Abstract
ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine dihydrochloride], a novel ligand at neuronal nicotinic acetylcholine receptors with reduced adverse effects and improved oral bioavailability relative to (−)-nicotine, was tested in a variety of cognitive tests in rats and monkeys. Administered acutely, ABT-089 only marginally improved the spatial discrimination water maze performance of septal-lesioned rats. However, more robust improvement (45% error reduction on the last training day) was observed when ABT-089 was administered continuously via subcutaneous osmotic pumps (minimum effective dose: 1.3 μmol/kg/day). Continuous infusion of (−)-nicotine produced comparable improvement in the spatial discrimination water maze performance of septal-lesioned rats, but a 40-fold higher dose of (−)-nicotine was required (62 μmol/kg/day). Continuous infusion of ABT-089 to aged rats enhanced spatial learning in a standard Morris water maze, as indexed by spatial bias exhibited during a probe trial conducted after 4 days of training, but not when they were subsequently trained in a two-platform spatial discrimination water maze. The compound induced a small impairment in young rats on the standard water maze, but not on the two-platform task. A probe trial conducted after additional training in the standard water maze revealed no age or drug effects. ABT-089 did not affect performance of either the aged or young rats during inhibitory (passive) avoidance training. Also, continuous infusion of ABT-089 did not affect responses to acoustic startle or prepulse inhibition of acoustic startle in young, aged or septal-lesioned rats and did not affect locomotor activity in either sham-lesioned or septal-lesioned rats. In monkeys, acute administration of ABT-089 modestly improved the delayed matching-to-sample performance of mature, adult monkeys and more robustly improved performance in aged monkeys. Improved performance in the aged monkeys was restricted to the longest delay intervals and was not accompanied by changes in response latencies.
Alzheimer’s disease is a degenerative brain disorder characterized by profound cognitive deficits. Although disruption of several neurotransmitter systems has been reported with AD, the most severe and best-substantiated neurotransmitter abnormality in AD involves the cholinergic system. In AD, basal forebrain cholinergic neurons innervating the cortex, amygdala and hippocampus degenerate (Coyleet al., 1983). Elaboration of the relationship between this cholinergic dysfunction and the cognitive deficits associated with this disease has been of central importance in efforts to understand AD and in efforts to develop therapeutic strategies. In these endeavors, the primary focus has traditionally been on muscarinic cholinergic function, an emphasis based partly on the greater abundance of muscarinic acetylcholine receptors relative to nicotinic acetylcholine receptors in the brain (Araujo et al., 1990), as well as the well-known amnestic effects of muscarinic antagonists in humans (Beattyet al., 1986; Christensen et al., 1992; Drachman, 1977) and in experimental animals (Bartus and Johnson, 1976; Flood and Cherkin, 1986; Rush, 1988; Willner et al., 1986).
Recently, there has been increased interest in the role of nicotinic neurotransmission in cognitive deficits associated with AD and in the therapeutic potential of agents that act at neuronal nAChRs. There is now substantial evidence that nicotinic neurotransmission has an important role in cognitive function (Levin, 1992). For example, in rodents, the nAChR channel blocker, mecamylamine, disrupts acquisition of spatial information in the Morris water maze and impairs inhibitory avoidance learning (Bammer, 1982; Decker and Majchrzak, 1992; Dilts and Berry, 1967; Riekkinen et al., 1992, 1993); and in monkeys, mecamylamine impairs delayed matching-to-sample performance (Elrodet al., 1988; Jackson et al., 1989). Similarly, mecamylamine impairs acquisition of new information but not retrieval of previous learning in humans (Newhouse et al., 1992,1994). Conversely, activation of nAChRs can improve performance in animals with cognitive impairments. The classical cholinergic channel activator, (−)-nicotine, reverses some of the cognitive deficits observed in rats with basal forebrain lesions that crudely model the cholinergic dysfunction characteristic of AD (Decker et al., 1992; Hodges et al., 1991; Tilson et al., 1988). (−)-Nicotine has also been reported to attenuate age-related cognitive impairments (Socci et al., 1995; Widzowski et al., 1994). The beneficial effects of (−)-nicotine can also be observed with other agents that act at nAChRs. (−)-Lobeline (Deckeret al., 1993) and ABT-418 (Decker et al., 1994b) have both been reported to ameliorate spatial learning deficits in rats with septal lesions. Similarly, GTS-21 can improve avoidance learning in rats with nucleus basalis magnocellularis lesions (Meyer et al., 1994) and reduces age-related learning deficits in both rats and rabbits (Arendash et al., 1995; Woodruff-Pak et al., 1994).
The potential for the development of therapies for AD that work through nAChRs is further suggested by recent findings that (−)-nicotine improves some aspects of cognitive function in AD patients (Joneset al., 1992; Newhouse et al., 1988; Wilsonet al., 1995). The utility of (−)-nicotine for this purpose, however, is severely limited by a variety of side effects, notably on gastrointestinal and cardiovascular systems. ABT-418 has been shown to improve aspects of cognitive performance in AD patients with fewer side effects than (−)-nicotine (Newhouse et al., 1996) but, like (−)-nicotine, cannot be given by the oral route.
(−)-Nicotine and ABT-418 are examples of a broader class of compounds that are capable of modulating the activity of nAChRs. Members of this broader class of compounds have been designated as ChCMs (Arnericet al., 1996a). The newer ChCMs, such as ABT-418 and GTS-21, act via nAChRs to improve cognitive performance in experimental animals and appear to be less potent than (−)-nicotine in producing many of the adverse side effects (Decker et al., 1995a). These findings suggest the possibility that selective compounds that target nAChRs mediating beneficial effects while avoiding many of the adverse side effects of (−)-nicotine might be developed.
ABT-089 is a newly developed potent and selective ChCM (Lin et al., 1997; Sullivan et al., 1997, companion paper) that, in contrast to previously described ChCMs, has excellent oral bioavailabitity (Arneric et al., 1996b). In addition, ABT-089 readily crosses the blood-brain barrier (Arneric et al., unpublished observations). This compound has an improved safety profile relative to (−)-nicotine in preclinical testing (Linet al., 1997), but like (−)-nicotine, improves retention of inhibitory avoidance training in mice (Lin et al., 1997). The current study was designed to characterize the cognitive-enhancing potential of ABT-089 by assessing its effects on spatial memory task performance in aged and septal-lesioned rats and by determining its effects on the DMTS performance of mature, adult and aged monkeys.
Materials and Methods
Animals
All animals were handled according to protocols approved by Institutional Animal Care and Use Committees at Abbott Laboratories or the Medical College of Georgia. Male, Long-Evans rats (2- to 3-months old) used in the septal lesion experiment were obtained from Charles River Laboratories (Portage, MI). Testing in aged rats was conducted with male, Long-Evans rats obtained from Harlan Laboratories (Indianapolis, IN). Aged animals were 20- to 22-months old and mature, control animals for these experiments were 4- to 6-months old at the beginning of the study. Rats were singly housed and maintained in a climate-controlled facility with a 12:12 light:dark cycle (lights on at 6:00 a.m.) with free access to food and water except during behavioral testing. Behavioral testing was conducted during the light portion of the day.
Six mature pigtail monkeys (3 male and 3 female; Macaca nemestrina), ages 6 to 12.4 years; four aged rhesus monkeys (1 male and 3 female; Macaca mulatta), ages 20 to 27.5 years; and two aged long-tail monkeys (2 female; Macaca fascicularis), ages 22 and 27.5 years, served as subjects. All monkeys were housed at the Animal Behavior Center of the Medical College of Georgia. The facilities of the Animal Behavior Center meet or exceed current federal standards for non-human primate housing. Monkeys are housed within individual stainless steel cages composed of 127 × 71 × 66 cm units. All monkeys used in the current study had similar previous experience with the testing procedures and had been used in previous experiments assessing the effects of compounds acting at nAChRs. In these previous experiments, no effects lasting more than 24 h had been noted. Before entering the current study, the monkeys were allowed a wash-out period of at least 4 weeks during which no drugs were administered. Monkeys were allowed ad libitum access to water and maintained on a feeding schedule that allowed approximately 15% of their normal daily food intake to be derived from 300-mg food pellets which served as rewards during experimental sessions (standard monkey chow and banana flakes, P.J. Noyes, Inc., Lancaster, NH) and the remainder obtained from standard laboratory monkey chow after completion of a test session. When not testing, monkeys were allowed to observe television programs and were given toys to provide environmental stimulation.
Surgery
Lesions of the septum were produced by delivering radiofrequency current. The rats were anesthetized with pentobarbital (55 mg/kg i.p.), and the electrode (0.7 mm diameter, 1.5 mm tip exposure) was placed in the septum under stereotaxic control. Rats were placed in the stereotaxic frame and the nose bar adjusted to level the skull between bregma and lambda and the electrode positioned with the coordinates: 0.5 mm anterior to bregma, 0.0 mm lateral to the midline and 6.5 mm ventral to the skull surface. Current sufficient to maintain a temperature of 63°C at the electrode tip was passed for 30 s by a model RFG-4A lesion maker (Radionics Inc., Burlington, MA). Sham surgery was conducted by lowering an electrode to a point 1.0 mm above the target location for the lesions, but passing no current. At the end of the behavioral experiments, the rats were decapitated under 3% halothane anesthesia. The brain was quickly removed and divided into two sections with a coronal cut just anterior to the hippocampus. The rostral section of the brain was saved in formalin and subsequently used for histological verification of the septal lesions by use of 60-μm-thick sections stained with thionin. The hippocampus was removed from the caudal section of the brain and stored at −80°C for later determination of choline acetyltransferase activity.
Osmotic minipumps (Alza Corp., Palo Alto, CA) were implanted subcutaneously between the shoulder blades under halothane anesthesia. A pump with a 2-ml reservoir (model 2 ML4) designed to provide continuous infusion for 4 weeks was used in the aged rat experiment, and a pump with a 200-μl reservoir (model 2002) designed to provide continuous infusion for 2 weeks was used in the septal lesion experiments.
Biochemical Determinations: ChAT Activity
ChAT activity was measured in the hippocampus by the procedure of Fonnum (1975) as modified by Bannon et al. (1996).
Behavioral Procedures
Submerged platform water maze task.
Rats were trained in a circular pool (180 cm in diameter and 60 cm high) that had white interior walls and was filled to a depth of 37 cm with 25 ± 1°C water clouded by the addition of powdered milk. Four start locations were equally spaced around the perimeter of the pool. A white escape platform, 13 cm in diameter and 36 cm high (i.e., its top surface was 1 cm below water level), was placed in the center of the northwest quadrant. The quadrants were defined by imaginary lines connecting start locations on opposite sides of the pool. The platform was kept in the same location for all training trials, and the same sequence of start locations was used for each animal. The start locations were varied across trials for the four trials conducted on each day, such that each start location was used once per day. Each training trial was initiated by placing the rat in the pool facing the wall at one of the start locations, and the trial ended when the rat found the escape platform. If the rat did not find the platform within 60 s, it was gently guided to the platform by the experimenter. Trials were separated by a 10 s stay on the platform. On the day after the fourth day of training (block 1), a 60-s probe trial (probe 1) was conducted during which no platform was present. Beginning 3 days after this probe trial, the rats received 3 additional days of training to the same platform location (block 2). The day after the additional training, a second 60-s probe trial (probe 2) was conducted.
Data were collected by a tracking system (San Diego Instruments, San Diego, CA) interfaced with a personal computer, and the distance traveled was recorded for each training trial. For probe trials, the average distance from the hidden platform location during the trial (after a correction for the starting location) was used to evaluate performance [the “Gallagher” measure (Gallagher et al., 1993)].
Cue training in a water maze.
In a separate room from that used during hidden platform training but in a maze identical with the one described above, animals were trained to locate an aluminum foil-covered platform that protruded 1.5 cm above the surface of the water. For each of the four trials conducted on a given day, the platform was moved to a different quadrant and a different start location was used. The latency to find the platform was recorded. If a rat had not located the platform before the 60-s cut-off, it was gently guided to the platform location. As during hidden platform training, rats were allowed to remain on the platform for 10 s, but during cue training rats were run in squads of six and were therefore allowed approximately 5 min in their home cages between trials. Two days of cue training were conducted.
Spatial-discrimination (two-platform) water maze task.
Beginning 3 days after cue training, and in the same room and the same maze as that used for cue training, a spatial-discrimination version of the Morris water maze task was conducted. In this procedure, two aluminum foil-covered platforms, similar in appearance to those used during cue training, protruded 1.5 cm above the surface of the water. One platform was stable whereas the other, made of polystyrene, floated and was easily tipped over when a rat attempted an escape. The locations of the platforms remained fixed during training. An error was recorded each time the rat contacted the floating platform with either its head or forepaws. If a rat did not find the correct platform by the 60-s cut-off, the rat was guided to the correct platform. In these cases the total errors committed in the 60-s period were recorded. Six trials on each of 4 consecutive days were conducted, with the starting location being varied between the two locations equidistant from the platforms across trials.
Inhibitory avoidance.
Inhibitory avoidance testing was conducted with an automated avoidance training system (San Diego Instruments, San Diego, CA). For the training trial, rats were placed in one of the chambers of the two-compartment apparatus. After a 10-s adaptation period, the chamber was illuminated and a gate to the second, darkened chamber was opened; and the rat was allowed 60 s to enter the dark chamber. If the rat entered the dark chamber, the gate closed, the rat received a footshock (0.5 mA, 1 s) through the grid floor and was then removed from the apparatus. A retention trial was conducted 72 h after the training trial with use of the same procedure except that no shock was delivered and the rat was allowed a maximum of 300 s to enter the dark chamber. The latency to enter the dark chamber was recorded for both trials.
Acoustic startle and prepulse inhibition.
Startle responses were recorded in startle chambers (SR-LAB, San Diego Instruments, San Diego, CA) as described previously (Decker et al., 1995b; Swerdlow and Geyer, 1993). The rat was placed in a Plexiglas cylinder contained in a sound-attenuating enclosure, and startle responses were measured for a period of 100 ms after the delivery of acoustic stimuli. On the first day, rats were placed into the chambers with 65 dB background noise for a 5-min habituation period and then received 30 acoustic startle trials, 10 trials each at 90, 95 and 105 dB noise of 30-ms duration presented in a quasirandom order. A 30-s variable intertrial interval separated each trial (minimum 5 s, maximum 45 s). On the next day, test sessions were conducted. During test sessions, two different trial types were used: a base-line startle trial, in which a 120 dB startle stimulus was presented, and a prepulse trial, in which this startle stimulus was preceded by 100 ms by a lower intensity auditory stimulus. Trials were presented in quasirandom order with a 20-s variable intertrial interval (minimum 5 s, maximum 30 s). The mean maximal startle response to the 120 dB stimulus presented alone and when preceded by 70, 75 and 80 dB prepulse stimuli (in the aged animal experiment) or by 67, 70 and 75 dB (in the septal lesion experiments) was measured. Twelve base-line trials and 12 trials at each prepulse intensity were conducted in a quasirandom order.
Open-field activity.
Spontaneous locomotor activity was assessed in a single session in an open field (41 × 41 cm). Horizontal activity was measured by a photobeam detector system (San Diego Instruments, San Diego, CA) and beam breaks were accumulated for four 6-min. blocks.
DMTS.
DMTS stimuli were 2.5-cm-diameter colored disks presented via light-emitting diodes located behind clear push-keys on panels attached to the front of home cages. A trial began with the illumination of the sample key by one of three colored stimuli. A press of this key initiated one of four preprogrammed delay intervals, during which no keys were illuminated. After the interval, two choice lights were then illuminated. One of the choice stimuli matched the hue of the sample light, whereas the nonmatching choice was one of the other two colors. Both choice stimuli remained illuminated until one was depressed. Responses to the choice key illuminated by color matching the sample color were rewarded by a 300-mg banana-flavored pellet. Four possible delay intervals were used. These include a 0-s delay and three longer delays (referred to as short, medium and long) chosen to produce the following levels of performance: short delay, 75 to 84%; medium delay, 65 to 74%, and long delay, 55 to 64%. Length of the delays was adjusted according to skill levels of individual monkey. These delays ranged from 0 to 15 s to a maximum of 0 to 240 s. Each possible stimulus and choice configuration occurred an equal number of times for each delay interval.
Experimental Protocols
Septal-lesioned young, adult rat experiments.
Acute ABT-089. In the acute ABT-089 experiments in septal-lesioned rats, cue training (2 days) in the water maze was initiated 8 to 10 days after the lesions were produced. These sessions served to habituate the animals to the maze and to train them to escape onto the visible platform. ABT-089 was not administered during these cue training sessions. Three days after the completion of cue training, training on the two-platform water maze was conducted for 4 days, with one session of six trials on each day. Each training session started 15 min after ABT-089 (1.9, 6.2 or 19 μmol/kg) or saline was administered i.p., a time when substantial plasma concentrations of ABT-089 are present (Arneric et al., unpublished observations). Septal-lesioned rats received either ABT-089 (n = 10 per dose) or saline (n = 9), and sham-lesioned rats (n = 8) received saline injections.
Continuous infusion of ABT-089 or (−)-nicotine. In the continuous infusion ABT-089 and (−)-nicotine studies, pumps were implanted 3 to 4 days after the lesions were produced. (−)-Nicotine was infused at 19 or 62 μmol/kg/day, with 9 lesioned and 9 sham rats receiving the lower dose and 10 lesioned and 10 sham rats receiving the higher dose. Saline infusions were administered to 9 lesioned and 9 sham rats as a control. In the ABT-089 experiment, 0.4, 4 and 13 μmol/kg/day were administered to lesioned (n = 14, 14 and 15, respectively) or sham (n = 14, 15 and 15, respectively) rats. Saline pumps were implanted in 15 lesioned and 12 sham rats as a control. Three days after pump implantation, open-field activity was assessed. During the next 2 days, testing of acoustic startle responses was conducted. On the day after the last startle session, cue training in the water maze was conducted for 2 days. Three days later, the rats began training on the two-platform water maze. Rats were sacrificed on the day after the completion of the two-platform water maze (i.e., 2 weeks after pump implantation).
Aged rat experiments.
Pumps administering 0, 1.3, 4 or 13 μmol/kg/day of ABT-089 were implanted in aged (n = 8, 8, 9 and 9, respectively) and young (n = 10 per group) rats. Hidden platform training was initiated 3 days after pump implantation. On the day after the second probe trial (i.e.,2 weeks after pump implantation), inhibitory avoidance training was conducted. Acoustic startle testing was initiated on the day after the inhibitory avoidance retention test, and cue training in the water maze was started on the day after completion of startle testing. Beginning 3 days after the cue training, rats were trained on the two-platform water maze for 4 days.
Monkeys experiments.
Mature monkeys. Doses of ABT-089 (4.0, 8.1, 16.2, 32.4, 64.8 and 129.6 nmol/kg) were administered in an ascending dose series in the gastrocnemius muscle within a volume of 0.1 ml saline/kg. Control data were obtained after vehicle administration with each monkey serving as its own control. One vehicle session was conducted during each week of testing. Test sessions began 10 min after vehicle or drug administration. A minimum “drug washout” period of 2 days was allowed between sessions. During this period, a return to base line DMTS performance was established in each monkey.
Aged monkeys. The behavioral testing procedures and analyses used with aged animals were identical with those used with mature monkeys. Doses of ABT-089 (2.0, 4.0, 8.1, 16.2 and 32.4 nmol/kg) were administered in an ascending dose series in the gastrocnemius muscle within a volume of 0.1 ml saline/kg. Administration of this lower dose range of ABT-089 to aged monkeys was used to avoid side effects often observed in aged animals receiving compounds with activity at nAChRs. A best dose (i.e., the dose at which maximal DMTS performance collapsed across delays was obtained) was selected for each individual monkey from the dose-response experiment. This dose was used in a subsequent experiment designed to determine whether ABT-089 effects could be blocked with the noncompetitive nAChR antagonist, mecamylamine. In this experiment, the effects of ABT-089 and mecamylamine (2.5 μmol/kg) were assessed separately and in combination.
Compounds
ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine dihydrochloride] was synthesized by Abbott Laboratories (Abbott Park, IL) with recently described procedures (Lin et al., 1997) and (−)-nicotine bitartrate and mecamylamine HCl were obtained from Sigma Chemical Co.(St. Louis, MO). Drugs used in minipump infusion experiments were dissolved in sterile water, and control infusions consisted of 0.9% sterile saline. Compounds used for i.p. and i.m. injections were dissolved in 0.9% sterile saline. Doses are expressed in micromoles per kilogram for rats and nanomoles per kilogram for monkeys.
Statistics
Data from the rat studies were analyzed by ANOVA or ANCOVA, followed by means comparison contrast analysis. Because inhibitory avoidance latencies were not distributed normally and because the variance of startle responsivity varied linearly with the mean startle response in the acoustic startle experiment with aged rats, log transformations of these data were performed before ANOVA. For statistical analyses of monkey DMTS performance, the percent correct during vehicle-treated sessions was compared with sessions after administration of ABT-089 and to sessions completed 24 h after drug administration by one-way repeated measures ANOVA.
Results
Verification of Septal Lesions
The septal lesions produced in the animals used in this study were fairly large, destroying most of the medial portion of the septal area and frequently involving portions of the lateral septum and portions of the diagonal band of Broca. Slight damage to the corpus callosum was observed, but very little damage to the anterior commissure was noted. Some lesions extended caudally into the most rostral aspects of the fimbria-fornix. Analysis of hippocampal choline acetyltransferase activity (table 1) revealed lesion-induced reductions of about 70 to 75% in each of the minipump infusion studies and about 60% in the i.p. injection study. Although sham-lesioned rats treated with continuous infusion of either (−)-nicotine or ABT-089 tended to have somewhat lower choline acetyltransferase activity, drug treatment did not significantly alter hippocampal choline acetyltransferase activity in either sham or lesioned animals in any of the experiments.
Hippocampal ChAT activity
Effects in Septal-lesioned Rats
Acute administration of ABT-089 did not consistently affect the two-platform water maze performance of rats with septal lesions (fig.1). One-way repeated measures ANOVA of these data, with use of the training session as the repeated measure, revealed a significant group effect [F(4,42) = 8.478, P < .0001] that was caused by a lesion-induced impairment. Lesioned rats treated with ABT-089 did not differ from lesioned rats treated with saline in this analysis, although independent analysis of the data from each session by ANOVA revealed a small, but statistically significant improvement in the 6.2 and the 19 μmol/kg groups on the third but not the fourth training day. Because this pattern of effects could be related to the development of decreased efficacy with repeated administration, a continuous infusion experiment was conducted.
Effects of acute (i.p.) administration before daily training sessions on the two-platform water maze performance of rats with septal lesions. Shown are the mean (± S.E.M.) number of errors on each training day. *significantly different from lesion saline group (P < .05).
In the continuous infusion experiment, neither ABT-089 [F(3,106) = 0.327, P = .806] nor septal lesions [F(1,106) = 1.316, P = .254] significantly affected latencies to find the single visible platform during the two cue training sessions conducted before the beginning of spatial discrimination (two-platform) training (data not shown). However, analysis of two-platform water maze data by a two-way repeated measures ANOVA revealed a significant lesion effect [F(1,106) = 56.175, P < .0001], a significant training session effect [F(3,318) = 74.218, P < .0001] and a significant lesion by training interaction [F(3,318) = 3.147, P < .05], with the lesion effect being more pronounced in the later training sessions. Separate two-way ANOVA for each training session revealed a significant drug effect on the last training day [F(3,106) = 2.743, P < .05]. This effect is illustrated in figure 2 and is caused by a significant improvement in the performance of lesioned rats who were treated with 1.3 μmol/kg/day of ABT-089 (P < .05) and a trend toward improved performance noted at 4.0 μmol/kg/day (P < .06).
Effects of continuously infused ABT-089 (A) or (−)-nicotine (B) on the two-platform water maze performance of rats with sham lesions or septal lesions. Shown is the mean (± S.E.M.) number of errors on the last day of training (day 4).+marginally different from lesion saline (P < .1). *different from lesion saline (P < .05).
As in the ABT-089 experiment, continuous infusion of (−)-nicotine [F(2,50) = 1.358, P = .266] and septal lesions [F(1,106) = 0.002, P = .966] did not significantly alter latencies to find the single visible platform during the two cue training session conducted before the beginning of spatial discrimination (two-platform) training (data not shown). Effects on two-platform water maze performance were noted. The repeated measures ANOVA revealed a significant lesion effect [F(1,50) = 28.178, P < .0001], a significant main effect of nicotine infusion [F(2,50) = 4.820, P < .02], a significant training session effect [F(3,150) = 53.987, P < .0001] and a significant lesion by training interaction [F(3,150) = 8.688, P < .0001], with the lesion effect again being more pronounced in the later training sessions. As in the ABT-089 experiment, separate two-way ANOVA for each training session revealed significant drug effects on the last training day in the (−)-nicotine experiment [F(2,50) = 3.306, P < .05]. This effect, shown in figure 2, is caused by a significant improvement in the performance of lesioned rats who were treated with 62 μmol/kg/day of (−)-nicotine (P < .05) and a trend toward improved performance noted at 19 μmol/kg/day (P < .06).
In contrast to the effects of continuous infusion of (−)-nicotine or ABT-089 on the water maze performance of septal-lesioned rats, these compounds did not alter lesion-induced changes in locomotor activity (fig. 3) or startle reactivity (table2). Septal lesions produced a biphasic effect on locomotor activity in both experiments [time by lesion interaction, F(3,150) = 15.207, P < .0001 for the (−)-nicotine experiment and F(3,318) = 47.099, P < .0001 for the ABT-089 experiment], with a lesion-induced decrease in horizontal activity early in the session being followed by a lesion-induced increase in activity later in the session. Neither (−)-nicotine nor ABT-418 altered this pattern of performance [main effects and interactions involving ABT-089 or (−)-nicotine were not statistically significant, P > .05]. Similarly, septal lesions were associated with increased startle responses in both experiments [for the response to the 120 dB startle stimulus, F(1,50) = 8.082, P < .01 for the (−)-nicotine experiment andF(1,106) = 9.363, P < .005 for the ABT-089 experiment], but there were no significant drug effects or drug by lesion interactions in these experiments (P > .4). Prepulse inhibition was not affected by the septal lesions or by either compound (data not shown).
Effects of continuously infused ABT-089 or (−)-nicotine on locomotor activity of rats with sham lesions or septal lesions. Shown are the mean number of horizontal activity counts in 6-min blocks during a 24-min session in a novel open field.
Startle response (expressed in arbitrary units) of sham- and septal-lesioned rats treated with ABT-089 or (−)-nicotine by subcutaneous osmotic pumps
Effects in Aged Rats
Analysis of the water maze data in the aged rats study was conducted by both standard ANOVA and ANCOVA. ANCOVA was used because a significant age effect was found on performance during cue training to a visible platform (see fig. 4C). Aged rats were impaired relative to young animals based on latency to escape to the visible platform. Because cue training to a visible platform has been used as an apparent measure of nonmneumonic factors (e.g., sensorimotor deficits) on water maze performance, cue training latency (averaged over 2 days) was used as a covariate for analyzing data from the hidden platform water maze task when a significant relationship was found between cue training latency and water maze parameters. This step was taken to reduce variability in performance of the hidden platform water maze task attributable to the age deficit observed during cue training to a visible platform. The between-subject measures were age and ABT-089 dose.
Effects of ABT-089 on Morris water maze performance of old and young rats. Values represent means (± S.E.M.). (A) Distance swam to reach the hidden platform in the water maze for young and aged rats during first 4 days of acquisition. (B) Average distance from the hidden platform location (Gallagher measure) during probe 1 [*significantly different from saline-treated young rats (P < .02); +significantly different from saline-treated old rats (P < .01)]. (C) Latency to reach visible platform during cue training averaged across the 2 days of testing. [#significantly different from young rats receiving the same dose of ABT-089 (P < .05)].
Hidden platform water maze.
Acquisition data are presented in figure 4. The average distance required to reach the platform was computed for the four trials per day. Data were analyzed separately for block 1 (days 1–4 of the experiment), and block 2 (days 8–10 of the experiment). Initial analysis of the mean escape distance across days in blocks 1 and 2 by standard repeated measures ANOVA revealed no differential effects of ABT-089 across days (i.e., no interactions involving ABT-089 dose and the repeated measure, days, were statistically significant; all P values > .4). The only interaction between age and training day was observed during block 1 [F(3,198) = 4.795, P = .003]. This interaction resulted from longer mean escape distances for young rats relative to older rats on the first day [F(1,66) = 4.616, P = .035] but no age-related differences on subsequent days. The longer mean escape latencies for young rats on the first day is likely an artifact because many animals failed to find the escape platform within the time allotted (60 s) on the first day, and young rats had faster swim speeds [F(1,66) = 95.27, P = .003] that allowed them to cover more distance before the time ran out. Given the lack of interaction between days within the blocks and the variables of interest, subsequent analysis of escape distance data with ANCOVA used escape distances averaged across days within each block.
Block 1.
Mean distance traveled from over trials in block 1 of acquisition is presented in figure 4A. In saline-treated animals, aged rats tended to swim a longer distance to find the platform than the young animals, and there was a trend for the lower doses of ABT-089 to reduce the difference between aged and young rats. However, these effects were not statistically significant.
Probe 1.
Analysis of data (fig. 4B) from probe 1 by ANCOVA indicated a significant age × drug interaction [F(3,65) = 4.223, P < .01]. Compared with young animals treated with saline, aged rats treated with saline remained significantly further from the hidden platform location during the probe trial (P < .01); and in aged rats, the 1.3 μmol/kg/day dose of ABT-089 significantly attenuated this deficit (P < .02), with a similar trend noted for the 4 μmol/kg/day dose (P = .08). In young rats, 1.3 μmol/kg/day and 13 μmol/kg/day ABT-089 significantly increased the average distance from the hidden platform location (P < .01 and P < .02, respectively). These data are presented in figure 4B.
Block 2.
During block 2 of acquisition training, both aged and young animals reduced the swim distance required to find the hidden platform compared with performance observed during block 1. Similar to the findings in block 1, no statistically significant age or drug effect was found (data not shown).
Probe 2.
Data from probe 2 also did not reveal statistically significant age or drug effects (data not shown).
Cue training.
The average latency to reach the visible platform during 2 days of cue training are presented in figure 4C. A significant age effect was found [F(1,66) = 31.713, P < .0001]. Comparable effects were observed whether escape latencies or distance required to escape (data not shown) were analyzed. Overall, aged rats took significantly longer to reach the visible platform than young animals. Although there was not a significant drug effect, in both aged and young animals the 4 μmol/kg/day dose of ABT-089 tended to decrease the latency to reach the visible platform.
Two-platform spatial discrimination.
There were no statistically significant effects found in the two-platform spatial discrimination version of the water maze when analyzed by ANCOVA with average escape latencies during cue training used as the covariate. On average, all animals were making less than two errors by the end of training [mean daily errors over 4 days ± S.E.M. for saline, 1.3, 4 and 13 μmol/kg/day, respectively = 1.20 ± 0.17, 0.90 ± 0.14, 0.89 ± 0.18 and 1.42 ± 0.25 for the young rats and 1.22 ± 0.22, 1.92 ± 0.49, 1.69 ± 0.35 and 1.75 ± 0.20 for the old rats].
Inhibitory avoidance.
Data from the inhibitory avoidance task are presented in figure 5. On the training trial, no significant differences were observed. On the retention trial, conducted 72 h after the training trial, a significant age effect was found, F(1,66) = 9.102, P < .01. Young rats treated with saline took significantly more time to enter the dark chamber during the retention trial than the aged rats treated with saline (P < .02). No significant effect of drug treatment and no significant drug by age interactions were noted in this experiment.
Data from the inhibitory avoidance task. The retention trial was conducted 72 hr after the training trial. Values represent the means (±S.E.M.) on a log scale. *significantly different from saline-treated young rats (P < .02).
Acoustic startle and PPI.
Acoustic startle data are presented in table 3. A significant age effect was found [F(1,66) = 51.310, P < .0001]. Overall, aged rats were less responsive to the acoustic startle stimulus. Both young and aged animals exhibited substantially lower startle responses when the 120 dB stimulus was preceded by prepulses (PPI), and this effect was not altered by drug treatment (data not shown).
Startle response of aged and mature rats to a 120 dB stimulus
Effects in Monkeys
Mature monkeys.
The six mature monkeys used in this study exhibited the following base-line DMTS performance (% correct) after vehicle administration: zero delay = 96.5 ± 2.73; short delay = 84.0 ± 3.47; medium delay = 62.5 ± 3.9; long delay = 54.2 ± 4.0. ABT-089 (4.0, 8.1, 16.2, 32.4, 64.8 or 129.6 nmol/kg) did not significantly alter performance on zero- or short-delay trials initiated 10 min or 24 h after drug administration. DMTS accuracy on medium-delay trials on average was maintained from 6 to 9% points greater than saline-derived base-line performance (fig. 6). Administration of the 64.8 nmol/kg dose resulted in significant enhancement of performance 10 min after administration [F(1,6) = 17.70, P < .01]. Thus, the improvement in performance at the medium delay at the 64.8 nmol/kg dose represented a 14.3% increase over base line. The elevation of performance was not observed 24 h after administration of this dose. DMTS accuracy on long-delay trials, although elevated 10 min after administration of the 32.40 nmol/kg dose (14.8% above base line), was not significantly greater than base-line performance (P < .08).
Effects of ABT-089 on DMTS performance on medium- and long-delay trials in six mature monkeys 10 min after drug administration (i.m.). Each value represents the % of DMTS trials completed correctly (mean ± S.E.M.). *Significantly different from saline-derived base-line performance (P < .05).
Two measures of response latency were recorded during DMTS testing: choice latency, the time between presentation of the two choice stimuli and depression of one of the choice keys, and sample latency, the time between initiation of a new trial (illumination of the stimulus lights behind the sample) and depression of the sample key by the animal. Examination of choice latency data indicated that the latency to make incorrect matching choices was significantly greater than the latency to make correct matching choices, 3.15 vs. 5.88 s, respectively [F(1,45) = 8.33, P < .05]. ABT-089 administration had no significant effect on this pattern of responding. Sample response latencies were similar for correct and incorrect trials and were not altered by drug administration.
Aged monkeys.
The six aged monkeys used in this study exhibited the following base-line DMTS performance (% correct ± S.E.M.) after vehicle administration: zero delay = 88.9 ± 3.0; short delay = 72.2 ± 3.0; medium delay = 65.3 ± 2.8; long delay = 58.3 ± 2.4. ABT-089 did not significantly alter DMTS performance on trials with zero, short or medium delay intervals. However, accuracy on long delay trials was markedly enhanced (18.0% above base-line performance) after administration of the 4.0 nmol/kg dose [F(1,5) = 9.39, P < .05; figure 7]. DMTS performance 10 min after administration of the 8.1 nmol/kg dose was similarly elevated, an effect which approached statistical significance (P = .059). Although performance 24 h following administration of the 4.0 nmol/kg dose was 6.5% above baseline, this effect did not achieve statistical significance.
Effects of ABT-089 on DMTS performance for medium- and long-delay trials in six aged monkeys 10 min after drug administration (i.m.). Each value represents the % of DMTS trials completed correctly (mean ± S.E.M.). *Significantly different from saline-derived base-line performance (P < .05).
Regarding response latencies to sample and choice stimuli, there were no statistically significant differences in either latency between correct and incorrect trials. However, latencies to respond to choice stimuli on incorrect trials were increased relative to correct trials (1.55 vs. 1.38 s, respectively), an effect which approached statistical significance (P = .054). ABT-089 did not alter latencies to respond to sample or choice stimuli.
When mecamylamine and the individualized best dose of ABT-089 were subsequently tested in separately and in combination, a significant effect was observed [F(3,12) = 3.66, P < .05] at the long delay interval. ABT-089 enhanced performance relative to saline treatment (mean % correct ± S.E.M.: Saline = 50.8 ± 1.6, ABT-089 = 62.5 ± 7.0, P < .05). By itself, mecamylamine did not significantly affect performance at the dose tested (mean % correct ± S.E.M.: 57.5 ± 2.8, P > .1); and the effect of combined administration of ABT-089 and mecamylamine was not significantly different from either the effect of ABT-089 alone or the effect of mecamylamine alone (mean % correct ± S.E.M.: 60.9 ± 2.8).
Discussion
Septal lesions, which reduce cholinergic input to the hippocampus, impaired the acquisition of a spatial discrimination water maze task in the present study. Although it is conceivable that the sham rats used a nonspatial strategy, such as odor cues associated with the platforms, to solve this task, this appears unlikely. We have, for example, demonstrated that changing the position of the prominent visual stimuli in the testing environment produces a corresponding change in the performance of normal, well-trained rats (unpublished observation). Thus, it would appear that the septal lesions interfered with the rats’ ability to make use of a spatial strategy. This lesion-induced impairment was significantly attenuated by continuous infusion of ABT-089 or (−)-nicotine. The results of these experiments suggest that with continuous infusion, ABT-089 was approximately 40-fold more potent than (−)-nicotine, because significantly improved performance at the end of training was observed with 1.3 μmol/kg/day of ABT-089 and 62 μmol/kg/day of (−)-nicotine. Significant improvements were not noted in sham-lesioned rats with either compound, although improved performance may have been difficult to detect given the rapid acquisition of this task by sham-lesioned rats.
Continuous infusion of improved performance in rats with septal lesions, whereas acute administration of ABT-089 did not produce a robust improvement of water maze performance in the present study. Thus, ABT-089 appeared to be more effective after several days of continuous s.c. administration than after acute administration. The doses effective with continuous infusion (1.3 and 4.0 μmol/kg/day) produce plasma levels in the range of 5 to 14 ng/ml (Arneric et al., 1996b). This is considerably less than the plasma levels required to observe improved performance with (−)-nicotine, because the 62 μmol/kg/day dose of (−)-nicotine that improved performance produces a plasma concentration of at least 150 ng/ml (J.P. Sullivan, personal communication). The failure to observe robust enhancement of water maze performance in acutely treated rats does not appear to be simply the result of pharmacokinetics. Substantial plasma levels of ABT-089 are achieved with i.p. administration; the highest dose tested in the current study (19 μmol/kg i.p.) producing plasma levels of several hundred nanograms per milliliter during the time when behavioral testing was initiated (Arneric et al., 1996b). Moreover, the half-life of ABT-089 in rats is 157 min (Arnericet al., 1996b), making it likely that reasonable plasma levels were maintained during any memory consolidation period immediately after training. Although it is possible that the acute doses reported here may have been too high (because of the U-shaped dose response), it is notable that we have also not observed robust effects with lower doses (as low as 0.19 μmol/kg i.p.) of acutely administered ABT-089 (data not shown). Thus, an explanation of the apparent potentiation of the cognitive-enhancing effects of ABT-089 with continuous administration is not yet available, but this observation finds a parallel in the increased potency of this compound in an in vitro cytoprotective assay seen with prolonged incubation (Sullivan et al., 1997, companion paper).
The improved two-platform water maze performance produced in septal-lesioned rats by continuous infusion of (−)-nicotine or ABT-089 contrasts with the much more modest effects we have observed with the cholinesterase inhibitor, tacrine, under identical test conditions. Tacrine, infused at 16 to 64 μmol/kg/day, only modestly improved performance in the two-platform water maze task, an effect that was actually more pronounced in sham rats than in lesioned rats (unpublished observations). This finding corresponds to the modest efficacy that has been observed with tacrine in AD patients (Freeman and Dawson, 1991; Sahakian et al., 1993). Cholinesterase inhibitors presumably work by prolonging the availability of endogenous acetylcholine. The failure of a cholinesterase inhibitor to produce the more robust effects of (−)-nicotine and ABT-089 in septal-lesioned rats suggests that the cognitive enhancing effects of these latter compounds depend less on intact cholinergic input to the hippocampus than does tacrine. These data, then, suggest that at least a component of the effects of both (−)-nicotine and ABT-089 is mediated by actions at a postsynaptic site.
The impaired performance of aged rats in the water maze experiments in the current study could largely be accounted for by deficits in cue training performance. When the cue training performance of the rats was used as a covariate in ANCOVA, age-related differences in most of the learning measures used to assess water maze performance were not found. This could be interpreted as evidence that age-related sensorimotor or other performance deficits account for age-related impairments in water maze performance. Similarly, age-related impairments in inhibitory avoidance performance could have been caused by diminished sensory acuity in the aged rats (i.e., reduced shock sensitivity). Consistent with this interpretation is our finding that aged animals displayed greatly decreased startle responses to acoustic stimuli, although it should be noted that the aged animals were apparently able to detect much less intense stimuli because prepulse inhibition in the aged rats was completely normal. It is also possible, however, that the cue training deficit might be the result of a cognitive impairment. Performance on cued learning tasks, for example, are impaired by striatal lesions that do not disrupt performance on spatial learning tasks with similar sensorimotor and motivational requirements (Kesneret al., 1993; Packard and McGaugh, 1992). This suggests that sensorimotor or other performance abnormalities are not necessarily the basis for impaired cue learning. Thus, cue-finding deficits may be correlated with spatial learning deficits without there being a causal relationship between the two.
Even when cue training performance was used as a covariate, however, an interaction between age and ABT-089 dose was noted on the first probe trial in the hidden platform task. This interaction resulted from ABT-089-induced improvements in the performance of aged animals but impaired performance in younger rats. These effects were not observed in the second probe trial in which all groups showed improved performance relative to that in the first probe. Although ABT-089 produced only a modest improvement in the performance of aged animals that was restricted to the first probe measure, it should be noted that this was the only measure on which an age-related deficit was detected with ANCOVA and that improvement was observed at the same doses that improved the two-platform water maze performance of septal-lesioned rats. The difference between the effects of ABT-089 in normal and impaired animals is not unexpected. As will be discussed below, it is difficult to improve the performance of normal animals in many learning and memory tasks unless steps are taken to produce suboptimal base-line performance. Furthermore, manipulations that improve the performance of a brain that functions abnormally might not improve the performance of a normal brain. For example, lobeline, a nicotinic cholinergic ligand, improves the two-platform water maze performance of septal-lesioned rats, but impairs the performance normal animals in this task (Deckeret al., 1993) and impairs the performance of normal animals in a sustained attention operant task (Turchi et al., 1995).
Although continuous infusion of ABT-089 or (−)-nicotine improved the water maze performance of rats with substantial damage to the septal hippocampal system, these treatments did not alter the effects of septal lesions on open-field activity or acoustic startle responses. As has been shown previously, septal lesions produced a biphasic effect on locomotor activity with decreased activity at the beginning of the session and increased activity later in the session (Decker et al., 1995b), a pattern of behavior that was not changed by drug treatment. Continuous infusion of ABT-089 or (−)-nicotine also did not reduce the lesion-induced increase in startle responses to an intense acoustic stimulus. The dissociation between the effects of drug treatment on the cognitive and noncognitive effects of septal lesions suggests that the mechanisms underlying these various septal lesion effects may differ. This dissociation also suggests that the drug-induced improvements in water maze performance were not the result of some generalized reduction in the septal syndrome. Alternatively, it is possible that differences between the effects of these compounds on these behaviors are related to the order in which the experiments were conducted because the locomotor activity and startle experiments were conducted before the two-platform water maze experiment.
The present data indicate that ABT-089 produced a significant enhancement of DMTS performance in both mature and aged monkeys on items requiring the protracted retention and recall of sample stimuli. In mature monkeys, significant enhancement of DMTS accuracy was observed on medium-delay trials after administration of the 64.8 nmol/kg dose. It is interesting to note a consistent elevation above base line (6–9%) for all doses of ABT-089 on medium-delay trials. The possibility exists that with an increase in sample size, ABT-089 may enhance DMTS performance over a broad range of doses. A moderate increase was observed on long-delay trials in these monkeys after administration of the 32.4 nmol/kg dose, although this effect only approached statistical significance (P < .08). Given that long-delay trials require a longer duration of sample stimulus retention and that ABT-089 did not significantly enhance DMTS accuracy on these trials, the possibility exists that ABT-089 may possess limited efficacy in facilitating retention and/or recall on the most difficult retention tasks in mature monkeys. In aged monkeys, however, ABT-089 produced a significant and selective enhancement of DMTS performance on long-delay trials. This was observed after administration of the 4.0 nmol/kg dose, and enhancement after administration of the 8.1 nmol/kg dose did approach statistical significance (P < .059). It should be noted, however, that because old monkeys perform more poorly than younger monkeys, the “long” delays for the old monkeys are actually shorter than the long delays for the younger animals. In general, the i.m. doses that improved DMTS performance in monkeys generated plasma levels [approximately 2–50 ng/ml; Arneric et al., 1996b] similar to those that were effective in the rat studies.
In general, data derived from the performance of both mature and aged monkeys are consistent with previous evidence demonstrating the ability of ChCMs to enhance performance on learning and memory tasks in monkeys (Buccafusco et al., 1995; Buccafusco and Jackson, 1991;Terry et al., 1993). Interestingly, ABT-089 appeared to be more potent in aged monkeys than in mature monkeys. In contrast, the beneficial effects of the ChCM, ABT-418, require higher doses in aged monkeys than in mature monkeys (Buccafusco et al., 1995;Prendergast et al., 1997). It should also be noted that ABT-089 was more potent in monkeys than it was in rats. This finding is consistent with the relative potencies of other ChCMs in these two species (Buccafusco et al., 1995; Buccafusco and Jackson, 1991; Decker et al., 1992, 1994a).
The selectivity of ABT-089 in enhancing retention and recall processes is suggested by its lack of influence on response latencies. Altered latencies to respond to sample and/or choice stimuli may be indicators of drug-induced side effects. Choice response latencies on incorrect trials were elevated, as compared with correct trials (possibly because of increased duration of attempted sample recall). ABT-089 had no effect on this pattern of responding. Sample latencies were not markedly different for correct and incorrect trials and were also unaffected by drug administration.
Mecamylamine did not attenuate the effects of ABT-089; the performance animals receiving mecamylamine combined with ABT-089 did not differ from that of animals given ABT-089 alone. However, the data from this experiment also indicate that ABT-089 significantly improved performance (11.7-point increase) when compared with the saline base line, whereas the mecamylamine/ABT-089 combination did not significantly improve performance (3.4-point increase) when compared with the mecamylamine baseline. Thus, although there is a suggestion that the effect of ABT-089 is not prevented by mecamylamine, the data are not completely clear on this point. A failure to block the effect of ABT-089 with mecamylamine would contrast with the ability of this dose of mecamylamine to prevent nicotine-induced improvements in performance (Elrod et al., 1988). Similarly, the discriminative stimulus properties of ABT-089 in rats trained to discriminate (−)-nicotine from saline (Brioni et al., 1997) and the ability of ABT-089 to stimulate cation efflux from mouse thalamic synaptosomes in vitro can be blocked by mecamylamine (Sullivan et al., 1997, companion paper). However, it is possible that the effects of ABT-089 on cognitive performance are mediated via a nAChR subtype with reduced sensitivity to mecamylamine. Alternatively, the cognitive effects of ABT-089 may not be mediated by nAChRs, although extensive in vitro characterization has not revealed significant affinity for a variety of other receptors (Sullivan et al., 1997, companion paper).
The failure to observe effects of ABT-089 and (−)-nicotine in sham-lesion animals in the water maze contrasts with the finding that acute administration of these compounds can improve DMTS performance in normal, mature monkeys. Similarly, both ABT-089 and (−)-nicotine can improve retention of inhibitory avoidance training in normal mice (Brioni and Arneric, 1993; Lin et al., 1997). Moreover, slight tendencies toward impaired performance were noted with continuous infusion of ABT-089 in the current study in mature adult animals in the invisible platform water maze and in the inhibitory avoidance task. These apparent discrepancies could be related to differences between acute and continuous administration, but it is also important to note that improved performance of normal animals treated with these compounds was observed under conditions designed to produce suboptimal performance. Specifically, retention of inhibitory avoidance training in the previous experiments was controlled by adjusting the shock parameters. Similarly, delayed matching-to-sample performance can be reduced in mature monkeys by increasing the delay interval. Thus, in these studies, control performance was reduced by parametric manipulations to assess memory-enhancing effects. Indeed, the effect of ABT-089 in mature monkeys was also less robust than the effect of the compound in aged monkeys. The failure to observe improved water maze performance in normal rats might then be related to the high level of performance in these animals under normal conditions. Similarly, drug-induced impairments in the cognitive performance of normal animals may result in animals performing optimally, as discussed previously.
In summary, ABT-089 appears to have cognitive-enhancing properties in some preclinical tests of learning and memory. When comparison with (−)-nicotine is possible, ABT-089 appears to be at least as potent and in some cases more potent than (−)-nicotine. ABT-089, however, has a significantly improved safety profile relative to (−)-nicotine (Arneric et al., 1996b; Lin et al., 1997). For example, ABT-089 is less potent than (−)-nicotine in producing seizures, death and reductions in locomotor activity and body temperature in mice and has reduced cardiovascular liabilities in dogs (Arneric et al., 1996b; Lin et al., 1997). This improved separation between potentially toxic and beneficial effects of ABT-089 may be related to different patterns of relative potencies of ABT-089 and (−)-nicotine in in vitro functional assays (Sullivan et al., 1997, companion paper). Although it is not yet clear which in vitro effects predict potential beneficial effects and which predict toxicities, characterization of the in vitro and in vivo profiles of novel ChCMs will be important in identifying these relationships.
Acknowledgments
The authors thank Dave Gunn, Yun He and William Arnold for their efforts in synthesizing ABT-089 and Mike Williams for his helpful comments.
Footnotes
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Send reprint requests to: Michael W. Decker, Dept. 47W, Bldg. AP-9A/LL, 100 Abbott Park Rd., Abbott Laboratories, Abbott Park, IL 60064-3500. .
- Abbreviations:
- AD
- Alzheimer’s Disease
- ANCOVA
- analysis of covariance
- ANOVA
- analysis of variance
- ChAT
- choline acetyltransferase
- ChCM
- cholinergic channel modulator
- DMTS
- delayed matching to sample
- nAChR
- nicotinic acetylcholine receptor
- PPI
- prepulse inhibition
- Received December 10, 1996.
- Accepted June 26, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
