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. Author manuscript; available in PMC: 2006 Feb 8.
Published in final edited form as: Neuropsychopharmacology. 2005 Dec;30(12):2135–2143. doi: 10.1038/sj.npp.1300761

Cholinesterase Inhibitors Ameliorate Behavioral Deficits Induced by MK-801 in Mice

John G Csernansky 1,2,✉, Maureen Martin 1,3, Renu Shah 1, Amy Bertchume 1, Jenny Colvin 1, Hongxin Dong 1
PMCID: PMC1361686  NIHMSID: NIHMS4600  PMID: 15956997

Abstract

Enhancing cholinergic function has been suggested as a possible strategy for ameliorating the cognitive deficits of schizophrenia. The purpose of this study was to examine the effects of acetylcholinesterase (AChE) inhibitors in mice treated with the non-competitive N-methyl-daspartate (NMDA) receptor antagonist, MK-801, which has been suggested as an animal model of the cognitive deficits of schizophrenia. Three separate experiments were conducted to test the effects of physostigmine, donepezil or galantamine on deficits in learning and memory induced by MK-801. In each experiment, MK-801 (0.05 mg/kg or 0.10mg/kg) or saline was administered i.p. 20 minutes prior to behavioral testing over a total of 14 days. Thirty minutes prior to administration of MK-801 or saline, one of three doses of the AChE inhibitor (i.e., physostigmine - 0.03, 0.10, or 0.30 mg/kg, donepezil - 0.10, 0.30, or 1.00 mg/kg, or galantamine - 0.25, 0.50, or 1.00 mg/kg) or saline was administered s.c. Behavioral testing was performed in all experimental animals using the following sequence: 1) spatial reversal learning, 2) locomotion, 3) fear conditioning and 4) shock sensitivity. Both doses of MK-801 produced impairments in spatial reversal learning and in contextual and cued memory, as well as hyperlocomotion. Physostigmine and donepezil, but not galantamine, ameliorated MK-801-induced deficits in spatial reversal learning and in contextual and cued memory in a dose-dependent manner. Also, physostigmine, but not donepezil or galantamine, reversed MK-801-induced hyperlocomotion. None of the AChE inhibitors altered shock sensitivity. These results suggest that AChE inhibitors may differ in their capacity to ameliorate learning and memory deficits produced by MK-801 in mice, which may be related to the cognitive effects of cholinomimetic drugs in patients with schizophrenia.

INTRODUCTION

There has been increasing interest in developing new drug treatments for the cognitive deficits of schizophrenia (Fenton et al, 2003), and among the CNS targets for such treatments is the cholinergic neurotransmitter system (Friedman, 2004). The cholinergic system projects widely throughout the brain, and includes dense projections to limbic and paralimbic areas (Mesulam and Geula, 1988). Pharmacological studies have shown muscarinic antagonists, such as scopolamine, can produce memory deficits in rodents and primates (Aigner and Mishkin, 1986; Rogers and Kesner, 2003; Rogers and Kesner, 2004). Also, acetylcholinesterase (AChE) inhibitors have been shown to reverse the memory deficits induced by such drugs (Higgins et al, 2002; Ballard and McAllister, 1999; Rupniak et al, 1997). Acetylcholine levels are increased in the hippocampus and cortex during cognitive processes in rats (Pepeu and Giovannini, 2004) and decreases in CSF acetylcholine concentrations are correlated with cognitive decline in Alzheimer’s disease (Jian-ping et al, 2004).

AChE inhibitors are now routinely used for the treatment of dementia in patients with probable Alzheimer’s disease (Knopman and Morris, 1997), and among such drugs, donepezil (Rogers et al, 1996) and galantamine (Raskind et al, 2000) are among the most widely prescribed in the United States. AChE inhibitors have also been tested for their capacity to ameliorate the cognitive deficits associated with schizophrenia, but with less than definitive results (Tugal et al, 2004; Buchanan et al, 2002; Lenzi et al, 2003).

The purpose of this study was to examine the effects of AChE inhibitors in mice treated with the non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801, which has been suggested as an animal model of the cognitive deficits of schizophrenia (Bardgett et al, 2003). Several recent hypotheses about the pathophysiology of schizophrenia have implicated NMDA-type glutamate receptors (Olney and Farber, 1995; Goff and Coyle, 2001; Coyle and Tsai, 2004). Early support for such hypotheses came from observations that the non-competitive NMDA receptor antagonist, phencyclidine (PCP) induced a psychosis similar to that observed in patients with schizophrenia (Luisada and Brown, 1976; Allen and Young, 1978). More recently, other non-competitive NMDA receptor antagonists, such as ketamine and MK-801, have been shown to produce complex syndromes that mimic positive and negative symptoms, as well as the cognitive deficits of schizophrenia (Adler et al, 1999; Buffalo et al, 1994; Moghaddam and Jackson, 2003; Newcomer and Krystal, 2001). Notably, antipsychotic drugs used in the treatment of schizophrenia, such as haloperidol, risperidone and clozapine, have been shown to block the hyperlocomotion, stereotypies and ataxia induced by MK-801 in rats (Andine et al, 1999); however, the effects of such drugs on the learning and memory deficits induced by MK-801 in rodents (Bardgett et al, 2003) has not been investigated.

The rationale for using AChE inhibitors to ameliorate cognitive deficits in mice induced by NMDA receptor blockade can be based on neuroanatomical and functional interactions between CNS cholinergic and glutamatergic systems (Everitt and Robbins, 1997). The tri-synaptic excitatory circuit within the hippocampus receives tonic excitatory cholinergic input from the medial septum and the diagonal band of Broca (Benes 1999; Colgin et al, 2003; Stewart and Fox, 1990). In turn, glutamate, acting on NMDA receptors located on inhibitory GABAergic interneurons within the septum, inhibits the activity of cholinergic neurons that project to the hippocampus (Giovannini et al, 1994). Muscarinic drugs, including both muscarinic (Friedman, 2004) and nicotinic agonists (Martin et al, 2004) have been investigated for their cognition enhancing effects in a number of animal species. Finally, based in part on post-mortem studies of the brains of subjects with schizophrenia, it has been proposed that abnormalities of the cholinergic system may underlie at least some of the cognitive deficits associated with schizophrenia (Friedman, 2004; Hyde and Crook, 2001; Freedman et al, 1995; Olney and Farber, 1995)

In the present study, groups of mice were pretreated with multiple doses of physostigmine, donepezil and galantamine prior to administration of the non-competitive NMDA antagonist, MK-801. Behaviors related to learning and memory were assessed using T-maze reversal learning and fear conditioning paradigms (Bardgett, et al, 2003). Physostigmine was selected for this study because it is considered the prototypical AChE inhibitor (Knopman and Morris, 1997), while donepezil and galantamine were selected because they are frequently used in the treatment of patients with Alzheimer disease and are being considered for use in patients with schizophrenia (Tugal et al, 2004; Buchanan et al, 2002; Lenzi et al, 2003). Moreover, while all three drugs inhibit AChE, they differ in their capacity to modulate nicotinic acetylcholine receptors (Samochocki et al, 2003).

METHODS

Animals and housing. A total of 404 male C57BL/6Hsd mice (10-12 animals per experiment condition) (Jackson Laboratories, Bar Harbor, Maine), seven weeks of age, were used for all experiments. The animals were housed in groups of 3 or 4, and food and water were available ad libitum. All procedures were performed according to NIH guidelines for the treatment of animal subjects and the Current Guide for the Care and Use of Laboratory Animals (USPHS) under a protocol approved by the Washington University Animal Studies Committee. Care was taken to minimize the pain, suffering and number of animals used.

Drug administration. The animals were divided into three approximately equal groups to conduct separate experiments to test the effects of physostigmine, donepezil or galantamine on behavioral deficits induce by MK-801. In each experiment, MK-801 (RBI, Natick, MA), at doses of 0.05 mg/kg or 0.10mg/kg, or saline was administered by intraperitoneal (i.p.) injection. Thirty minutes prior to administration of MK-801 or saline, one of three doses of the AChE inhibitor (i.e., physostigmine salicylate (Sigma, St. Louis), donepezil (Eisai Co Ltd, Tokyo, Japan) or galantamine (Sigma, St. Louis)) or saline was administered subcutaneously. Three doses of each drug were tested: i.e., physostigmine (0.03, 0.10, or 0.30 mg/kg), donepezil (0.10, 0.30, or 1.00 mg/kg), and galantamine (0.25, 0.50, or 1.00 mg/kg). All drugs were administered in a constant volume of 1 ml/kg of body weight. Behavioral testing was performed 50 minutes after AChE inhibitor pretreatment and 20 minutes after administration of MK-801 or saline.

The doses of MK-801 and the timing of MK-801 dosing prior to behavioral testing was based on previous studies utilizing the same behavioral paradigms in our laboratory (Bardgett, et al, 2003). Drug doses and the timing of dosing prior to MK-801 administration and behavioral testing for physostigmine (Ohno, et al, 1996), donepezil (Wang and Tang, 1998) and galantamine (Barnes, et al, 2000) were similarly based on prior studies of these drugs in rodents. Also, the maximal doses of each of the three AChE inhibitors were tested in preliminary experiments to exclude the possibility that they would have deleterious effects on any of the behaviors tested.

BEHAVIORAL AND COGNITIVE TESTING

Behavioral testing was performed in all experimental animals using the following sequence: 1) spatial reversal learning, 2) locomotion, 3) fear conditioning and 4) shock sensitivity over a total of 14 days. This sequence of testing was not randomized, but was determined instead to minimize interference of the testing procedures across the four paradigms, as previously described (Bardgett, et al, 2003). Locomotion and shock sensitivity testing followed spatial reversal learning and fear conditioning testing, respectively, as control conditions. Drug administration was administered in an identical manner on each day of behavioral testing.

Spatial Reversal Learning. Spatial reversal learning was assessed on days 1-5 using a water T-maze. A four-armed maze was constructed of 1-cm thick clear Plexiglas; each arm was 21.5 cm long, 10.5 cm wide, and 34.5 cm deep. One of the arms was blocked to create a “T” configuration, and an 8.8 by 8.8 cm rectangular platform was placed at the end of the target arm. Warm water made opaque with non-toxic white paint was added to the maze to conceal the platform submerged 0.5 cm below the surface.

The animals were habituated to the apparatus on days 1-3 by placing them in the water near the platform and allowing it to climb onto the platform. Once the mouse was able to climb onto the platform in less than 10 seconds, the animals were repeatedly placed at the entrance of the T-maze until they successfully located and climbed onto the platform six times. Then, the platform was moved to the opposite arm and testing was repeated until the animals again located and climbed onto the platform six times.

On day 4, the platform was placed either in the right or the left arm for all trials (animals in each group were equally distributed between the two sides), and the animals were tested until they successfully located the platform six out of eight times; latency to select the correct arm of the maze was also recorded. On day 5, the location of the platform was reversed to the other side of the T-maze, and the animals were again tested until they successfully located the platform six out of eight times (i.e., reversal learning).

Locomotor Activity. Locomotor activity was tested on day 6 for a period of 60 minutes as previously described (3). Animals were tested in clear polycarbonate cages (46 cm long, 25.5 cm wide, and 21.5 cm high) within a darkened room. Locomotor activity was assessed by counting the number of times animals interrupted twelve beams of red light evenly spaced along the length and width of the activity cage. The total number of beam breaks that occurred during the testing period was recorded.

Fear Conditioning. The assessment of contextual and cued memory within a fear conditioning paradigm was performed on days 7-9 again using methods previously described (3). Animals were trained and tested in two Plexiglas conditioning chambers (Med Associates, Inc., Georgia, VT). The chambers were 26 cm long, 18 cm high and 18 cm wide, with a metal grid floor, and contained within a larger sound-attenuating chamber with a 24-watt white light and a fan to provide 75 dB background noise.

On day 7, training took place in the first chamber, which contained a cup containing mint extract beneath the grid floor. The animals were acclimated to the chamber for five minutes, and freezing behavior, defined as no movement (ambulation, sniffing or stereotypy) other than respiration, was recorded every ten seconds. After two minutes, a twenty second, 80 dB, 2800 Hz tone was presented, and during the last two seconds of the tone, the animals received a 1.0 mA continuous footshock. This procedure was repeated two more times at one-minute intervals.

On day 8, memory the context was tested by returning the animals to the first chamber (scented with mint) and recording freezing behavior again every ten seconds for eight minutes. On day 9, the animals were placed in a second chamber scented with coconut and in which the grid floor was covered with a sheet of smooth polyurethane. Freezing behavior was again recorded for two minutes, and then the 80 dB, 2800 Hz tone was represented continuously for eight minutes; freezing behavior in response to the tone was recorded as a measure of cued memory.

Footshock Sensitivity. Sensitivity to the footshock administered during the fear conditioning paradigm was tested on day 10. The animals were returned to the first conditioning chamber for two minutes and exposed to a series of two-second shocks, beginning with an intensity of 1.0 mA. The shock intensity was increased by 0.5 mA every 20-30 seconds until a jumping response was evoked.

DATA ANALYSIS

In the spatial reversal learning paradigm, the number of trials required to reach criterion (6 of 8 successful trials) for acquisition of the task and reversal learning were used in data analyses. In the fear conditioning paradigm, the percent of time spent freezing during the period of exposure to context, exposure to altered context, and exposure to cue were used in data analyses. Finally, the number of light beam interruptions over 60 minutes and the amperage necessary to elicit a jumping response were used as measures of locomotion and shock sensitivity in data analyses. Data analyses were conducted separately for each drug treatment and each behavioral paradigm using two-way ANOVA and MK-801 dose groups and AChE inhibitor dose groups as factors. Post-hoc analyses were conducted using Fisher’s PLSD test.

RESULTS

MK-801 EFFECTS ON BEHAVIOR

The capacity of the three AChE inhibitors, physostigmine, donepezil and galantamine, to reverse the behavioral effects of MK-801 was studied in Experiments 1, 2 and 3, respectively. Across all three experiments, MK-801 generally produced the predicted effects on behavior. In all three experiments, MK-801 significantly impaired acquisition of the spatial learning task (Experiment 1 – F=18.3, df=2,132, p<.0001; Experiment 2 – F=42.2, df=2,132, p<.0001; Experiment 3 – F=6.4, 2,104, p=.002). Also, in two of three experiments, MK-801 significantly impaired reversal learning (Experiment 1 – F=15.9, df=2,132, p<.0001; Experiment 2 – F=49.1, df=2,132, p<.0001; Experiment 3 – F=2.5, 2,104, p=.08). Notably, there were also significant MK-801-induced increases in locomotion (Experiment 1 – F=46.0, df=2,130, p<.0001; Experiment 2 – F=15.9, df=2,129, p<.0001; Experiment 3 – F=19.0, 2,85, p<.0001).

In all three experiments, MK-801 significantly impaired memory for context (Experiment 1 – F=52.6, df=2,132, p<.0001; Experiment 2 – F=106.4, df=2,132, p<.0001; Experiment 3 – F=59.2, 2,104, p<.0001), memory for altered context (Experiment 1 – F=24.3, df=2,132, p<.0001; Experiment 2 – F=32.9, df=2,132, p<.0001; Experiment 3 – F=9.5, 2,104, p=.0002), and memory for cue (Experiment 1 – F=43.7, df=2,132, p<.0001; Experiment 2 – F=41.9, df=2,132, p<.0001; Experiment 3 – F=43.0, 2,104, p<.0001). In two of three experiments, MK-801 did not significantly alter footshock sensitivity (Experiment 1 – F=1.3, df=2,132, p=.29; Experiment 2 – F=0.2, df=2,132, p=.80; Experiment 3 – F=8.2, 2,104, p=.001).

EXPERIMENT 1 – PHYSOSTIGMINE.

The effects of physostigmine on MK-801-induced impairments in behavior are summarized in Figure 1. Physostigmine significantly improved the acquisition of the spatial reversal learning task (physostigmine effect - F=12.1, df=2,132, p<.0001), and reversed the impairments in acquisition of the spatial reversal learning task produced by MK-801 (interaction between physostigmine and MK-801 - F=4.6, df=2,132, p=.0003). Physostigmine also significantly improved reversal learning (physostigmine effect - F=10.0, df=2,132, p<.0001), and reversed MK-801-induced impairments in reversal learning (interaction between physostigmine and MK-801 - F=3.8, df=2,132, p=.002). Physostigmine also decreased locomotion (effect of physostigmine - F=12.4, df=2,130, p<.0001), and reversed MK-801-induced hyperlocomotion (interaction between physostigmine and MK-801 - F=3.0, df=2,130, p=.008).

FIGURE 1.

FIGURE 1.

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Effects of Physostigmine on Changes in Behavior Induced by MK-801 in Mice. One of three doses of physostigmine were administered (s.c.) prior to administration of one of two doses of MK-801 in separate groups of mice (n = 10-12 per experimental group). Each panel represents the results of a different behavioral measure; i.e., panel a - the number of trials required for acquisition of a spatial reversal learning task; panel b - the number of trials required for reversal learning; panel c – spontaneous locomotion; panel d – freezing behavior in response to a remembered context; panel e – freezing behavior in response to an altered context, and panel f – freezing behavior in response to the cue used in conditioning. See the narrative for ANOVA results. Significant differences between individual groups pretreated with one of the three doses of physostigmine prior to administration of saline or one of two doses of MK-801 as compared to the groups pretreated with saline prior to administration of saline or one of two doses of MK-801 are denoted by * (p<.05), **(p<.01), or ***(p<.001).

Physostigmine also significantly improved memory for context (physostigmine effect - F=19.8, df=2,132, p<.0001), and reversed contextual memory impairments produced by MK-801 (interaction between physostigmine and MK-801 - F=3.1, df=2,132, p=.007). Physostigmine also significantly increased the freezing response to the altered context (physostigmine effect -F=44.3, df=2,132, p<.0001), and increased MK-801-induced decreases in freezing in responses to the altered context (interaction between physostigmine and MK-801 - F=2.9, df=2,132, p=.001). Finally, physostigmine improved memory for cue (effect of physostigmine - F=74.7, df=2,132, p<.0001), and reversed cued memory impairments produced by MK-801 (interaction between physostigmine and MK-801 - F=3.0, df=2,132, p=.009). However, physostigmine did not have a significant effect on shock sensitivity (physostigmine effect – F=0.9, df=2,132, p=.44).

EXPERIMENT 2 - DONEPEZIL

The effects of donepezil on MK-801-induced impairments in behavior are summarized in Figure 2. Donepezil significantly improved the acquisition of the spatial reversal learning task (donepezil effect - F=16.3, df=2,131, p<.0001), and reversed the impairments in acquisition of the spatial reversal learning task produced by MK-801 (interaction between donepezil and MK-801 - F=5.1, df=2,131, p<.0001). Donepezil also significantly improved reversal learning (donepezil effect - F=14.0, df=2,131, p<.0001), and reversed MK-801-induced impairments in reversal learning (interaction between donepezil and MK-801 - F=3.0, df=2,131, p=.009). However, donepezil did not have a significant effect on locomotion (effect of donepezil - F=0.9, df=2,129, p=.45), nor did it reverse MK-801-induced hyperlocomotion (interaction between donepezil and MK-801 - F=1.1, df=2,129, p=.34).

FIGURE 2.

FIGURE 2.

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Effects of Donepezil on Changes in Behavior Induced by MK-801 in Mice. One of three doses of donepezil were administered (s.c.) prior to administration of one of two doses of MK-801 in separate groups of mice (n = 10-12 per experimental group). Each panel represents the results of a different behavioral measure (see Figure 1 legend for panel descriptions). See the narrative for ANOVA results. Significant differences between individual groups pretreated with one of the three doses of donepezil prior to administration of saline or one of two doses of MK-801 as compared to the groups pretreated with saline prior to administration of saline or one of two doses of MK-801 are denoted by * (p<.05), **(p<.01), or ***(p<.001).

Donepezil also significantly improved memory for context (donepezil effect - F=28.0, df=2,132, p<.0001), and reversed contextual memory impairments produced by MK-801 (interaction between donepezil and MK-801 - F=8.7, df=2,132, p<.0001). Donepezil also significantly increased the behavioral response to the altered context (donepezil effect - F=24.4, df=2,132, p<.0001), and increased MK-801-induced decreases in the behavioral response to the altered context (interaction between donepezil and MK-801 - F=3.5, df=2,132, p=.003). Finally, donepezil improved memory for cue (effect of donepezil - F=15.9, df=2,132, p<.0001), and reversed cued memory impairments produced by MK-801 (interaction between donepezil and MK-801 - F=7.0, df=2,132, p<.0001). However, donepezil did not have a significant effect on shock sensitivity (donepezil effect – F=2.6, df=2,132, p=.054).

EXPERIMENT 3 - GALANTAMINE

The effects of galantamine on MK-801-induced impairments in behavior are summarized in Figure 3. Galantamine did not significantly improve the acquisition of the spatial reversal learning task (galantamine effect - F=1.5, df=2,104, p=.21), nor did it reverse the impairments in acquisition of the spatial reversal learning task produced by MK-801 (interaction between galantamine and MK-801 - F=0.9, df=2,104, p=.51). Galantamine also did not significantly improve reversal learning (galantamine effect - F=2.0, df=2,104, p=.11), not did it reverse MK-801-induced impairments in reversal learning (interaction between galantamine and MK-801 -F=0.9, df=2,104, p=.52). However, galantamine produced a significant increase in locomotion (effect of galantamine - F=3.7, df=2,85, p=.01), and accentuated MK-801-induced hyperlocomotion (interaction between galantamine and MK-801 - F=5.1, df=2,85, p=.0002).

FIGURE 3.

FIGURE 3.

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Effects of Galantamine on Changes in Behavior Induced by MK-801 in Mice. One of three doses of galantamine were administered (s.c.) prior to administration of one of two doses of MK-801 in separate groups of mice (n = 10-12 per experimental group). Each panel represents the results of a different behavioral measure (see Figure 3 for panel descriptions). See the narrative for ANOVA results. Significant differences between individual groups pretreated with one of the three doses of galantamine prior to administration of saline or one of two doses of MK-801 as compared to the groups pretreated with saline prior to administration of saline or one of two doses of MK-801 are denoted by * (p<.05), **(p<.01), or ***(p<.001).

Galantamine did not significantly improve memory for context (galantamine effect - F=1.1, df=2,104, p=.36), but did tend to reverse contextual memory impairments produced by MK-801 (interaction between galantamine and MK-801 - F=2.2, df=2,104, p=.04). Similarly, galantamine did not significantly increase the behavioral response to the altered context (galantamine effect - F=2.5, df=2,104, p=.06), but did reverse MK-801-induced decreases in the behavioral response to the altered context (interaction between galantamine and MK-801 -F=3.2, df=2,104, p=.006). Finally, galantamine tended to decrease memory for cue (effect of galantamine - F=5.7, df=2,104, p=.001) (see Figure 3, panel f), and did not reverse cued memory impairments produced by MK-801 (interaction between galantamine and MK-801 - F=1.3, df=2,104, p=.28). Interestingly, galantamine decreased shock sensitivity (galantamine effect –F=11.6, df=2,104, p<.0001), and reversed the slight MK-801-induced increases in shock sensitivity observed in this experiment (interaction between galantamine and MK-801 - F=4.0, df=2,104, p=.001).

DISCUSSION

The results of this study suggest that physostigmine and donepezil, but not galantamine, can ameliorate deficits in learning and memory produced by the NMDA antagonist, MK-801, in mice. Both doses of MK-801 (0.05 and 0.10 mg/kg) produced the expected impairments in spatial reversal learning and in contextual and cued memory. In addition, MK-801 produced hyperlocomotion, as has been observed in previous studies (Bardgett et al, 2003; Dall’Igna et al, 2003). Physostigmine and donepezil were both effective in ameliorating the deficits in spatial reversal learning and in contextual and cued memory produced by MK-801 administration in a dose-dependent manner. In contrast, galantamine was not effective in ameliorating the behavioral deficits produced by MK-801 administration, and even appeared to further interfere with cued memory at the highest dose of galantamine tested (1.0 mg/kg). Further, physostigmine, but not donepezil, reversed the hyperlocomotion produced by MK-801. None of the AChE inhibitors altered sensitivity to the shock used in the fear conditioning paradigm.

Although these findings suggest that at least two of the three AChE inhibitors tested had the capacity to ameliorate deficits in learning and memory produced by MK-801, other findings suggested that MK-801 and the AChE inhibitors might have had non-specific effects on behavior. For example, MK-801 produced decreases in freezing responses to the altered context in the fear conditioning paradigm, and physostigmine and donepezil appeared to reverse this effect. While the altered context condition of the fear conditioning paradigm was intended as a control condition not related to learning or memory, elements of the altered context may have been sufficiently similar to the context in which the fear conditioning took place so that memory for context was involved in the animals’ responses. Also, MK-801 produced hyperlocomotion, which could have had an effect on the animals’ performance in the spatial reversal learning paradigm. However, while both physostigmine and donepezil ameliorated the effects of MK-801 in the spatial reversal learning and fear conditioning paradigms, only physostigmine reversed the hyperlocomotion produced by MK-801. Galantamine also produced small, but significant, increases in locomotion when administered alone and in combination with MK-801. Finally, physostigmine produced increases in freezing associated with the cue and alternate context even when administered in combination with saline. These findings suggest that the behavioral effects of the AChE inhibitors tested might have involved alterations in motor behavior that were unrelated to the actions of MK-801.

It should be kept mind that the order of behavioral testing was predetermined to minimize interference between testing paradigms rather than randomized, which could have influenced the results. However, similar differences across the three AChE inhibitors were observed in spatial reversal learning and contextual and cued memory, which is not consistent with the presence of order effects. Also, it is possible that galantamine might have been effective at doses that were either higher or lower than the ones tested in this study. The highest dose of galantamine actually seemed to interfere with the capacity for cued memory, which may be related to the fact that modulators of nicotinic ACh receptors can interfere with ACh at high doses (see below). Finally, the behavioral effects of MK-801 were not identical in the three separate experiments in which the capacity of physostigmine, donepezil and galantamine to reverse the effects of MK-801 was evaluated. In particular, the high dose of MK-801 had different effects on locomotion in the three experiments, and only in Experiment 3, where galantamine was evaluated, did MK-801 have an effect on shock sensitivity. However, the effects of MK-801 were highly similar and dose-dependent in the two major behavioral paradigms that were used to evaluate the cognition-enhancing effects of the AChE inhibitors.

Our findings that MK-801 produced impairments in performance in two behavioral paradigms related to learning and memory are consistent with previous studies examining the behavioral effects of non-competitive NMDA receptor antagonists in rodents (Walker and Gold, 1992; Bardgett et al, 2003; Modgaddam and Jackson, 2003). While the circuitry underlying learning and memory in the mammalian brain remains under investigation, proper functioning of the NMDA receptor within the septo-hippocampal pathway appears to be a critical element of this circuitry (Newcomer and Krystal, 2001). Moreover, our findings that at least two of the three AChE inhibitors ameliorated the effects of NMDA receptor blockade are in keeping with earlier observations of the effects of physostigmine on cognitive impairments produced by the NMDA antagonist, NPC 12626 (Walker and Gold, 1992), as well as what is known about the interconnections between cholinergic neurons in the medial septum and excitatory glutamatergic circuits within the hippocampus. Cholinergic projections to the hippocampus generate and sustain the rhythmic activity of excitatory glutamatergic pathways within the hippocampus (Colgin et al, 2003), and development of new memory traces during encoding appears to be modulated by cholinergic input into specific hippocampal subregions. Also, NMDA receptors on GABA interneurons modulate acetylcholine release in the hippocampus and cortex (Giovanni et al, 1994; Kim et al, 1999). MK-801 has been reported to increase acetylcholine release in the hippocampus and cortex (Hutson and Hogg, 1996; Kim et al, 1999; Hasegawa et al, 1993), but not in the striatum (Hutson and Hogg, 1996) or the pontine reticular formation (Lydic and Badhdoyan, 2002). In a recent review, Gold (2004) noted that the influence of cholinergic mechanisms on learning and memory is complex, and that particular behaviors can be optimized by changing the levels of acetylcholine release in one structure relative to another, rather than by changing the absolute level of acetylcholine release throughout the brain.

Our finding that physostigmine and donepezil, but not galantamine, were effective in ameliorating the deficits in learning and memory produced by MK-801 raises the question of differences in the pharmacology of the three drugs. Galantamine is a weak inhibitor of ACh E (IC 50 is in the mM range) as compared to the most prescribed AChE inhibitors (Samochocki et al, 2003). However, both galantamine and physostigmine act as allosteric modulators at the nicotinic acetylcholine receptor (Samochocki et al, 2003). At lower doses of such modulators, nicotinic receptors on the terminals of acetylcholine neurons are activated, intracellular calcium levels are increased, and neurotransmitter release is facilitated. However, at higher doses, nicotinic receptor function and neurotransmitter release can be inhibited (Samochocki et al, 2000). Considering these differences in the pharmacology of the three drugs, our findings suggest that the capacity of physostigmine and donepezil to ameliorate cognitive deficits produced by Mk-801 may be the result of AChE inhibition and increases in synaptic levels of ACh, rather than allosteric modulation of the nicotinic acetylcholine receptor. In turn, however, increases in synaptic levels of ACh would increase the stimulation of both muscarinic and nicotinic ACh receptors, and direct stimulation of both subtypes of ACh receptors in the hippocampus has been shown to enhance memory (Kim and Levin, 1996). In future studies, it would be informative to determine whether direct stimulation of muscarinic or nicotinic ACh receptors can ameliorate deficits in learning and memory produced by MK-801.

There is increasing evidence that NMDA receptor hypofunction, in this study produced by non-competitive receptor blockade, may be a useful model of the pathophysiology of schizophrenia. In addition to clinical studies showing that the administration of non-competitive NMDA receptor antagonists can produce schizophrenia-like symptoms in humans (Goff and Coyle, 2001), there is evidence of abnormal NMDA receptor expression in the brains of subjects with schizophrenia (Coyle and Tsai, 2004). Recent work suggests the NMDA NR1 subunit mRNA is decreased in the CA3 while the NMDA NR2A subunit mRNA is increased in the CA2 of schizophrenics compared to controls (Gao et al, 2000). Also, increases in kynurenic acid, an endogenous antagonist at the glycine modulatory site on the NR1 subunit of the NMDA receptor, have been reported in numerous brain regions of subjects with schizophrenia, including the medial temporal lobe (Schwarcz et al, 2001). Thus, to the extent that MK-801-induced blockade of the NMDA receptor can be interpreted as a pharmacological model of at least one element of the pathophysiology of schizophrenia, the results of this study may be useful for predicting which AChE inhibitors may be most effective for ameliorating the cognitive deficits associated with schizophrenia.

As mentioned in the Introduction, donepezil and galantamine have been preliminarily tested for their capacity to enhance cognition in patients with schizophrenia. In two open-label trials where donepezil was added to the ongoing treatment of schizophrenia subjects with atypical antipsychotic drugs, an improvement in manual dexterity (Buchanan et al, 2002) and in clinical measures of dementia (e.g., MMSE) (Stryjer et al, 2003) was observed. However, in double-blind studies of donepezil added to treatment with risperidone (Friedman et al, 2002) or clozapine (Stryjer et al, 2004), no beneficial effects were observed. Preliminary results with galantamine added to ongoing atypical antipsychotic drug treatment have been similarly inconclusive (Ochoa and Clark, 2004; Schubert et al, 2004). In addition, direct stimulation of both muscarinic (Shannon et al, 2000; Dean et al, 2003) and nicotinic (Martin et al, 2004) ACh receptors has been investigated as a means of enhancing cognition in patients with schizophrenia. Earlier concepts of the use of cholinomimetic drugs in schizophrenia focused on the potential of such drugs to relieve the antipsychotic symptoms (Dean et al, 2003). Certainly, testing them for their specific effects on specific elements of cognition that might contribute to the complex presentation of schizophrenia is a refinement. However, given the expense of clinical trials of AChE inhibitors and other cholinomimetic drugs in combination with various antipsychotic drugs in patients with schizophrenia, further experimentation in animal models of cognition that may be analogous to the cognitive deficits of schizophrenia may be highly useful.

Footnotes

This research was supported by Public Health Service grant MH 60883.

REFERENCES

  1. Adler CM, Malhotra AK, Elman I, Goldberg T, Egan M, Pickar D, Breier A. Comparison of ketamine-induced thought disorder in healthy volunteers and thought disorder in schizophrenia. Am J Psychiatry. 1999;156:1646–1649. doi: 10.1176/ajp.156.10.1646. [DOI] [PubMed] [Google Scholar]
  2. Aigner TG, Mishkin M. The effects of physostigmine and scopolamine on recognition memory in monkeys. Behav Neural Biol. 1986;45:81–7. doi: 10.1016/s0163-1047(86)80008-5. [DOI] [PubMed] [Google Scholar]
  3. Allen RM, Young SJ. Phencyclidine-induced psychosis. Am J Psychatry. 1978;135:1081–1084. doi: 10.1176/ajp.135.9.1081. [DOI] [PubMed] [Google Scholar]
  4. Andine P, Widermark N, Axelsson R, Nyberg G, Olofsson U, Martensson E, Sandberg M. Characterization of MK-801-induced behavior as a putative rat model of psychosis. J Pharm Exp Ther. 1999;290:1393–1408. [PubMed] [Google Scholar]
  5. Ballard TM, McAllister KH. The acetylcholinesterase inhibitor, ENA 713 (Exelon), attenuates the working memory impairment induced by scopolamine in an operant DNMTP task in rats. Psychopharmacology. 1999;146:10–18. doi: 10.1007/s002130051082. [DOI] [PubMed] [Google Scholar]
  6. Barnes CA, Meltzer J, Houston F, Orr G, McGann K, Wenk GL. Chronic treatment of old rats with donepezil or galantamine: Effects on memory, hippocampal plasticity and nicotinic receptors. Neuroscience. 2000;99:17–23. doi: 10.1016/s0306-4522(00)00180-9. [DOI] [PubMed] [Google Scholar]
  7. Bardgett ME, Boeckman R, Krochmal D, Fernando H, Ahrens R, Csernansky JG. NMDA receptor blockade and hippocampal neuronal loss impair fear conditioning and position habit reversal in C57Bl/6 mice. Brain Res Bull. 2003;60:131–142. doi: 10.1016/s0361-9230(03)00023-6. [DOI] [PubMed] [Google Scholar]
  8. Benes FM. Evidence for altered trisynaptic circutry in schizophrenic hippocampus. Biol Psychiatry. 1999;46:589–599. doi: 10.1016/s0006-3223(99)00136-5. [DOI] [PubMed] [Google Scholar]
  9. Buchanan RW, Summerfelt A, Tek C, Gold J. An open-labeled trial of adjunctive donepezil for cognitive impairments in patients with schizophrenia. Schizophrenia Research. 2002;59:29–33. doi: 10.1016/s0920-9964(01)00387-5. [DOI] [PubMed] [Google Scholar]
  10. Buffalo EA, Gillam MP, Allen RR, Paule MG. Acute behavioral effects of MK-801 in rhesus monkeys: assessment using an operant test battery. Pharma Biochem Beh. 1994;48:935–940. doi: 10.1016/0091-3057(94)90203-8. [DOI] [PubMed] [Google Scholar]
  11. Colgin LL, Kubota D, Lynch G. Cholinergic plasticity in the hippocampus. PNAS. 2003;100:2872–2877. doi: 10.1073/pnas.0530289100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Coyle JT, Tsai G. NMDA receptor function, neuroplasticity, and the pathophysiology of schizophrenia. Intl Rev Neurobiol. 2004;59:491–515. doi: 10.1016/S0074-7742(04)59019-0. [DOI] [PubMed] [Google Scholar]
  13. Dall #x2019;Igna OP, Da Silva AL, Dietrich MO, Hoffman A, de Oliveira RV, Souze DO, Lara DR. Chronic treatment with caffeine blunts the hyperlocomotor but not the cognitive effects of the N-methyl-D-asparate receptor antagonist MK-801 in mice. Psychopharmacology. 2003;166:258–263. doi: 10.1007/s00213-002-1362-1. [DOI] [PubMed] [Google Scholar]
  14. Dean B, Bymaster FP, Scarr E. Muscarinic receptors in schizophrenia. Curr Molec Med. 2003;3:419–426. doi: 10.2174/1566524033479654. [DOI] [PubMed] [Google Scholar]
  15. Everitt BJ, Robbins TW. Central cholinergic systems and cognition. Annu Rev Psychol. 1997;48:649–684. doi: 10.1146/annurev.psych.48.1.649. [DOI] [PubMed] [Google Scholar]
  16. Fenton W, Stover EL, Insel TR. Breaking the log-jam in treatment development for cognition in schizophrenia. Psychopharmacology. 2003;169:365–366. doi: 10.1007/s00213-003-1564-1. [DOI] [PubMed] [Google Scholar]
  17. Freedman R, Hall M, Adler LE, Leonard S. Evidence in postmortem brain tissue for decreased numbers of hippocampal nicotinic receptors in schizophrenia. Biol Psychiatry. 1995;38:22–33. doi: 10.1016/0006-3223(94)00252-X. [DOI] [PubMed] [Google Scholar]
  18. Friedman JI, Adler DN, Howanitz E, Harvey PD, Brenner G, Temporini H, White L, Parrella M, Davis KL. A double blind placebo controlled trial of donepezil adjunctive treatement to risperidone for the cognitive impairment of schizophrenia. Biol Psychiatry. 2002;51:349–357. doi: 10.1016/s0006-3223(01)01342-7. [DOI] [PubMed] [Google Scholar]
  19. Friedman JI. Cholinergic targets for cognitive enhancement in schizophrenia: Focus on cholinesterase inhibitors and muscarinic agonists. Psychopharmacology. 2004;174:45–53. doi: 10.1007/s00213-004-1794-x. [DOI] [PubMed] [Google Scholar]
  20. Gao X-M, Sakai K, Roberts RC, Conley RR, Dean B, Tamminga CA. Ionotropic glutamate receptors and expression of N-methyl-D-aspartate receptor subunits in subregions of human hippocampus: Effects of schizophrenia. Am J Psychiatry. 2000;157:1141–1149. doi: 10.1176/appi.ajp.157.7.1141. [DOI] [PubMed] [Google Scholar]
  21. Giovannini MG, Mutolo D, Bianchi L, Michelassi A, Pepeu G. NMDA receptor antagonists decrease GABA outflow from the septum and increse acetylcholine outflow from the hippocampus: A microdialysis study. J Neurosci. 1994;14:1358–1365. doi: 10.1523/JNEUROSCI.14-03-01358.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Goff DC, Coyle JT. The emerging role of glutamate in the pathophysiology and treatment of schizophrenia. Am J Psychiatry. 2001;158:1367–1377. doi: 10.1176/appi.ajp.158.9.1367. [DOI] [PubMed] [Google Scholar]
  23. Gold PE. Coordination of multiple memory systems. Neurobiol Learn Mem. 2004;82:230–242. doi: 10.1016/j.nlm.2004.07.003. [DOI] [PubMed] [Google Scholar]
  24. Hasegawa M, Kinoshita M, Amano M, Hasegawa T, Kameyama T, Nabeshima T. MK-801 increases endogenous acetylcholine release in the rat parietal cortex: a study using brain microdialysis. Neurosci Lett. 1993;150:53–56. doi: 10.1016/0304-3940(93)90106-u. [DOI] [PubMed] [Google Scholar]
  25. Higgins GA, Enderlin M, Fimbel R, Haman M, Grottick AJ, Soriano M, Richards JG, Kemp JA, Gill R. Donepezil reverses a mnemonic deficit produced by scopolamine but not by perforant path lesions or transient cerebral ischaemia. Eur J Neurosci. 2002;15:1827–1840. doi: 10.1046/j.1460-9568.2002.02018.x. [DOI] [PubMed] [Google Scholar]
  26. Hutson PH, Hogg JE. Effects of and interaction between antagonists for different sites on the NMDA receptor complex on hippocampal and striatal acetylcholine efflux in vivo. Eur J Pharmacol. 1996;295:45–52. doi: 10.1016/0014-2999(95)00634-6. [DOI] [PubMed] [Google Scholar]
  27. Hyde TM, Crook JM. Cholinergic systems and schizophrenia: Primary pathology or epiphenomena? J Chem Neuroanat. 2001;22:53–63. doi: 10.1016/s0891-0618(01)00101-6. [DOI] [PubMed] [Google Scholar]
  28. Jian-ping J, Jian-min J, Wei-dong Z, Min X, Chang-biao C, Xin Y, Yong-xin S. Differential acetylcholine and choline concentrations in the cerebrospinal fluid of patients with Alzheimer’s disease and vascular dementia. Chin Med J. 2004;117:1161–1164. [PubMed] [Google Scholar]
  29. Kim SH, Price MT, Olney JW, Farber NB. Excessive cerebrocortical release of acetylcholine induced by NMDA antagonists is reduced by GABAergic and á2-adrenergic agonists. Mol Psychiatry. 1999;4:344–352. doi: 10.1038/sj.mp.4000529. [DOI] [PubMed] [Google Scholar]
  30. Kim JS, Levin ED. Nicotinic, muscarinic and dopaminergic actions in the ventral hippocampus and the nucleus accumbens: Effects on spatial working memory in rats. Brain Res. 1996;725:231–240. doi: 10.1016/0006-8993(96)00213-2. [DOI] [PubMed] [Google Scholar]
  31. Knopman D, Morris JC. An update on primary drug therapies for Alzheimer’s disease. Arch Neurol. 1997;54:1406–1409. doi: 10.1001/archneur.1997.00550230073020. [DOI] [PubMed] [Google Scholar]
  32. Lenzi A, Maltinti E, Poggi E, Fabrizio L, Coli E. Effects of rivastigmine on cognitive function and quality of life in patients with schizophrenia. Clin Neuropharmacol. 2003;26:317–321. doi: 10.1097/00002826-200311000-00011. [DOI] [PubMed] [Google Scholar]
  33. Luisada PV, Brown BL. Clinical management of the phencyclidine psychosis. Clin Toxicol. 1976;9:539–545. doi: 10.3109/15563657608988155. [DOI] [PubMed] [Google Scholar]
  34. Lydic R, Baghdoyan HA. Ketamine and MK-801 decreases acetylcholine release in the pontine reticular formation, slow breathing, and disrupt sleep. Sleep. 2002;25:617–622. [PubMed] [Google Scholar]
  35. Martin LF, Kem WR, Freedman R. Alpha-7 nicotinic receptor agonists: Potential new candidates for the treatment of schizophrenia. Psychopharmacol. 2004;174:54–64. doi: 10.1007/s00213-003-1750-1. [DOI] [PubMed] [Google Scholar]
  36. Mesulam MM, Geula C. Acetylcholinestase-rich pyramidal neurons in the human neocortex and hippocampus: absence at birth, development during the life span, and dissolution in Alzheimer’s disease. Ann Neurol. 1988;24:765–773. doi: 10.1002/ana.410240611. [DOI] [PubMed] [Google Scholar]
  37. Moghaddam B, Jackson ME. Glutamatergic animal models of schizophrenia. Ann NY Acad Sci. 2003;1003:131–137. doi: 10.1196/annals.1300.065. [DOI] [PubMed] [Google Scholar]
  38. Newcomer JW, Krystal JH. NMDA receptor regulation of memory and behavior in humans. Hippocampus. 2001;11:529–542. doi: 10.1002/hipo.1069. [DOI] [PubMed] [Google Scholar]
  39. Ochoa ELM, Clark E. Abstract Viewer/Itinerary Planner. Society for Neuroscience; Washington DC: 2004. Galantamine as an adjuvant treatment for negative symptoms in schizophrenia: a pilot study. [Google Scholar]
  40. Olney JW, Farber NB. Glutamate receptor dysfunction and schizophrenia. Arch Gen Psychiatry. 1995;12:998–1007. doi: 10.1001/archpsyc.1995.03950240016004. [DOI] [PubMed] [Google Scholar]
  41. Ohno M, Kishi A, Watanabe S. Effect of cholinergic activation by physostigmine on working memory failure caused in rats by pharmacological manipulation of hippocampal glutamatergic and 5-HTergic neurotransmission. Neurosci Lett. 1996;217:21–24. doi: 10.1016/0304-3940(96)13057-3. [DOI] [PubMed] [Google Scholar]
  42. Pepeu G, Giovannini MG. Changes in acetylcholine extracellular levels during cognitive processes. Learn Mem. 2004;11:21–27. doi: 10.1101/lm.68104. [DOI] [PubMed] [Google Scholar]
  43. Raskind SL, Peskind ER, Wessel T, Yuan W. Galantamine in AD: A 6-month, randomized, placebo-controlled trial with a 6-month extension. Neurology. 2000;54:2261–2268. doi: 10.1212/wnl.54.12.2261. [DOI] [PubMed] [Google Scholar]
  44. Rogers JL, Kesner RP. Cholinergic modulation of the hippocampus during encoding and retrieval. Neurobiol Learn Memory. 2003;80:332–342. doi: 10.1016/s1074-7427(03)00063-7. [DOI] [PubMed] [Google Scholar]
  45. Rogers JL, Kesner RP. Cholinergic modulation of the hippocampus during encoding and retrieval of tone/shock-induced fear conditioning. Learning and Memory. 2004;11:102–107. doi: 10.1101/lm.64604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rupniak NM, Tye SJ, Field MJ. Enhanced performance of spatial and visual recognition memory tasks by the selective acetylcholinesterase inhibitor E2020 in rhesus monkeys. Psychopharmacology. 1997;131:406–410. doi: 10.1007/s002130050310. [DOI] [PubMed] [Google Scholar]
  47. Samochocki M, Hoffle A, Fehrenbacher A, Jostock R, Ludwig J, Christner C, Radina M, Zerlin M, Ullmer C, Pereira EFR, Lubbert H, Albuquerque EX, Maelicke A. Galantamine is an allosterically potentiating ligand of neuronal nicotinic but not of muscarinic acetylcholine receptors. J Pharm Exp Ther. 2003;305:1024–1036. doi: 10.1124/jpet.102.045773. [DOI] [PubMed] [Google Scholar]
  48. Samochocki M, Zerlin M, Jostock R, Kormelink PJG, Luyten WHML. Galantamine is an allosterically potentiating ligand of the human a4b2 nAChR. Acta Neurol Scand. 2000;176:68–73. doi: 10.1034/j.1600-0404.2000.00310.x. [DOI] [PubMed] [Google Scholar]
  49. Schubert MH, Young KA, Hicks PB. Abstract Viewer/Itinerary Planner. Society for Neuroscience; Washington DC: 2004. Cognitive improvement in schizophrenia and schizoaffective disorder by augmentation of risperidone with galantamine. [Google Scholar]
  50. Schwarcz R, Rassoulpour A, Wu HQ, Medoff D, Tamminga CA, Roberts RC. Increased cortical kynurenate content in schizophrenia. Biol Psychiatry. 2001;50:521–530. doi: 10.1016/s0006-3223(01)01078-2. [DOI] [PubMed] [Google Scholar]
  51. Shannon HE, Rasmussen K, Bymaster FP, Hart JC, Peters SC, Swedberg MDB, Jeppesen L, Sheardown MJ, Sauerberg P, Fink-Jensen A. Xanomeline, an M1/M4 preferring muscarinic receptor agonist, produces antipsychotic-like activitiy in rats and mice. Schizophrenia Res. 2000;42:249–259. doi: 10.1016/s0920-9964(99)00138-3. [DOI] [PubMed] [Google Scholar]
  52. Stewart M, Fox SE. Do septal neurons pace the hippocampal theta rhythm? Trends Neurosci. 1990;13:163–168. doi: 10.1016/0166-2236(90)90040-h. [DOI] [PubMed] [Google Scholar]
  53. Tugal O, Yazici KM, Yagcioglu AE, Gogus A. A double blind, placebo controlled, cross over trial of adjunctive donepezil for cognitive impairment in schizophrenia. Int J Neuropsychopharmacol. 2004;7:117–123. doi: 10.1017/S1461145703004024. [DOI] [PubMed] [Google Scholar]
  54. Walker DL, Gold PE. Impairment of spontaneous alternation performance by an NMDA antagonist: attenuation with non-NMDA treatments. Behav Neural Biol. 1992;58:69–1. doi: 10.1016/0163-1047(92)90952-z. [DOI] [PubMed] [Google Scholar]
  55. Wang T, Tang XC. Reversal of scopolamine-induced deficits in radial arm maze performance by (-)-huperzine A: comparison with E2020 and tacrine. Eur J Pharmacol. 1998;349:137–142. doi: 10.1016/s0014-2999(98)00199-x. [DOI] [PubMed] [Google Scholar]

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