade impairs 12A4,IT'1s memory in experimental animals suggests that nicotinic cholinergic neurotransmission might also play an important role in learning and memory. Recent findings that nicotinic but not muscarinic receptors are decreased in the brains of A D victims also suggest that nicotinic cholinergic dysfunction might be particularly important in the m e m o r y deficits associated with AD 21'23. Thus, nicotinic receptor stimulation might be effective in attenuating some of the m e m o r y deficits as-
sociated with A D . Consistent with this suggestion are findings that nicotine appears to improve matching to sample performance in aged monkeys 1 and to enhance radial maze performance in rats with N B M lesions 22. Relatively little additional data regarding the effects of nicotine in animal models of A D is available. Therefore, in the current study, we assessed the effects of nicotine on spatial m e m o r y performance in a water maze in rats with lesions of the medial septum. Radiofrequency lesions of the medial septal nucleus were produced in male, L o n g - E v a n s rats under pentobarbital anesthesia using the coordinates: 0.5 m m anterior to bregma, 0.0 m m lateral to the midline, and 6.5 m m ventral to the skull surface. Radiofrequency current sufficient to maintain a temperature of 61°C at the electrode tip was passed for 25 s. Sham surgery consisted of lowering an electrode to a point 1.0 m m above the target location for the lesions, but passing no current. At the end of the behavioral experiments, lesions were evaluated by measuring choline acetyltransferase (CHAT) activity in the hippocampus using an adaptation of the procedure of Fonnum 6. Behavioral testing was conducted using a round pool (180 cm in diameter and 60 cm high) filled to a depth of 37 cm with water (26 + I°C) rendered opaque by the addition of powdered milk. Two weeks after surgery, rats were habituated to the water maze by training them to swim to a visible escape platform (13 cm in diameter). Four trials per day were conducted for two days.
Correspondence: M.W. Decker, Department 47W, AP-10, Abbott Laboratories, Abbott Park, IL 60064, U.S.A. Fax: (1) (708) 937-9195.
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Fig. 1. Errors during initial training and retention test sessions on the spatial discrimination water maze. A: mean errors (_+S.E.M.) during the first 4 days of training in septal-lesioned and sham rats,
Each session was preceded by an injection of saline or nicotine, Rats with septal lesions made more errors than sham rats, an effect that was attenuated by nicotine treatment (see text for statistical analysis; n = 9-10 per group). B: mean errors (+S.E.M.) for these same animals during a retention test session conducted one day after initial training. No injections were made before this session. Rats with septal lesions who had been trained under saline were impaired, but rats with septal lesions who had been trained under nicotine (0.3 mg/kg) were not (see text for statistical analysis). C: Mean errors (+S.E.M.) for a subset of these animals during a retention test session conducted two weeks later. No injections were made before this session. Enhanced performance in rats with septal lesions who had been trained under nicotine (0.3 mg/ kg) was largely maintained during this retention test (see text for statistical analysis; n = 5 per group).
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E a c h trial was initiated by placing the animal in the pool at one of four start positions that were located equidistantly a r o u n d the p e r i m e t e r of the pool. The escape platform was located in the center of one of the four quadrants of the pool defined by the start locations. Different platform positions and start positions were used for each trial administered on these habituation days. T h r e e days
later, the rats were trained on the spatial discrimination water maze. F o r the spatial discrimination water maze, two visible platforms similar in a p p e a r a n c e to the platform used in cue training were provided. H o w e v e r , only one of the platforms provided a means of escape from the water; the other was m a d e of e x p a n d e d polystyrene and thus was unstable. The platforms could be distinguished by their spatial locations, since the position of each platform r e m a i n e d constant in relation to extramaze cues available in the r o o m throughout training. Rats were given 6 trials p e r day, with the start location being changed from trial to trial. Contacts with the incorrect platform were scored as errors. On each of the first 4 days of training, ( - ) n i c o t i n e (0.1 or 0.3 mg/kg, i.p.) or saline was administered 15 rain before training. A retention 6-trial test session was conducted on the fifth day, and half of the animals who had received either saline or the higher dose of nicotine during initial training received an additional retention test session two weeks later. No additional drug was injected before these retention test sessions. The rats who had received either saline or the higher dose of nicotine but who did not receive the second retention test were retrained with the platform locations reversed beginning 10 days after the original training. During reversal training, the rats received the drug t r e a t m e n t opposite to that received during initial training (i.e. rats originally trained under nicotine were retrained under saline during the reversal phase). The general p r o c e d u r e s used during reversal training were the same as those used for initial training. Performance during drug t r e a t m e n t was analyzed using a 3-way r e p e a t e d measures A N O V A , with session as the r e p e a t e d measure and drug dose and lesion as between group measures. Performance during retention sessions (sessions not immediately p r e c e d e d by drug in-
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Fig. 2. Mean errors (+S.E.M.) during the test day following reversal training on the spatial discrimination water maze. These rats were trained in the spatial discrimination task with the platform locations reversed from those used during initial training. Drug treatment was also reversed from that during original training. Nicotine or saline injections preceded training on the first 4 days of training, but no injections were made before the retention test session illustrated in this figure. Rats with septal lesions were impaired but nicotine improved performance (see text for statistical analysis; n = 4-5 per group).
jection) were analyzed separately using a 2-way ANOVA. (-)Nicotine (hydrogen tartrate salt; Sigma) was dissolved in sterile 0.9% saline. Doses were calculated based on the weight of the base. Measurement of ChAT activity in the hippocampus revealed no overlap in the ChAT activity measured in the hippocampi of sham and septal rats. Behavioral data from one septal animal were discarded, however, because the hippocampal ChAT activity measured in its hippocampus was within 2 standard deviations of the control mean. Comparison of the 28 remaining septal animals with the 29 sham rats revealed an average decrease in ChAT activity of 65.1% in septal animals (33.84 + 2.45 nmol acetylcholine (ACh)/mg protein/40 min) relative to sham rats (98.03 + 1.40 nmol ACh/mg protein/40 min) (t55 = 22.89, P < 0.001). Although substantial decreases in ChAT activity were noted in the septal animals, radiofrequency lesions such as ours are likely to disrupt other systems projecting to the hippocampus arising from or coursing through the septal area. The most notable of these non-cholinergic systems is probably the rather extensive GABAergic input to the hippocampus originating in the medial septum 13. Unfortunately, despite the early promise of the neurotoxin AF64A, no method for producing specific cholinergic lesions is yet available. During initial training under nicotine (Fig. 1A), rats with medial septal lesions made significantly more errors than rats with sham lesions (F~,51 = 28.44, P < 0.0001).
Nicotine administration prior to each of these training sessions, however, produced a dose-dependent improvement in the performance of the septal rats (F2,51 = 4.61, P < 0.02 for the main effect of nicotine and F2,51 = 5.77, P < 0.01 for the nicotine by lesion interaction). Furthermore, this improvement was maintained during the oneday retention test, which was not preceded by the administration of nicotine (F1,51 = 31.70, P < 0.0001 for the lesion effect; F2,51 = 5.05, P < 0.01 for the nicotine effect; F2,51 = 7.12, P < 0.002 for the nicotine by lesion interaction; Fig. 1B). Long-term effects of training under nicotine (P < 0.05) were also apparent when half of the animals who had received either saline or 0.3 mg/kg of nicotine were retested after a 2 week delay without further drug administration (Fig. 1C). Thus, nicotine treatment improved acquisition of this spatial memory task in septal rats; and once the septal rats learned the task, they retained the information without additional administration of nicotine. Nicotine effects were also observed during reversal training. Fig. 2 illustrates the performance of these rats on the one-day reversal retention test (during which no drugs were administered). During this retention test session, a significant lesion effect was again noted (F1,15 = 9.98, P < 0.01), but animals who had been trained on this reversal task under nicotine during the previous 4 days performed significantly better than rats trained under saline (F1,15 = 5.49, P < 0.05). Septal animals who had n o t been impaired when initially trained under nicotine were impaired when trained under saline in this reversal task. Conversely, septal animals who had originally been impaired when trained under saline were not impaired when given nicotine during reversal training. Thus, although septal rats who learned the initial task under nicotine continued to perform this discrimination correctly without additional drug treatment, rats initially trained under nicotine did not perform normally on a new spatial discrimination in the absence of additional nicotine treatment. While these experiments strongly suggest that nicotine administration may ameliorate spatial memory deficits in medial septal animals, the mechanisms and site(s) of action are unclear. Nicotine could act directly on postsynaptic nicotinic cholinergic receptors in the hippocampus or other structures involved in learning and memory processing. Alternatively, the effects we observed with nicotine could be mediated presynaptically. Nicotine administration affects the release of several neurotransmitters believed to play a role in memory modulation, including dopamine, norepinephrine and serotonin 7, effects that might underlie the memory enhancement we observed. In addition, nicotine enhances the release of acetylcholine7, which might then mediate memory en-
284 h a n c e m e n t via muscarinic or nicotinic mechanisms. It is perhaps unlikely, however, that enhanced release of A C h in the hippocampus underlies our effects given the rather substantial reduction of cholinergic input to hippocampus produced by our lesions. Interestingly, tetrahydroaminoacridine ( T H A ) , a cholinesterase inhibitor, improves but does not completely reverse the reference m e m o r y deficits exhibited by rats with medial septal lesions in the hidden platform version of the Morris water maze 19'2°. The partial attenuation of reference m e m o r y deficits found with T H A in these studies contrasts with a more nearly complete reversal of deficits by nicotine in the current study. However, it is not possible to determine the relative efficacy of these two drugs by comparing across studies. The extent and placement of the lesions likely differed in these studies. In addition, we used a spatial discrimination version of the water maze which differs significantly from the hidden platform task used in the evaluation of T H A . Only a direct comparison of the effects of nicotine with those of T H A will be able to determine if there are substantial differences in the efficacy of these two treatment approaches.
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This work was supported by Abbott Laboratories research funds. The authors would like to thank Dr. Stephen P. Arneric and Dr. Jorge D. Brioni for their comments on an earlier draft of this paper.
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