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NEUROPHARMACOLOGY

Effects of a Novel Cognitive Enhancer, Spiro[imidazo-[1,2-a]pyridine-3,2-indan]-2(3H)-one (ZSET1446), on Learning Impairments Induced by Amyloid-_1–40 in the Rat

Research Laboratory, Zenyaku Kogyo Co., Ltd., Tokyo, Japan (Y.Y., H.M., K.S., T.M., S.K.); Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University School of Medicine, Nagoya, Japan (H.T., A.M., T.N.); and Neurotoxicology Program, Department of Pharmacy, College of Pharmacy, Kangwon National University, Korea Institute of Drug Abuse, Chunchon, South Korea (H.-C.K.)

Received November 14, 2005; accepted February 8, 2006.

	Abstract

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We have previously shown that intracerebroventricular (i.c.v.) infusion of amyloid- (A)_1–40 produces oxidative stress and cholinergic dysfunction, as well as learning and memory deficits, in rats. In the present study, effects of a newly synthesized azaindolizinone derivative, spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ZSET1446), were assessed in rats with learning deficits induced by A_1–40 or scopolamine. The i.c.v. infusion of A_1–40 caused impairments in spontaneous alternation behavior in a Y-maze task, spatial reference and short-term memory in a water-maze task, and retention of passive-avoidance learning. A_1–40-infused rats also showed reduction in choline acetyltransferase (ChAT) activity in the medial septum and hippocampus, but not in the basal forebrain and cortex, and a decrease in glutathione S-transferase (GST)-like immunoreactivity in the cortex. Nicotine-stimulated acetylcholine (ACh) release in A_1–40-infused rats was lower than that in vehicle-infused rats. Oral administration of ZSET1446 at the dose range of 0.01 to 1 mg/kg ameliorated A_1–40-induced learning impairment in Y-maze, water-maze, and passive-avoidance tasks. ZSET1446 reversed the decrease of ChAT activity in the medial septum and hippocampus, GST-like immunoreactivity in the cortex, and nicotine-stimulated ACh release of A_1–40-treated rats to the levels of vehicle-infused control rats. Furthermore, 0.001 to 0.1 mg/kg ZSET1446 showed ameliorative effects on learning impairments caused by scopolamine in a passive-avoidance task. These results suggest that ZSET1446 may be a potential candidate for development as a therapeutic agent to manage cognitive impairment associated with conditions such as Alzheimer's disease.

On the other hand, it has been shown that oxidative stress is also involved in A-induced neurotoxicity (Schubert et al., 1995) and the progression of AD (Yankner, 1996). It is known that glutathione S-transferase (GST) plays an important role in cellular protection against oxidative stress, and a decrease in GST activity and GST levels is observed in AD compared with controls in the brain and cerebrospinal fluid (Lovell et al., 1998).

An animal model induced by a chronic intracerebroventricular (i.c.v.) infusion of A is particularly attractive for evaluation of drugs for AD because change is similar to those known for AD patients: 1) dysfunction of learning and memory (Nitta et al., 1994), 2) decrease of choline acetyltransferase (ChAT) activity (Nitta et al., 1994) and nicotine-induced acetylcholine (ACh) release (Itoh et al., 1996; Tran et al., 2003) in the hippocampus and cortex, 3) impairment of LTP induced by tetanic stimulation in CA1 pyramidal cells in the slices from A_1–40-infused rats (Itoh et al., 1999), 4) robust peroxinitrite formation and subsequent tyrosine nitration of synaptophysin in the hippocampus (Tran et al., 2003), and 5) reduction of the immunoreactivity of antioxidant substances, such as manganese-superoxide dismutase, glutathione, glutathione peroxidase, and GST in the hippocampus and cortex (Kim et al., 2003). It has been shown that the solution of A at the concentration of 15 mg/ml (the concentration is 10 times higher than ours) does not precipitate for 24 h at 37°C in 35% acetonitrile containing 0.1% trifluoroacetic acid (Waite et al., 1992). Because we have used the same solvent, it is unlikely that A aggregates in the pump (Nitta et al., 1994). Therefore, it is likely that A oligomers rather than A fibrils cause these changes.

In a separate study, a novel azaindolizinone derivative, ZSET845, shows ameliorative effects on impaired performance caused by scopolamine and A_25–35 in passive avoidance and increases the ChAT activity in the hippocampus in rats (Yamaguchi and Kawashima, 2001; Yamaguchi et al., 2002). Further search for more potent compounds with less toxicity and good bioavailability led to a second compound, ZSET1446, which also has ameliorating effects on cognitive impairment induced by scopolamine and dizocilpine (Yamaguchi et al., 2002). In addition, ZSET1446 has ameliorating effects on impairment of performance in passive-avoidance task caused by a single i.c.v. injection of A_25–35 or lesions with the nucleus basalis magnocellularis by ibotenic acid (Yamaguchi et al., 2003). Moreover, we have shown that p.o. administration of ZSET1446 increases the extracellular ACh in the cortex and hippocampus (Yamaguchi et al., 2002, 2003) and enhanced nicotine-stimulated ACh release in the hippocampus in normal rats (Y. Yamaguchi, unpublished data), although the mechanism of action of ZSET1446 is not clear. In the present study, we further verified the ameliorating effects of ZSET1446 on the impairment of performance caused by chronic i.c.v. infusion with A_1–40 in Y-maze, water-maze, and passive-avoidance tasks and on the decrease of ChAT activity, GST immunoreactivity, and nicotine-stimulated ACh release in discrete areas of the brain.

	Materials and Methods

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Animals. Male Wistar rats at age 7 weeks were obtained from Japan SLC (Shizuoka, Japan) or Japan Laboratory Animals Inc. (Tokyo, Japan). They were housed in groups of two or three in a temperature- and light-controlled room (23°C; 12-h light cycle starting at 9 AM) and had free access to food and water. All of the experiments were performed in accordance with the Guidelines for Animal Experiments of the Nagoya University School of Medicine, the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society, and National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Surgery. Rats were anesthetized with pentobarbital (50 mg/kg, i.p.), and the infusion cannula for A was implanted into the right ventricle (A, –0.8; L, 1.4; V, 4.5), according to the atlas of Paxinos and Watson (1982). Continuous infusion of A_1–40 (300 pmol/day) was maintained for 15 days by attaching an infusion cannula to a miniosmotic pump (Alzet 2002; Alza, Palo Alto, CA) (Nitta et al., 1994). The day when the A_1–40 infusion started was designated as day 0 (Fig. 1). The control rats were infused with vehicle or A_40–1. A_1–40 and A_40–1 were purchased from Bachem (Torrance, CA) and dissolved in 35% acetonitrile containing 0.1% trifluoroacetic acid at a concentration of 25 µM.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Experimental schedule and chemical structure of ZSET1446.

The behavioral test and the administration of ZSET1446 were started on day 2 (2 days after the start of A_1–40 infusion). A_1–40-infused rats were administered ZSET1446 at doses of 0.01, 0.1, and 1 mg/kg or 1% CMC solution p.o. by gavage for 12 consecutive days 1 h before the behavioral test throughout the experimental period. Vehicle- and A_40–1-infused rats received 1% CMC only. These experimental schedules (Fig. 1) were followed by our preliminary observation that ZSET1446 reached a peak in its plasma and brain tissue levels 1 h after the p.o. administration, and half-life of ZSET1446 was approximately 3.5 h (Y. Yamaguchi, unpublished data). On day 14, rats were killed 1 h after the last administration of ZSET1446, and the discrete areas of the brain were dissected out for the measurement of ChAT activity and GST-like immunoreactivity.

Measurement of Locomotor Activity. Locomotor activity was measured on day 2. The experimental apparatus was a locomotor cage (25 x 42 x 20 cm) equipped with photobeams placed 2 cm above the floor at 1-inch intervals along two sides of the cage (Scanet SV-10 LD; Melquest, Toyama, Japan). Locomotor activity was measured during a 10-min period.

Y-Maze Task. The Y-maze task was carried out on day 3. The experimental apparatus consisted of a Y-maze made of plywood. Each arm of the Y-maze was 35 cm long, 25 cm high, and 10 cm wide and positioned at an equal angle (labeled A, B, and C). The room (20 m²) was filled with distinctive visual cues such as desks, chairs, and pictures. The apparatus was placed on the floor of the experimental room and illuminated with a 100-W bulb from 200 cm above. Each rat was placed at the end of one arm and allowed to move freely through the maze during an 8-min session. The sequence of arm entries was recorded manually (i.e., ABCBAC). An actual alternation was defined as entries into all three arms on consecutive occasions. Therefore, the number of maximal alternation was the total number of arm entries minus 2, and the percentage of alternation was calculated as (actual alternations/maximal alternations) x 100. In addition, the total number of arms entered during the sessions was also determined.

Water-Maze Task. The water-maze task of Morris (1984), with some modification, was carried out from day 4 to day 11. The experimental apparatus consisted of a black circular water tank (140 cm in diameter and 45 cm high). A transparent platform (10 cm in diameter and 25 cm high), which could not be seen by rats, was set inside the tank, which was filled to a height of 27 cm with water of temperature approximately 23°C; the surface of the platform was 2 cm below the surface of the water. The water tank was located in a test room (15 m²), in which there were many cues external to the maze. The room had adjustable indirect illumination, and a camera was fixed to the ceiling. The position of the cues remained unchanged throughout the water-maze task.

Reference Memory Test. Reference memory test was carried out for five consecutive days from day 4 to day 8 and consisted of two trials per day. Intertrial interval was approximately 30 min. For each training trial, the rat was put into the water tank at one of five starting positions. The platform was located in a constant position throughout the test period in the middle of one quadrant, equidistant from the center and edge of the water tank. In each training session, the latency to escape onto the hidden platform was recorded. If a rat was unable to find the platform within 90 s, the rat was guided to the platform, and a maximal score of 90 s was assigned. The escape latency and swimming speed were analyzed using Target/2 system (Neuroscience Inc., Tokyo, Japan). After each training session, the rat was allowed to remain on the platform for 15 s and then was returned to its home cage.

Probe Trial. Immediately after the 10th training trial on day 8, the platform was removed from the water tank, and animals were tested on a 30-s spatial probe trial. The time spent in the target quadrant, where the platform had been located during training, and the time spent in other quadrants were measured.

Short-Term Memory (Repeated Acquisition) Test. Short-term memory test was performed for three consecutive days from day 9 to day 11 and consisted of five trials per day. The procedure for short-term memory test was the same as the standard water-maze training, except that the platform location was rotated clockwise to a new quadrant in each day of the short-term memory task. For each trial, the rat was put into the water tank at one of the five starting positions. The first trial of each session was an informative sample trial in which the rat was allowed to swim to the platform in its new location and to remain there for 15 s. The rat was then placed in a home cage for an intertrial interval of 1 min. The platform remained in the same location following the remaining four trials of the day. Spatial short-term memory was designated as the mean escape latency of the second to fifth trials. The short-term memory in each rat was assessed by the mean performance for three consecutive days (Yamada et al., 1999b).

Passive-Avoidance Task. The multiple-trial passive-avoidance task was carried out from day 12 to day 13. A two-chamber step-through passive-avoidance apparatus was used. The apparatus consisted of illuminated and dark chambers. Two chambers were separated by a guillotine door. In the acquisition trial, each rat was placed in the illuminated chamber. Immediately after entering the dark chamber, the door was closed, and an inescapable scrambled electric shock (100 V, 0.3 mA, 5 s) was delivered through the floor grid. The rat was removed after receiving the foot shock and was placed back into the illuminated chamber by the experimenter. The door was opened 30 s later again to start the next trial. Training continued in this manner until the rat stayed in the illuminated chamber for 120 s on a single trial. Twenty-four hours later, each rat was placed in the illuminated chamber for retention trial. The interval between the placement in the illuminated chamber and the entry into the dark chamber was measured as step-through latency (maximum 300 s).

Rats were administered ZSET1446 at doses of 0.0001, 0.001, 0.01, and 0.1 mg/kg or 1% CMC p.o. 60 min before acquisition trial. Scopolamine hydrobromide (Sigma-Aldrich Japan, Tokyo, Japan), which was dissolved in saline, or saline was injected i.p. at the dose of 2 mg/kg 40 min after the administration of ZSET1446 or 1% CMC. The procedure for passive-avoidance test was the same as the A experiment, except that the interval of electric shock was 3 s.

ChAT Activity Measurement. Rats were killed by decapitation 1 h after the last administration of ZSET1446 on day 14, and brains were quickly removed and placed on an ice-cooled glass plate. The brain was placed on its dorsal surface, and a coronal section was made through the optic chiasm. The medial septum, readily visible and situated between the lateral ventricles, was pinched out with fine dissecting forceps, referring to the atlas of Paxinos and Watson (1982). The basal forebrain, the region directly ventral to the anterior commissure containing the nucleus basalis, was then removed from the remaining anterior portion of the brain. A piece of the cerebral cortex was dissected out. Then, the hippocampus was dissected out, and a piece of hippocampus was separated. Theses tissues were homogenized in 1.5-ml tubes containing 400 µl of iced 50 mM phosphate buffer (pH 6.8) with 10 mM EDTA and 0.5% Triton X-100. The tubes were centrifuged at 18,000g for 5 min at 4°C, and the supernatants were used as the enzyme solution. They were rapidly frozen and kept in a deep freezer at –80°C until assayed. Measurement of a ChAT activity was carried out as reported previously (Yamaguchi and Kawashima, 2001).

Western Blot Analysis. The remaining tissues of cortex and hippocampus used for ChAT assay as described in a previous section were homogenized in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM sodium orthovanadate, 2 mM EDTA, 50 mM NaF, 0.1% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, 10 µg/ml aprotinin, and 10 µg/ml leupeptin, pH 7.4). The homogenate was centrifuged at 18,000g for 20 min. The protein concentration in the supernatant was determined using Protein Assay Rapid Kit (Bio-Rad Laboratories, Hercules, CA). The sample was boiled in a sample buffer [0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol, and 20% 2x Tris borate/EDTA (90 mM Tris, 64.6 mM boric acid, and 2.5 mM EDTA, pH 8.4)], electrophoresed by SDS-polyacrylamide gel electrophoresis on a 12% separating gel, and then transferred electrophoretically to polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA). The same concentration (20 µg) of protein per lane was located in all of the Western blot analyses. The membrane was incubated in blocking buffer (Kirkegaard and Perry Laboratories, Gaithersburg, MD) for 30 min at room temperature. Each blot was incubated overnight at 4°C with the antibody against GST- (Kim et al., 2003) at a 1:1000 dilution. After washing, membranes were incubated with horseradish peroxidase-linked anti-rabbit IgG (Kirkegaard and Perry Laboratories) at a 1:2000 dilution for 1 h at room temperature. Immunoreactive materials on the membrane were detected using the enhanced chemiluminescence Western blot detection reagents (Amersham Biosciences Inc., Piscataway, NJ) and exposed to X-ray film (Hyperfilm, Amersham Biosciences, Buckinghamshire, UK). The band intensities on the film were analyzed using densitometric analysis and the ATTO Densitograph Software Library Lane Analyzer (ATTO, Tokyo, Japan). The same membranes were stripped with stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7) at 50°C for 20 min, incubated with primary antibody of -actin (Santa Cruz Biotechnology Inc., Beverly, MA) at 1:1000, and detected as described above. The optical density of each sample was corrected using the optical density of the corresponding -actin band.

Measurement for ACh. A separate set of rats from the other experiments was used in this study. Control rats were infused with vehicle only because we have previously shown that A_40–1 does not induce an impairment of nicotine-stimulated ACh release (Tran et al., 2003). A_1–40-infused rats were administered ZSET1446 at doses of 0.01, 0.1, and 1 mg/kg or 1% CMC solution p.o. from day 2 to day 14 and were not subjected to any behavioral tests. The cannula delivering A was removed, and a dialysis probe was implanted on day 14 after starting infusion of A. In brief, rats were anesthetized with pentobarbital (50 mg/kg, i.p.) and fixed in a stereotaxic apparatus. A dialysis probe (A-I-8-03; membrane length, 3 mm; EICOM, Kyoto, Japan) was implanted into the hippocampus (A, –5.8; L, 4.8; V, 4.0 mm) (Paxinos and Watson, 1982). Approximately 24 h after the implantation of the dialysis probe, Ringer's solution (147 mM NaCl, 4.02 mM KCl, and 2.25 mM CaCl₂) was perfused at a flow rate of 1.0 µl/min; dialysate was collected every 20 min; and ACh level was detected by high-performance liquid chromatography system with electrochemical detection. ACh was separated from the dialysates using a column (Eicopac AC-Gel 2.0 x 150 mm). The enzymatic reactor contains acetylcholinesterase (AChE) and choline oxidase, which catalyzes the formation of hydrogen peroxide from ACh and choline. The resultant H₂O₂ was detected by electrochemical detection, with a platinum electrode at 450 mV. After ACh level in dialysate became stable, Ringer's solution containing 3 mM nicotine to stimulate release of ACh was perfused for 20 min and then replaced with normal Ringer's solution.

AChE Activity Measurement. Effect of ZSET1446 on AChE was measured in in vitro experiments. Rat brain homogenates (100 mg of brain/ml of 0.1 M sodium potassium phosphate buffer, pH 8.0) were used as sources of AChE. Acetylthiocholine was used as the substrates for measurement of AChE activity. AChE activity was measured by the spectrophotometric method of Ellman et al. (1961).

Statistical Analysis. Results were expressed as mean ± S.E.M. For the results of locomotor activity, Y-maze, probe trial of Morris water maze, ChAT activity, and microdialysis, data were analyzed using one-way analysis of variance (ANOVA), which was followed by Dunnett's multiple comparison test. For the results of reference memory test, data were analyzed by two-way ANOVA, which was followed by Tukey analysis. For the results of short-term memory test of water-maze task, passive-avoidance task, and GST-like immunoreactivity, data were analyzed using the Kruskal-Wallis ANOVA by rank, which was followed by the nonparametric analysis of Mann-Whitney U test. The criterion for significance was p < 0.05 in all of the statistical evaluations.

	Results

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Locomotor Activity. There were no significant differences in locomotor activity counts on day 2 (2 days after the start of A infusion) among all of the groups [F(5,47) = 0.57, p > 0.05] (Fig. 2A).

View larger version (23K): [in this window] [in a new window]   Fig. 2. Effects of ZSET1446 on locomotor activity and spontaneous alternation behavior in A1–40-infused rats. Locomotor activity (A) was measured during 10-min period on day 2 (2 days after the start of A infusion). Daily p.o. administration of ZSET1446 was started on day 2, and locomotor activity was measured 1 h after the administration. Spontaneous alternation behavior (B) and the number of arm entries (C) during an 8-min session in the Y-maze task were measured on day 3. Vertical bars show mean ± S.E.M. The number within each column shows the number of rats used. ##, p < 0.01 compared with vehicle-infused control rats. ++, p < 0.01 compared with A40–1 -infused rats. **, p < 0.01 compared with A1–40-infused rats given 1% CMC.

Water-Maze Task. Changes in escape latency to find the hidden platform produced by training trials in each group of rats are shown in Fig. 3A. Two-way ANOVA with all of the treatment groups revealed significant main effects of group [F(5,470) = 4.73, p < 0.01] and training [F(9,470) = 35.19, p < 0.01] but no significant effect of group by trial interactions [F(45,470) = 0.51, p > 0.05]. Post hoc analysis indicated that performance in the A_1–40-infused group was significantly impaired compared with that in vehicle- (p < 0.05) or A_40–1-infused (p < 0.01) control group. Repeated daily administration of ZSET1446 at the doses of 0.01(p < 0.05), 0.1 (p < 0.01), and 1 mg/kg (p < 0.05) significantly ameliorated the impairment of performance caused by A_1–40 (Fig. 3A). Changes in path length produced by training trials in each group of rats showed similar pattern with the escape latency. There were also no significant differences in swim speed among six groups of animals during the course of the 10 training trials [F(5,470) = 0.72, p > 0.05].

View larger version (32K): [in this window] [in a new window]   Fig. 3. Effect of ZSET1446 on performance in the training trials (A) and in the probe trial (B) of the water-maze task in A1–40-infused rats. The training trials were carried out from day 4 to day 8. The probe trial was carried out on day 8, immediately after the last training trial. Daily p.o. administration of ZSET1446 was started on day 2, and the training was carried out 1 h after the administration. #, p < 0.05 compared with vehicle-infused control rats. ++, p < 0.01 compared with A40–1-infused rats. *, p < 0.05; **, p < 0.01 compared with A1–40-infused rats given 1% CMC.

A 30-s spatial probe trial was carried out on day 8 following the 10th training trial (Fig. 3B). One-way ANOVA indicated that there was a significant group effect on the time spent in the quadrant in which the platform had been located at the same place during training (target quadrant) [F(5,47) = 3.43, p < 0.05]. The A_1–40-infused rats searched the target quadrant for a significantly less amount of time than the A_40–1-infused group (p < 0.05) but not the vehicle-infused group. ZSET1446 at the doses of 0.01 (p < 0.05), 0.1 (p < 0.05), and 1 (p < 0.05) mg/kg significantly attenuated the impairment caused by A_1–40 (Fig. 3B).

The escape latency in the first trial (sample trial) and in the second to fifth trials (test trials) of the short-term memory test is shown in Fig. 4. Although there was no difference in the escape latency in the sample trial (Fig. 4A), marked differences in the test trials were observed among all of the treatment groups (Fig. 4B) [H = 18.53, p < 0.01]. Post hoc analysis revealed that escape latency in the A_1–40-infused group was significantly less than that in the vehicle- or A_40–1-infused control group (p < 0.01). ZSET1446 at the doses of 0.01(p < 0.01) and 0.1 (p < 0.01) mg/kg, but not 1 (p > 0.05) mg/kg, significantly attenuated the impairment caused by A_1–40 (Fig. 4B).

View larger version (24K): [in this window] [in a new window]   Fig. 4. Effect of ZSET1446 on performance in the sample trial (A) and in the test trials (B) of the short-term memory test of the water-maze task in the A1–40-infused rats. The short-term memory test (five trials per day) was carried out from day 9 to day 11. Daily p.o. administration of ZSET1446 was started on day 2, and the test was carried out 1 h after the administration. ##, p < 0.01 compared with vehicle-infused control rats. ++, p < 0.01 compared with A40–1-infused rats. **, p < 0.01 compared with A1–40-infused rats given 1% CMC.

View larger version (25K): [in this window] [in a new window]   Fig. 5. Effect of ZSET1446 on step-through latency (A) and the number of training trials (B) in the multiple-trial passive-avoidance task in the A1–40-infused rats. The task was carried out from day 12 to day 13. Daily p.o. administration of ZSET1446 was started 2 days after the start of A infusion, and the passive-avoidance task was carried out 1 h after the administration. #, p < 0.05 compared with vehicle-infused control rats. +, p < 0.05 compared with A40–1-infused rats. **, p < 0.01 compared with A1–40-infused rats given 1% CMC.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Effect of ZSET1446 on step-through latency (A) and the number of training trials (B) in the multiple trial passive-avoidance task in the scopolamine-treated rats. ##, p < 0.01 compared with vehicle-treated control rats. **, p < 0.01 compared with scopolamine-treated rats given 1% CMC.

ChAT Activity. ChAT activities in the medial septum [F(5,47) = 3.40, p < 0.05] and hippocampus [F(5,47) = 5.36, p < 0.01], but not in the cortex [F(5,47) = 1.87, p > 0.05] and basal forebrain [F(5,47) = 0.26, p > 0.05], were significantly changed among all of the treatment groups (Fig. 7). The ChAT activities in the medial septum and hippocampus in the A_1–40-infused rats were significantly reduced compared with vehicle- (p < 0.05) or A_40–1-infused rats (p < 0.01). ChAT activity was increased by ZSET1446 at the doses of 0.01 (p < 0.05), 0.1 (p < 0.01), and 1 (p < 0.05) mg/kg in the medial septum and at the doses of 0.01 (p < 0.01), 0.1 (p < 0.01), and 1 (p < 0.05) mg/kg in the hippocampus.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Effect of ZSET1446 on ChAT activity in four brain regions of A1–40-infused rats. ChAT activity is expressed as nanomoles of ACh produced per hour per milligram protein. Rats were killed 1 h after the last administration of ZSET1446 on day 14. #, p < 0.05 compared with vehicle-infused control rats. ++, p < 0.01 compared with A40–1-infused rats. *, p < 0.05 and **, p 0.01 compared with A1–40-infused rats given 1% CMC.

View larger version (27K): [in this window] [in a new window]   Fig. 8. Effects of ZSET1446 on GST-like immunoreactivity in the cortex (A) and hippocampus (B) in A1–40-infused rats. Rats were killed 1 h after the last administration of ZSET1446 on day 14. The number within each column shows the number of rats used. ##, p < 0.01 compared with vehicle-infused control rats. *, p < 0.05 and **, p < 0.01 compared with A1–40-infused rats given 1% CMC.

Microdialysis. As shown in Fig. 9, the extracellular level of ACh in the hippocampus was elevated approximately 2.9-fold by perfusion of Ringer's solution containing nicotine in vehicle-infused rats. There was a significant group effect in all of the treatment groups [F(4,20) = 8.27, p < 0.01] (Fig. 9). In A_1–40-infused rats, however, nicotine-stimulated release of ACh was significantly lower than that in vehicle-infused rats (p < 0.05). However, repeated p.o. administration of ZSET1446 significantly increased nicotine-stimulated release of ACh in A_1–40-infused rats at the doses of 0.01 (p < 0.01) and 1 (p < 0.01) mg/kg, but not 0.1 mg/kg (p > 0.05), although the level of ACh is almost the same of vehicle-infused rats.

View larger version (24K): [in this window] [in a new window]   Fig. 9. Effects of ZSET1446 on nicotine-stimulated ACh release in the hippocampus in A1–40-infused rats. #, p < 0.05 compared with vehicle-infused control rats. **, p < 0.01 compared with A1–40-infused rats given 1% CMC.

AChE Activity. Effect of ZSET1446 on AChE activity was determined according to the method of Ellman et al. (1961) using brain homogenate as the AChE source. However, ZSET1446 failed to inhibit the activity at the concentration up to 40 µM (data not shown).

	Discussion

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The present study showed that continuous i.c.v. infusion of A_1–40 into rat brain induced a significant learning disturbance in Y-maze and water-maze tasks, both of which are considered to involve spatial memory. Furthermore, the experiment in the retention of passive-avoidance learning by continuous infusion of A_1–40 confirmed the results of previous reports of our laboratory (Nitta et al., 1994, 1997; Yamada et al., 1999b). We observed that the locomotor activity and the number of total arm entries in the Y-maze in the A_1–40-infused rats were not different from those in the vehicle- or A_40–1-infused rats, indicating no changes in motor function. Moreover, there were also no significant differences among vehicle-, A_40–1-, and A_1–40-infused rats in the number of training trials in the passive-avoidance task and the escape latency onto the platform in the first training trial in the short-term memory test in the water maze. Collectively, these findings suggest that the continuous infusion of A_1–40 into the brain causes learning and memory deficits without change in spontaneous activity throughout the experiment.

In the present study, p.o. administration of ZSET1446 significantly ameliorated impairment of spontaneous alternation behavior in the Y-maze task, reference memory, and probe trial in the water-maze task at the doses of 0.01, 0.1, and 1 mg/kg. Moreover, p.o. administration of ZSET1446 at the doses of 0.01 and 0.1 mg/kg, but not 1 mg/kg, significantly shortened escape latency in short-term memory test of water-maze task and prolonged step-through latency in the retention trial of passive-avoidance task in A_1–40-infused rats. Furthermore, p.o. administration of ZSET1446 at the doses of 0.001, 0.01, and 0.1 mg/kg, but not 0.0001 mg/kg, significantly prolonged step-through latency in the retention trial of passive-avoidance task in scopolamine-treated rats. As described above, ZSET1446 did not affect motor and sensory activities. These results indicate that this compound has the ameliorating effect on cognitive impairment caused by A_1–40.

We have shown previously that nefiracetam at doses of 1, 3, and 10 mg/kg (Yamada et al., 1999b) and idebenone at doses of 10 and 20 mg/kg (Yamada et al., 1999a) improved cognitive deficits in the A-infused rats. Present results showed that ZSET1446 at doses of 0.01, 0.1, and 1 mg/kg ameliorated cognitive impairment induced by A infusion. Furthermore, we have previously shown that the potency of ZSET1446 is 100 times greater than that of donepezil in scopolamine-treated rats and more than 10 times greater than that of donepezil in A_25–35-treated rats in the passive avoidance when minimal effective doses are compared (Yamaguchi et al., 2001, 2002, 2003). Furthermore, ZSET1446 has shown a broader effective dose range (0.001–0.1 mg/kg p.o.) on learning impairment caused by scopolamine and dizocilpine. In the present study, ZSET1446 also showed broader dose range (0.01–1 mg/kg in the experiment of A, 0.001–0.1 mg/kg in the experiment of scopolamine) in both behavioral and chemical parameters. The broader "window" observed in ZSET1446 may be advantageous in therapy.

We observed that i.c.v. infusion of A_1–40 reduced ChAT activity in the medial septum and hippocampus and impaired nicotine-induced increase in extracellular ACh in the hippocampus. Several studies have shown that A causes cholinergic dysfunction and impairment of learning and memory (Nitta et al., 1994; Kar and Quirion, 2004). Chronic i.c.v. infusion of A_1–40 causes impairment of performance in the passive-avoidance and water-maze tasks and a decrease in the ChAT activity in the frontal cortex and hippocampus in the rat (Nabeshima and Nitta, 1994; Nitta et al., 1994). Pederson et al. (1996) have confirmed that A peptides reduce ChAT activity and intracellular concentration of ACh in a cell line that expresses cholinergic characters (generated by the fusion of neuroblastoma cells with primary mouse septal neurons). Likewise, Harkany et al. (1999) have reported that injection of A_1–42 into the nucleus basalis magnocellularis causes impairment in passive-avoidance learning and reduction in the ChAT activity in the cerebral cortex. We have previously shown that a single i.c.v. injection of A_25–35 results in impairment in passive-avoidance performance and a decrease in ChAT activity in the medial septum, cortex, and hippocampus (Yamaguchi and Kawashima, 2001). Itoh et al. (1996) have shown that nicotine-induced increase in extracellular ACh in the hippocampus/cortex is markedly impaired by the continuous infusion of A_1–40. Likewise, soluble A_1–42 has been shown to have an immediate dysfunctional effect on nicotinic ACh receptors in hippocampal CA1 interneurons (Pettit et al., 2001). Collectively, these results suggest that A induces the dysfunction of cholinergic neuronal system, and the dysfunction of cholinergic neuronal system is related to the learning and memory impairments in A-infused rats.

In the present study, repeated p.o. administration of ZSET1446 recovered the decrease of ChAT activity in the medial septum and hippocampus in A_1–40-treated rats to the level of vehicle-treated control rats. Moreover, our separate studies have shown that p.o. administration of ZSET1446 increases the extracellular ACh measured by in vivo microdialysis in the cortex and hippocampus in normal rats (Yamaguchi et al., 2002, 2003). The increase in extracellular ACh may be the result of the enhancement of ACh release because ZSET1466 has no inhibitory action on AChE in this study. Furthermore, ZSET1446 recovered an impairment of nicotine-induced increase in extracellular ACh in the hippocampus in the A_1–40-infused rats. Therefore, it is likely that the ameliorating effects of ZSET1446 on learning impairment induced by A_1–40 are caused by the stimulation of the cholinergic neuronal system. In the present study, the high dose of ZSET1446 (1 mg/kg) was effective in the behavioral experiments performed before day 8, but not effective on day 9. On the contrary, ChAT activity was recovered at all of the doses of ZSET1446 even on day 14. One possible explanation for this discrepancy is that ZSET1446 may show the different effects between short-time administration and long-time administration. Indeed, ZSET1446 did not increase ChAT activity in the brain after a single p.o. administration in normal rats (Y. Yamaguchi, unpublished observation). Therefore, further study is required to clarify the time course effect of ZSET1446 on ChAT activity and nicotine-stimulated ACh release.

Further, the ameliorating effects of ZSET1446 may also be partially because of the protection against A-induced neurotoxicity. In the present study, decrease in GST-like immunoreactivity was observed in the cortex in A_1–40-infused rats, and repeated p.o. administration of ZSET1446 protected the decrease of GST-like immunoreactivity in the cortex. It is known that a decrease in GST activity and GST level is observed in AD compared with controls in the brain and cerebrospinal fluid (Lovell et al., 1998). Furthermore, pretreatment of hippocampal cultures with GST before exposure to toxic doses of 4-hydroxy-2-noneal, which is significantly elevated in the brain (Markesbery and Lovell, 1998) and ventricular fluid (Lovell et al., 1997) of patients with AD, leads to a statistically significant enhancement in cell survival (Xie et al., 1998). In addition, overexpression of GST in neuroblastoma cells significantly increases resistance to ferrous sulfate/hydrogen peroxide, A-peptide, and peroxynitrite (Xie et al., 2001). These results suggest that ZSET1446 produces ameliorative effects through elevation of GST expression to promote resistance to the neurotoxic effect of A_1–40.

ZSET1446 increased ChAT activity, but not GST immunoreactivity, in the hippocampus, whereas ZSET1446 increased GST immunoreactivity, but not ChAT activity, in the cortex. The concentration of ZSET1446 in the hippocampus and cortex after p.o. administration was approximately the same level (Y. Yamaguchi, unpublished observation). Moreover, a single p.o. administration of ZSET1446 caused an increase in extracellular ACh in both the hippocampus and cortex (Yamaguchi et al., 2002, 2003). These results suggest that ZSET1446 did not affect in a region-dependent manner. On the contrary, repeated administration of ZST1446 may affect ChAT activity and GST immunoreactivity in a region-dependent manner. Therefore, further study is required to examine the time course effects of ZSET1446 on region dependence. Moreover, in the present results, the ChAT activity in the cortex and GST immunoreactivity in the cortex did not significantly decrease by infusion of A_1–40. Under these circumstances, there may be no detectable net effect of ZSET1446 on the cortex.

In binding study, ZSET1446 (10^–5 M) showed affinity for neither muscarinic and nicotinic receptors, nor the other central receptors (data not shown). A lack of receptor affinity is a characteristic feature of the nootropic compounds, such as piracetam. These drugs do not act at any well-characterized receptor system without any toxicity and side effects (Gouliaev and Senning, 1994). ZSET1446 produced no apparent adverse effects at any of the tested doses, and the acute and subacute preliminary toxicity tests with much greater doses showed no toxicity (data not shown). It has been shown that the effects of nootropic compounds on the cholinergic system might be secondary (Muller et al., 1999; Ghelardini et al., 2002). Therefore, even if the precise molecular mechanism is still unknown, it seems likely that ZSET1446 elicits its cognitive enhancing action through an indirect enhancement of the central cholinergic system. Work is in process to collect more information on the molecular mechanism of action of this compound. To conclude, the new compound ZSET1446 would be a useful candidate for further preclinical study aimed for the management of AD.

	Acknowledgements

 
We thank Kazuhiro Hashimoto, President of Zenyaku Kogyo Co., Ltd., for encouragement and constant support.

	Footnotes

 
This study was supported by a grant-in-aid for Science Research (no. 14370031), a COE Grant, and Special Coordination Funds for Promoting Science and Technology, Target-Oriented Brain Science Research Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

doi:10.1124/jpet.105.098640.

ABBREVIATIONS: AD, Alzheimer's disease; A, amyloid-; APP, amyloid precursor protein; LTP, long-term potentiation; GST, glutathione S-transferase; ChAT, choline acetyltransferase; ACh, acetylcholine; ZSET845, 3,3,-dibenzylimidazo[1,2-a]pyridin-2-(3H)-one; ZSET1446, spiro-[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one; CMC, carboxymethyl cellulose; AChE, acetylcholinesterase; ANOVA, analysis of variance.

Address correspondence to: Dr. Yoshimasa Yamaguchi, Research Laboratory, Zenyaku Kogyo Co., Ltd., 2-33-7 Ohizumi-machi, Nerima-ku, Tokyo 178-0062, Japan. E-mail: yoshimasa_yamaguchi{at}mail.zenyaku.co.jp

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