Abstract
In this rodent study, we evaluated the effects of different time periods (7, 14, 45, and 90 days) of oral treatment with haloperidol (HAL; 2.0 mg/kg/day) or ziprasidone (ZIP; 12.0 mg/kg/day) on nerve growth factor (NGF) and choline acetyltransferase (ChAT) levels in the hippocampus, and we subsequently assessed water maze task performance, prepulse inhibition (PPI) of the auditory gating response, and several NGF-related proteins and cholinergic markers after 90 days of treatment. Seven and 14 days of treatment with either HAL or ZIP resulted in a notable increase in NGF and ChAT immunoreactivity in the dentate gyrus (DG), CA1, and CA3 areas of the hippocampus. After 45 days, NGF and ChAT immunoreactivity had abated to control levels in ZIP-treated animals, but it was markedly reduced in HAL-treated subjects. After 90 days of treatment, NGF and ChAT levels were substantially lower than controls in both antipsychotic groups. Furthermore, after 90 days of treatment and a drug-free washout period, water maze performance (but not PPI) was impaired in both antipsychotic groups, although the decrement was greater in the HAL group. Several NGF-related and cholinergic proteins were diminished in the brains of subjects treated with either neuroleptic as well. These data support the premise that, although ZIP (given chronically) seems somewhat superior to HAL due to less pronounced behavioral effects and a more delayed appearance of neurochemical deficits, both antipsychotics produce time-dependent deleterious effects on NGF, cholinergic markers (i.e., important neurobiological substrates of memory), and cognitive function.
The newer pharmacological treatments for schizophrenia, now commonly referred to as second generation antipsychotics (SGAs), offer several advantages over first generation antipsychotics (FGAs) such as greater improvements in negative symptoms, prevention of relapse, increased functional capacity and quality of life, and fewer movement-related side effects (for review, see Miyamoto et al., 2005). It is also generally believed that SGAs are superior to FGAs when their effects on cognition are considered (for reviews, see Keefe et al., 1999; Purdon, 1999), and some studies suggest that SGAs improve cognition in schizophrenia. This suggestion is of particular importance given that the degree of cognitive impairment in schizophrenia is recognized as an important predictor of social functioning, unemployment, and even relapse of psychiatric symptoms (for review, see Castner et al., 2004). It should be noted, however, that such conclusions regarding antipsychotics and cognitive function rely primarily on meta-analyses and short clinical trials (i.e., they rarely exceed a few months to 1 year in length). Therefore, since schizophrenic patients are often treated with antipsychotics for decades, the aforementioned conclusions regarding SGAs may be premature. Since multiyear, prospective clinical studies designed specifically to identify neuroleptics that have optimal effects on cognition have not been conducted (and may be cost-prohibitive), animal studies designed to investigate such issues may be especially important. Furthermore, animal studies allow for rigorous investigations of the effects of chronic neuroleptic treatment on the neurobiological substrates of cognitive function.
Previous work in our laboratories demonstrated that, in contrast to certain SGAs such as risperidone and clozapine, chronic oral haloperidol (HAL) treatment in rats resulted in sustained impairments in spatial learning performance as well as decrements in an important cholinergic marker, choline acetyltransferase (ChAT), in brain regions such as the cortex and hippocampus (Terry et al., 2002, 2003). This finding is potentially very important, because cholinergic activity in these brain areas is well documented to modulate a number of cognitive processes (for review, see Perry et al., 1999). Further work in our laboratories indicated that, although HAL treatment was associated with decrements in brain levels of the endogenous neurotrophin, nerve growth factor (NGF), and ChAT, this did not seem to be the case with the SGAs risperidone, clozapine, or olanzapine (Parikh et al., 2004a,b) at least up to a treatment period of 90 days. More recently, however, we detected decrements in NGF associated with HAL, risperidone, and olanzapine treatment for 180 days (Pillai et al., 2006), leading to the conclusions that such growth factor changes, although dependent on the length of treatment, may be common to several antipsychotics. Because the survival and function of adult mammalian cholinergic neurons (particularly those projecting from the basal forebrain to the cortex and hippocampus) are dependent on NGF (for review, see Counts and Mufson, 2005), we hypothesize that some of the unfavorable effects of HAL (and potentially other neuroleptics) on memory function may be related to time-dependent impairments in cholinergic activity due to reduced levels of NGF and/or its receptors.
The novel SGA ziprasidone (ZIP) has a unique pharmacological profile with high affinity at a number of neurotransmitter receptors, including D2, 5HT1A, 5HT2A, and 5HT2C,as well as 5HT1B/1D receptors (Seeger et al., 1995; Schmidt et al., 2001). It has proven efficacy in schizophrenia and related disorders (Goff et al., 1998; Daniel et al., 1999), as well as a low liability for certain adverse reactions, such as extrapyramidal symptoms and weight gain (for review, see Weiden et al., 2003). However, as in the cases highlighted above, the effects of long-term treatment with ZIP (particularly on cognitive function and neurobiological substrates of cognitive function) have not been evaluated. Therefore, the purpose of this study was to compare ZIP to the archetypal FGA HAL for effects on NGF and NGF receptors, key cholinergic proteins, and memory-related task performance in rats. The overall hypothesis was that chronic treatment with HAL or ZIP leads to sustained memory-related behavioral changes that are due (at least in part) to effects on NGF and/or its receptors and the regulation of key cholinergic marker proteins (i.e., for memory function). The two experimental approaches used to test this hypothesis were 1) to investigate a time course of exposure to either HAL or ZIP for effects on the cholinergic marker protein ChAT and the neurotrophin NGF and 2) to measure the effects of the neuroleptics on memory-related behavioral tasks, as well as the levels of NGF and cholinergic proteins after 90 days of treatment followed by a significant drug-free washout period. The latter experiments were designed to investigate the residual effects of prior chronic treatment with these agents (i.e., effects not associated with acute exposure).
Materials and Methods
Test Subjects
Male albino Wistar rats (Harlan, Indianapolis, IN) (2-3 months old) were housed individually in a temperature-controlled room (25°C), maintained on a 12-h light/dark cycle with free access to food (Teklad Rodent Diet 8604 pellets; Harlan Teklad, Madison, WI). Water was allowed ad libitum for the first week in all test animals, but then it was replaced with solutions that contained neuroleptics for the animals that were placed in the chronic antipsychotic studies (see below). Table 1 provides the details for the all study cohorts, the numbers of animals tested per group, and the experiments conducted with each group. All procedures used during this study were reviewed and approved by the Medical College of Georgia Institutional Animal Care and Use Committee and are consistent with Association for Assessment and Accreditation of Laboratory Animal Care guidelines. Measures were taken to minimize pain or discomfort in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publications 80-23) revised 1996. Significant efforts were also made to minimize the total number of animals used while maintaining statistically valid group numbers.
Drug Dosing for Chronic Antipsychotic Experiments
Oral antipsychotic dosing was based several factors: 1) for HAL, previous rodent studies in our laboratory in which time-dependent behavioral and neurochemical effects were detected and plasma drug levels were achieved that approximated those often associated with antipsychotic effects in humans (Terry et al., 2002, 2003, 2005a); and 2) for ZIP, previous studies using oral dosing in rodents in which notable behavioral effects were observed (Mansbach et al., 2001). Furthermore, for both HAL and ZIP, the doses selected (see below) would be expected to achieve comparable (and therapeutically) relevant D2 receptor occupancy values in vivo (i.e., in the range 65-80%; see Kapur et al., 2003) based on the recent work of Barth et al. (2006). Rats were thus treated with 2.0 mg/kg/day HAL (Sigma-Aldrich, St. Louis, MO) or 12.0 mg/kg/day ZIP (Pfizer, Inc., New York, NY) orally for periods of 7, 14, 45, or 90 days. The antipsychotics were dissolved in 0.1 M acetic acid and subsequently diluted (1:100) with distilled, deionized water for daily drug administration in drinking water. Drug dosing was based on the average daily fluid consumption and the weight of the animals. Animals that were evaluated for residual neuroleptic-related behavioral effects were administered antipsychotics at the doses described above (or vehicle) for 90 days and then given a 1-week, drug-free washout period (i.e., returned to normal drinking water), behaviorally tested for 1 week, and then sacrificed for neurochemical studies (i.e., 2 weeks after the last drug exposure).
Immunohistochemistry
In the first series of immunohistochemical experiments, rats were administered the antipsychotics (or vehicle) as described above and sacrificed at different time points (7, 14, 45, or 90 days of treatment; Table 1) to determine whether there are time-dependent effects of antipsychotic treatment on NGF and ChAT.
Nerve Growth Factor. Rats were deeply anesthetized with ketamine/xylazine and perfused with ice-cold 0.01 M phosphate-buffered saline (PBS) through the left cardiac ventricle to remove circulating blood elements. Brains were quickly removed and cryoprotected in embedding media. Coronal sections (20 μm in thickness) were cut at interaural, 4.84 mm; bregma, -4.16 mm to obtain sections from hippocampus [dentate gyrus (DG); CA1 and CA3] (Paxinos and Watson, 1998) using a cryostat microtome (Leica CM 3050S; Leica Microsystems Inc., Chantilly, VA) at -20 ± 2°C. Fresh frozen sections were fixed in ice-cold acetone for 30 min and airdried. Then, sections were rinsed in 0.01 M PBS containing Tween 20 (PBST). After blocking with 10% normal goat serum for 1 h, sections were washed and incubated overnight at 4°C with rabbit anti-mouse polyclonal NGF antibody (1:100) (Chemicon International, Temecula, CA). Endogenous peroxidase was blocked for 30 min with 0.1% H2O2 and 100% methanol. Sections were then washed and incubated for 2 h with biotinylated anti-rabbit IgG antibody made in goat (1:50) followed by incubation for 1 h with avidin-biotin-horseradish peroxidase complex. The avidin-HRP complex was then detected with 3-3′-diaminobenzidine tetrahydrochloride in the presence of 0.02% H2O2 and nickel chloride (Vectastain kit; Vector Laboratories, Burlingame, CA).
Choline Acetyltransferase. For ChAT immunohistochemistry, the method used has been described previously (Parikh et al., 2004a,b). In brief, rats were anesthetized and perfused with 100 ml of saline followed by 300 ml of ice-cold 4% paraformaldehyde in 0.1 M PBS. After perfusion, all brains were postfixed in paraformaldehyde for 2 h (with shaking) at 4°C followed by storage at 30% sucrose solution in 0.1 M PBS for 48 h. The tissues were embedded with OCT in liquid nitrogen and kept at -80°C until further use. A cryostat was used to cut 40-μm coronal sections at specific anatomical landmarks: interaural, 4.84 mm; bregma, -4.16 mm to obtain sections from hippocampus (Paxinos and Watson, 1998). Cryoprotected fixed sections were washed three times in PBST, blocked with 10% normal horse serum for 1 h, and then incubated with 10 μg/ml mouse monoclonal anti-ChAT antibody (Chemicon International) overnight at 4°C. The sections were washed three times with PBST and then incubated for 2 h with 1:20 diluted rat-adsorbed biotinylated horse anti-mouse IgG (Vector Laboratories) containing 1% horse serum. After washing, the sections were incubated with avidin-HRP for 1 h, and the avidin-HRP complex was subsequently detected with diaminobenzidine tetrahydrochloride.
Quantitative Image Analysis. Photomicrographs from each treatment group were obtained with a Zeiss Axioplan-2 microscope equipped with a charge-coupled device camera, PC computer, and Zeiss KS-300 image analysis software by an experimenter blinded to the study code. The analysis for NGF was performed on live acquired images of dimensions 582 × 455 μm2 in DG granule cell layer and 582 × 228 μm2 each in CA1 and CA3 cell layer pyramidal neurons. For quantitation, densitometric assessments were made measuring the optical density (OD) of immunostained cells. The cells were considered positive if their OD values were higher than a defined threshold OD value above which only cell bodies and not processes were detectable. Three rectangles per section with dimensions 582 × 455 μm2 for DG and 582 × 228 μm2 for CA1 and CA3 region were delineated. NGF-immunoreactive neurons were identified in these regions, and staining intensity was expressed as mean OD obtained by averaging OD values of all stained profiles in analyzed field and subtracting the background OD of each section. OD range 0 to 2 was divided into 256 (0-255) digitized values. ChAT-immunoreactive nerve fibers in the hippocampus were analyzed as described previously (Parikh et al., 2004a,b). Three rectangles per section for DG, CA1, and CA3 subfields with similar dimensions as described above were selected for analysis. All the digitalized images of ChAT immunoreactive nerve fibers were converted to gray scale, and the brightness, contrast, and masking were adjusted to enhance the visibility of fibers (Photoshop 5.0; Adobe Systems, San Jose, CA). Quantitative data for ChAT-immunoreactive fibers are expressed as fiber pixel density.
Behavioral Experiments
In the behavioral studies, two series of experiments were conducted 1) behavioral task validation experiments and 2) comparisons of HAL and ZIP for residual effects on memory-related behavior performance after 90 days of treatment (i.e., a treatment period previously associated with HAL-induced memory impairments) beginning 1 week into a drug-free washout period.
Behavioral Task Validation Experiments. In each of the validation experiments, identical conditions were used in each procedure as that used for the antipsychotic behavioral studies (see methodological details below). Thus, in spatial learning experiments, the water maze task used was assessed for its sensitivity to cholinergic antagonism (a central issue in this study) with a reference dose of scopolamine (0.1 mg/kg s.c. determined in preliminary experiments). Thereafter, two doses of the commonly used acetylcholinesterase inhibitor (AChEI) and cognitive-enhancing agent donepezil (Table 1) were evaluated for their ability to attenuate the impairing effects of scopolamine. Thereafter, the anxiety test used, the light-dark box test, was evaluated for its sensitivity to published reference doses (Table 1) of the anxiolytic agent diazepam and the anxiogenic agent meta-chlorophenylpiperazine (mCPP; see Chaouloff et al., 1997 and Bilkei-Gorzo et al., 1998, respectively). Finally, the prepulse inhibition (PPI) method used was evaluated for its sensitivity to known PPI impairing agents (and compounds known to attenuate the effects of such agents). Thus, dose-effect curves were established for the FGA haloperidol, the SGA clozapine, and the AChEI donepezil to reverse the impairing effects of reference doses of the dopamine agonist apomorphine, the N-methyl-d-aspartate antagonist MK801, and the muscarinic antagonist scopolamine on PPI, respectively. Drug doses (Table 1) were selected from previous work in our laboratory (Terry et al., 2005b) and other published PPI studies (Stanhope et al., 2001).
Water Maze Testing.Testing apparatus. To determine the effects of the test drugs on spatial learning, water maze experiments were performed in a circular pool (180 cm in diameter, 76 cm in height) made of black plastic. For the task validation experiments, reference compounds (see above) were administered by subcutaneous injection acutely, and for the chronic (oral) neuroleptic studies, testing was begun on day 7 of a drug-free washout period (i.e., after the 90 days of oral drug dosing). The pool was filled to a depth of 35 cm with water (maintained at 25.0 ± 1.0°C). The pool was located in a large room with a number of extramaze visual cues, including geometric images (e.g., squares, triangles, and circles) hung on the wall, ambient lighting of approximately 25 to 30 lux (lumens per square meter), and black curtains, used to hide the experimenter (visually) and the resting test subjects. Swimming activity of each rat was monitored via a television camera mounted overhead, which relayed information, including latency to find the platform, total distance traveled, time, and distance spent in each quadrant, to a video tracking system (Actimetrics, Evanston, IL).
Visible platform task. On the day before water maze hidden platform testing, a visible platform test was performed to ensure that the study subjects were visually capable of performing the task and that they demonstrated normal search/escape behaviors. To accomplish this task, a highly visible (white) cover fitted with a small white flag was attached to the platform (dimensions with cover attached 12 × 12 cm), which raised the surface approximately 1.0 cm above the surface of the water. Each rat was gently lowered into the water in the quadrant diametrically opposite to the platform quadrant and given one or more trials with a 90-s time limit to locate and climb on to the platform. If unsuccessful after 90 s, it was physically placed on the platform for 30 s and then given a new trial. Once a rat was successful on its own accord, it was then given a series of four additional trials (with a 1.0-min intertrial interval) and the latency (in seconds) to locate the platform was recorded. The platform was moved on each trial to a different quadrant (the subject was always entered from the opposite quadrant) until the test was conducted once in all four quadrants.
Hidden platform task. For these experiments, an invisible (black) 10- × 10-cm square platform was submerged approximately 1.0 cm below the surface of the water and placed in the center of the northeast quadrant (which remained constant throughout hidden platform training). Each rat was given two trials per day for 6 consecutive days to locate and climb on to the hidden platform. A trial was initiated by placing the rat in the water directly facing the pool wall (i.e., nose approximately 2 cm from the wall) in one of the four quadrants. The daily order of entry into individual quadrants was pseudorandomized such that all four quadrants were used once every two training days. For each trial, the rat was allowed to swim a maximum of 90 s, to find the platform. When successful the rat was allowed a 30-s rest period on the platform. If unsuccessful within the allotted time period, the rat was given a score of 90 s and then physically placed on the platform and also allowed the 30-s rest period. In either case, the rat was given the next trial after an additional 1.5-min rest period (i.e., intertrial interval 2.0 min).
Probe trials (transfer tests). Twenty-four hours following the last hidden platform trial, a probe trial was conducted in which the platform was removed from the pool to measure spatial bias for the previous platform location. This was accomplished by measuring the percentage of time spent in the previous target quadrant and the number of crossings over the previous platform location, and it provided a second estimate of the strength and accuracy of the memory of the previous platform location.
Locomotor Activity and the Light/Dark Preference Test. To assess the effects test drugs on general locomotor activity as well as anxiety levels, a light/dark preference test (also referred to as light/dark exploration or emergence neophobia test) was conducted. In this test, we were interested in determining whether the neuroleptics (i.e., day 10 of a drug-free washout) had significant motor effects that might have influenced performance in the memory-related tests. We were also interested to learn whether the neuroleptics (or reference compounds; see above) had any effects on anxiety levels (a factor that could at least theoretically influence performance in the water maze). The light/dark preference test is one of the most commonly used rodent models of anxiety (Holmes et al., 2001), and avoidance of the lighted portion of the chamber reflects elevated anxiety, whereas significantly reduced time spent in the dark area reflects an antianxiety effect of a test drug. MED Associates (St. Albans, VT) rat open field activity monitors (43.2 × 43.2 cm) were used for these experiments. They were fitted with dark box inserts (which are opaque to visible light) to cover one-half the open field area, thus separating the apparatus into two zones of equal area (i.e., a brightly lit zone and a darkened zone). Desk lamps located above the activity monitors were used to provide an illumination level of approximately 1000 lux in the brightly lit zone, whereas the illumination level in the darkened zone was approximately 5 lux. The following parameters were recorded for the 5 min test session: horizontal activity (horizontal photobeam breaks or counts), number of stereotypy movements, vertical activity (vertical photobeam breaks) as well as the time spent in the light and dark zones of the apparatus. Thus, spontaneous locomotor activity, olfactory activity (rearing and sniffing movements), stereotypical movements, and emergence neophobia were assessed.
Prepulse Inhibition Procedure. To assess the effects of the reference compounds (see above) and previous neuroleptic exposure (i.e., day 12 of a drug-free washout) on auditory gating (an important behavioral process that is often disrupted in schizophrenic patients), a PPI procedure was conducted as described previously (Terry et al., 2005a). Four startle chambers (San Diego Instruments, San Diego, CA) were used that consisted of a Plexiglas tube (8.2 cm in diameter, 25 cm in length) placed in a sound-attenuated chamber, in which the rats were individually placed. The tube is mounted on a plastic frame, under which a piezoelectric accelerometer is mounted, which records and transduces the motion of the tube. Two days before drug testing, the experimental subjects were each placed in one of the startle test chambers for a period of 10 min (without any startle stimuli) as an initial period of acclimation to the apparatus. One day before drug testing the animals were again placed in the test chamber and then exposed to 12 startle stimuli and to each prepulse level 3 times (see below). This procedure was done to reduce the highly variable responses to the initial exposures to the startle stimuli as well as to ensure that the prepulse stimuli (alone) had no significant effect on the startle response. On the day of drug testing, experimental subjects were transported to the startle chamber room and left undisturbed for at least 30 min. Afterward, the rats were placed in the chamber and then allowed to habituate for a period of 5 min, during which a 70-dB background white noise was present. After this period, the rats received 12 startle trials, 12 no-stimulus trials, and 12 trials of each of the prepulse/startle trials (see below) for a total of 60 trials. The intertrial interval ranged from 10 to 30 s, and the total session lasted approximately 25 to 30 min. The startle trials consisted of single 120-dB white noise bursts lasting 20 ms.
The prepulse inhibition trials consisted of a prepulse (20-ms burst of white noise with intensities of 75, 80, or 85 dB) followed, 100 ms later, by a startle stimulus (120-dB, 20-ms white noise). During the no-stimulus trial, no startle noise was presented, but the movement of the rat was recorded. This represented a control trial for detecting differences in overall activity. The 60 different trials were presented pseudorandomly, ensuring that each trial was presented 12 times and that no two consecutive trials were identical. The resulting movement of the rat in the startle chamber was measured during 100 ms after startle stimulus onset (sampling frequency 1 kHz), rectified, amplified, and fed into a computer that calculated the maximal response over the 100-ms period. Basal startle amplitude was determined as the mean amplitude of the 12 startle trials. Prepulse inhibition was calculated according to the formula: [100 - (startle amplitude on prepulse-pulse trials)/(startle amplitude on pulse alone trials) × 100]. The mean level of PPI (i.e., averaged across the three prepulse intensities) was also analyzed.
Enzyme-Linked Immunosorbent Assay Experiments
After behavioral testing, some of the test subjects (Table 1) were anesthetized with KetaVed (ketamine hydrochloride injection; Vedco, Inc., St. Joseph, MO), intracardially perfused with PBS, pH 7.4, and then decapitated. Brains were quickly harvested, immediately frozen in dry ice-cooled 2-methylbutane (isopentane), and stored at -70°C until dissected for subsequent analyses. The basal forebrain, hippocampal formation, cortex, and prefrontal cortex were dissected and the homogenized in radioimmunoprecipitation assay buffer containing protease inhibitors and glycerol. Brain lysates were evaluated using ELISA methods to measure the relative levels of ChAT, vesicular acetylcholine transporter (VAChT), p75 neurotrophin receptor (p75NTR), TrkA (nerve growth factor receptor), phospho-TrkA (P-TrkA), and NGF. The brain dissections, preparation of brain lysates, protein assay, and ELISA methods (except NGF) were performed according to Gearhart et al. (2006), except that in the present study, different quantities of brain protein were analyzed by ELISA (see Table 2 for details). As an internal control for day-to-day variation in the ELISA methods, brain lysates (same amount of protein per well) from vehicle-, HAL-, and ZIP-treated rats were always assayed at the same time on the same ELISA plate. NGF was measured using the NGF Emax ImmunoAssay System (catalog no. G7631; Promega, Madison, WI) according to the kit instructions. The brain lysates were not acid-treated before the NGF ELISA. On the day of the NGF assay, the brain lysates were diluted in sample buffer (SB) as follows: basal forebrain lysate (30 μl) + SB (90 μl), hippocampus lysate (30 μl) + SB (90 μl), cortex lysate (60 μl) + SB (30 μl), and prefrontal cortex lysate (60 μl) + SB (30 μl). The diluted lysates were mixed thoroughly, and then 100 μl of each diluted lysate was analyzed in the NGF ELISA. In addition, after preliminary experiments indicated that there were no significant differences in ELISA results between PBS-perfused animals and those sacrificed by decapitation (see below), some half-brains left from the autoradiography experiments were dissected and used in increase the sample size (n) in ELISA experiments.
Quantitative Receptor Autoradiography
To assess the effects of prior chronic antipsychotic administration on cholinergic receptor densities, autoradiographic analyses of brain tissues harvested from rats previously exposed to HAL, ZIP, or vehicle were conducted with subtype-specific cholinergic radioligands to nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs), i.e., receptors that have been found to play important roles in learning and memory processes (van der Zee and Luiten, 1999; Rezvani and Levin, 2001). After behavioral testing, some of the test subjects (n = 6-9) were immediately sacrificed by decapitation, the brains flash frozen. Using a Microm HM 505N cryostat (-18°C; Richard Alan Scientific, Kalamazoo, MI), the left hemisphere of each brain was serially sectioned (16 μm) up to the midline onto chrome alum/gelatin-coated slides. Low-affinity (homomeric α7) nAChRs and high-affinity (heteromeric α/β subunit complexes) were labeled with 125I-α-bungarotoxin (BTX) and [3H]epibatidine (EPB), respectively. The density of M1 and M2 mAChRs, i.e., the mAChRs expressed in highest quantities in mammalian brain (van der Zee and Luiten, 1999), were quantified using [3H]pirenzipine (PRZ) and [3H]AFDX384 (AFX), respectively. For each radioligand, the receptor subtype target, incubation time, concentration, and duration of film exposure are listed in Table 3. Please refer to previous publications for more detailed descriptions of the autoradiographic procedures (Hernandez and Terry, 2005; Terry et al., 2005a).
Statistical Analyses
All statistical analyses were performed using either SigmaStat 2.03 (SPSS Inc., Chicago, IL) or InStat, version 3.06 for Windows (GraphPad Software Inc., San Diego CA). Statistical significance was assessed using an alpha level of 0.05. A one or two-way analysis of variance (ANOVA) (with repeated measures when indicated) was used for all treatment comparisons. In some cases, ranked data were used when the particular data set was non-normally distributed. For post hoc analysis, the Bonferroni method was used for multiple comparisons and Dunnett's test was used for cases when comparisons were made against vehicle control only. Raw ELISA data (optical density at 450 nm) were compared for all of the proteins with the exception of NGF in which picograms of nerve growth factor per milligram of protein were compared. For graphing purposes, the raw ELISA data were converted to percentage of the vehicle-treated group. Specifically, each OD (n = 7-10 per HAL or ZIP group) from each ELISA (ChAT, VAChT, p75NTR, TrkA, and P-TrkA) was divided by the mean OD for the vehicle-treated group (n = 6-12) that was tested on the same ELISA plate. Similar calculations were used for the NGF results, except that the units were picograms of NGF per milligram of protein (rather than OD). The resulting quotients were multiplied by 100 to yield percentage of the vehicle-treated group, and then InStat was used to determine the mean ± S.E.M. For autoradiographic comparisons, a repeated measures two-factor ANOVA was used to examine differences in densitometry values between each treatment (HAL and ZIP) versus control within area of the brain. Animal was nested within the treatment and considered a random effect. Fixed effects were treatment and area of the brain. All two-factor interactions were included in the model. An α level of 0.05 was used to examine post hoc differences on the adjusted least square means of the two-factor interaction between treatment from control and area of the brain.
Results
Immunohistochemistry
NGF. Treatment comparisons (i.e., for effects on NGF immunoreactivity) made by measuring the mean optical density of immunostained cells are provided in Table 4. As indicated, NGF immunoreactivity was somewhat higher in the DG compared with the CA1 and CA3 regions of the hippocampus (which were similar). Interestingly, there was a notable increase in NGF immunoreactivity at 7 and 14 days of treatment with either HAL or ZIP in the DG, CA1, and CA3 areas of the hippocampus. After 45 days, however, animals treated with HAL had markedly reduced NGF immunoreactivity, whereas immunoreactivity in ZIP-treated subjects had returned to control levels. After 90 days of exposure to either HAL or ZIP, NGF levels were substantially lower than controls.
ChAT. Varicose ChAT immunoreactive fibers were distributed in the granule cell layer of DG and in the pyramidal neurons of CA1 and CA3 subfields. The density of ChAT was highest in the hippocampal CA3 pyramidal cell layer (Table 5). Representative images illustrating significant time-dependent, treatment-related effects on ChAT immunoreactivity (fiber pixel density) are provided in Fig. 1. The effects of the neuroleptics on ChAT immunoreactivity followed a similar pattern as the effects on NGF. Specifically, there was a significant increase in ChAT immunoreactivity at 7 and 14 days of treatment with either HAL or ZIP in the DG, CA1, and CA3 areas of the hippocampus. After 45 days, animals treated with HAL had markedly reduced ChAT immunoreactivity, whereas immunoreactivity in ZIP-treated subjects was similar to control levels. After 90 days of exposure to either HAL or ZIP, ChAT levels were substantially lower than controls.
Behavioral Test Validation Experiments
Water Maze Hidden Platform Test.Figure 2A illustrates the results of experiments in which a reference dose (0.1 mg/kg) of the muscarinic cholinergic receptor antagonist scopolamine was evaluated for its ability to impair spatial learning and for the effects of two doses of donepezil to reverse or attenuate these effects of scopolamine. The latency (number of seconds) of each experimental group to locate the hidden platform over 6 consecutive days of testing is depicted. There was a highly significant treatment effect (F3,45 = 8.8; p < 0.001), a significant day effect (F5,15 = 14.1; p < 0.001), without a significant treatment × day interaction (F225,293 = 1.6; p = 0.8). Post hoc analyses indicated that performance was clearly impaired by scopolamine and that the higher (but not the lower) dose of donepezil attenuated this impairment. Specifically, the performance of controls (vehicle + vehicle) and those administered scopolamine + donepezil (2.0 mg/kg) was superior (p < 0.05) to performance of the animals administered scopolamine + vehicle on days 4, 5, and 6 and days 4 and 6, respectively.
Light/Dark Box Experiments.Figure 2B illustrates the results of experiments in which reference doses of the anxiolytic agent diazepam and the anxiogenic agent mCPP were evaluated in the light/dark box test. There was a highly significant differences in response to the different drugs (F4,45 = 13.1; p < 0.001). Post hoc analyses indicated that 1) both doses of diazepam significantly (p < 0.05) increased the amount of time spent in the illuminated arena (and that there was a dose-related difference in response) compared with vehicle controls and 2) the higher (but not the lower) dose of mCPP was associated with a strong trend (p = 0.06) toward decreased time spent in the illuminated arena.
PPI Experiments.Figure 2, C to E, illustrates the results of experiments in which known PPI-impairing agents (and compounds known to attenuate the effects of such agents) were evaluated. The following results were observed (overall treatment effect, p < 0.001 in all three PPI validation studies): 1) apomorphine, MK801, and scopolamine at the doses evaluated (0.5, 0.1, and 0.33 mg/kg s.c., respectively) clearly diminished the effects of the prepulse stimuli on the acoustic startle response; 2) the FGA haloperidol (0.03, and 0.10 mg/kg i.p.) clearly attenuated the effects of apomorphine; 3) the SGA clozapine (5.0 mg/kg i.p.) clearly attenuated the effects of MK801; and 4) the AChEI donepezil (1.0 mg/kg s.c.) clearly attenuated the impairing effects of scopolamine (post hoc effects p < 0.05 in all cases).
Water Maze (Chronic Antipsychotic Studies)
Visible Platform Test.Figure 3A illustrates the effect of the neuroleptics on the visible platform test (mean ± S.E.M. of four trials) in the water maze. This test was used as a method to ensure that the test subjects were not impaired visually and that they did not exhibit other (nonmnemonic) behaviors such as thigmotaxis that might have confounded the analyses. There were no significant treatment-related effects observed in this procedure (i.e., all p values were >0.05).
Swim Speeds. Swim speeds were also analyzed (Fig. 3B) in an effort to further investigate treatment related differences in task performance. Average swim speeds ranged between 15 and 20 cm/s across the groups for the 6 days of hidden platform testing. Statistical analyses revealed that there was not an overall treatment (group) effect (F2,33 = 1.3; p = 0.39) or day effect (F5,10 = 1.4; p = 0.25); however, there was a significant treatment × day interaction (F165,215 = 2.1; p = 0.02). Post hoc analyses indicated that the only treatment-related difference in swim speeds occurred on day 2 of testing in which HAL-treated animals swam slightly (but significantly) faster than vehicle- and ZIP-treated animals.
Hidden Platform Test.Figure 3C illustrates the efficiency of each experimental group to locate a hidden platform in a water maze task on six consecutive days of testing. For the latency comparisons, there was a highly significant treatment effect (F2,33 = 8.0; p < 0.001), a significant day effect (F5,10 = 14.9; p < 0.001), without a significant treatment × day interaction (F165,215 = 1.6; p = 0.1). Post hoc analyses (for overall treatment effect across all days of testing) indicated that performance was superior in the vehicle-treated animals compared with both the HAL and ZIP animals (p < 0.001 and p < 0.05, respectively). In the case of ZIP, all p values in the individual day comparisons were >0.05 (compared with vehicle controls), whereas in the case of HAL, there were several days where the performance was significantly different from controls (p < 0.05; Fig. 2C).
Probe Trials.Figure 4, A and B, illustrates the performance of probe trials by the various treatment groups. There were statistically significant (treatment-related) effects on performance as indicated by the percentage of the total time spent in the previous target quadrant [treatment effect (F2,33 = 3.4; p < 0.05)] and the number of crossing over the previous 10- × 10-cm target area [treatment effect (F2,33 = 7.5; p < 0.01)]. Post hoc analysis indicated that performance was superior in the vehicle-treated animals compared with HAL-treated animals in both measures (p < 0.001 and p < 0.01, respectively). In the case of ZIP, there was a not a significant difference (compared with control), although there was a trend toward a performance decrement in the platform crossing analysis (p = 0.1).
Locomotor Activity and the Light/Dark Preference Test (Chronic Antipsychotic Studies)
Figure 5, A to D, illustrates the effects of the drug treatments on horizontal and vertical locomotor activity, stereotypical movements as well as fear/anxiety-related behaviors (i.e., time spent in the lighted zone of the test apparatus). There were no significant treatment-related differences (p > 0.05) in horizontal activity, vertical activity, or stereotypical movements. There were also no significant drug related effects on the light/dark preference test, although there was a strong trend toward a reduction in anxiety-related behaviors in the ZIP group (treatment effect: F2,30 = 3.3; p = 0.053).
PPI Experiments (Chronic Antipsychotic Studies)
The effects of 90 days of prior exposure to the neuroleptics on PPI testing assessed on day 12 of the drug-free washout are presented in Fig. 6, A to C. There was a highly significant (prepulse intensity-dependent) reduction in the startle response when the prepulse stimuli preceded the startle stimulus, prepulse level effect (F2,4 = 41.1; p < 0.001) (Fig. 5A). Post hoc analysis indicated that a graded response to the increasing prepulse intensities was present (i.e., 75 < 80 < 85 dB) in all treatment groups; however, there were no significant differences in responses to the various drug treatments (F2,27 = 0.9; p = 0.44). Likewise, the treatment × prepulse level interaction was not significant (F54,89 = 0.9; p = 0.49). Additional analyses revealed that the drugs also had no significant effects on the startle response (F2,27 = 0.4; p = 0.70) (Fig. 5B). Figure 5C depicts the overall effects of the treatments on PPI (i.e., averaged across prepulse intensity).
ELISA Experiments
Figure 7 summarizes ELISA results for ChAT, VAChT, p75NTR, TrkA, phospho-TrkA, and NGF in memory-associated brain regions from rats treated with HAL (Fig. 7, A-D) or ZIP (Fig. 7, E-H). Each plot is presented in the same format. ELISA results from drug-treated rats are presented as a percentage of vehicle-treated rats; each column is labeled with the name of the protein measured (sample number in parentheses); the left two columns show levels of the cholinergic marker proteins ChAT and VAChT; the middle three columns depict data for the NGF receptors (p75NTR, TrkA, and phospho-TrkA); and the far right column represents the amount NGF protein. The horizontal dashed line is drawn at 100% of the vehicle-treated group (i.e., columns near this line indicate similar protein levels between vehicle- and drug-treated groups).
HAL-Treated Rats. HAL treatment did not change ChAT levels in any of the four brain regions tested; however, VAChT protein was significantly decreased in the hippocampus (-25%; Fig. 7B) and prefrontal cortex (-10%; Fig. 7D). HAL-treatment had modest effects on the levels of NGF-receptors (p75NTR, TrkA, and phospho-TrkA), except for an ∼50% decrease in phospho-TrkA protein in the hippocampus (Fig. 7B). It is noteworthy that HAL-treated rats had significantly less NGF protein in the basal forebrain (-25%; Fig. 7A), cortex (-20%; Fig. 7C), and prefrontal cortex (-20%; Fig. 7D).
ZIP-Treated Rats. In contrast to HAL, ZIP treatment significantly decreased (by 15-25%) ChAT and VAChT in the hippocampus (Fig. 7F) and prefrontal cortex (Fig. 7H). ZIP treatment had little to no effect on the levels of NGF receptors (p75NTR, TrkA, and phospho-TrkA) in the basal forebrain (Fig. 7E) and cortex (10% increase in TrkA; Fig. 7D). In the hippocampus from ZIP-treated rats (Fig. 7F), p75NTR and TrkA were slightly decreased, whereas phospho-TrkA protein decreased by 35%. In the prefrontal cortex from ZIP-treated rats (Fig. 7H), p75NTR and TrkA were reduced by ∼25 and 10%, respectively. Like HAL, ZIP-treated rats had significantly less NGF protein in the basal forebrain (-30%; Fig. 7E) and prefrontal cortex (-35%; Fig. 7H).
Autoradiographic Data
Autoradiographic results are depicted in Tables 6 and 7. Representative autoradiograms are illustrated in Fig. 8. For each of the radioligands used in this study, the pattern of binding site distribution was similar to that observed in a previous study (Hernandez and Terry, 2005). Statistical analyses revealed that for each of the ligands there were highly significant regional binding differences (area effect p < 0.001) as expected; however, there were no significant, overall treatment-related differences or treatment × brain area interactions (i.e., all p values were >0.05). Post hoc analyses on individual brain areas did reveal a few select, treatment-related differences, however (see below).
Nicotinic Receptor Expression.125I-BTX. Binding of 125I-BTX was widely distributed across all regions of the brain, with the exception of the striatum and cerebellum (Table 6; Fig. 8). The highest 125I-BTX binding densities were observed in the accessory olfactory bulb, supraoptic nuclei, mammillary nuclei, dorsal raphe, and medial vestibular nuclei. Moderate binding was observed in the superior colliculus, hippocampus, hypothalamus, and tegmental nuclei. Lower binding densities were observed in the cerebral cortex and amygdala. There were no significant differences in 125I-BTX binding associated with the different antipsychotic treatments (all p values were >0.05 in post hoc analyses).
[3H]EPB. The highest [3H]EPB binding densities were observed in the medial habenular nuclei, interpeduncular nuclei, and pineal gland (Table 6; Fig. 8). Moderate binding was observed in the anterior thalamus and subicular complex, whereas lower binding densities were observed in the cerebral cortex and individual cortical layers. Of the 19 areas measured, antipsychotic-treated animals exhibited a statistically significant difference from vehicle controls (in post hoc analyses) in two areas, the cingulate cortex and the anteroventral thalamic nucleus. In the cingulate cortex both HAL and ZIP were associated with a decrease in binding sites, whereas in the anteroventral thalamic nucleus binding was significantly increased by HAL (compared with vehicle controls).
Muscarinic Receptor Expression.[3H]AFX. The highest [3H]AFX binding densities were observed in olfactory areas, the caudate putamen, and accumbens nuclei, whereas moderate binding was found in the cortex, basolateral amygdala, and hippocampal formation (Table 7; Fig. 8). Of the 24 areas measured, antipsychotic-treated animals exhibited a statistically significant difference from vehicle controls (in post hoc analyses) in only one area. Specifically, binding was higher in the HAL-treated rats in the pontine nuclei compared with vehicle-treated rats.
[3H]PRZ. [3H]PRZ binding was widely distributed in the cortex and hippocampal formation and minimally represented in the thalamus, hypothalamus, and midbrain (Table 7; Fig. 8). The highest [3H]PRZ binding densities were observed in the telencephalic regions such as CA1 region of the hippocampus, dentate gyrus, nucleus accumbens, basolateral amygdala, neocortex caudate putamen, and anterior olfactory nuclei. There were no significant differences in [3H]PRZ binding associated with the different antipsychotic treatments (all p values were >0.05).
Discussion
In the initial phase of this study, we observed that during the early time points of treatment (i.e., 7 and 14 days), both HAL and ZIP were associated with marked increases in NGF and ChAT immunoreactivity in the DG, CA1, and CA3 regions of the hippocampus. Such increases in NGF protein in the hippocampus have been previously observed in association with 14 days of HAL treatment (Ozaki, 2000). After 45 days, however, a very different pattern was observed; NGF and ChAT levels had abated to control levels in the ZIP-treated animals and had dropped significantly below control in HAL-treated animals. Moreover, after 90 days, NGF and ChAT levels were substantially lower than controls in both antipsychotic groups. The basis for this time-dependent (biphasic), growth factor and cholinergic response to the neuroleptics in the hippocampus is unclear, since such observations have not been reported previously. The pattern could reflect some compensatory (but unsustainable) growth factor response to a neurotoxic effect of the antipsychotics, or alternatively, some time-dependent reaction to their inhibitory D2 receptor effects on cholinergic neurons. Interestingly, observations of time-dependent (biphasic), cholinergic responses to FGAs in the striatum of animals (similar to our observations in the hippocampus) have been discussed as a potential mechanism of their adverse motor effects in humans. Namely, an increase in the activity of cholinergic interneurons in the striatum, in response to FGAs, initially seemed to parallel the extrapyramidal side effects in humans, whereas longer treatment periods were associated with decreases in cholinergic activity below baseline (i.e., effects that correspond with the emergence of tardive dyskinesia; Miller and Chouinard, 1993; Kelley and Roberts, 2004). This phenomenon may reflect the inhibitory D2 receptor effects on cholinergic interneurons by FGAs, which results in excessive neuronal activity, intracellular accumulation of calcium, and subsequent cell damage. Since D2 receptors are relatively sparse in the septohippocampal pathway, it is unclear whether an analogous process would occur here. However, dopamine regulation (via the D1 receptor) of septohippocampal cholinergic activity has been described previously (Day and Fibiger, 1994), and both HAL and ZIP have significant antagonist activity at D1 receptors (Miyamoto et al., 2005). Our detection of sustained (HAL- and ZIP-related) decreases in cholinergic markers in the cortex (described further below) may reflect the involvement of other dopamine-acetylcholine interactions. For example, it has been hypothesized that dopamine in the nucleus accumbens inhibits the activity of GABAergic projections to the basal forebrain, thereby modulating the excitability of cholinergic neurons that project to the cortex (Sarter and Bruno, 1999). Therefore, chronic exposure to drugs that antagonize mesolimbic dopamine receptors could indirectly lead to imbalances in cholinergic activity in basal forebrain neurons (and thus projection areas such as the cortex).
In the second (behavioral testing) phase of the study, the task validation experiments indicated that 1) the water maze task used was sensitive to cholinergic manipulation (a central issue in this study), 2) reference anxiolytic and anxiogenic agents were active in the light/dark box procedure, and 3) the PPI task was sensitive to the effects of three known PPI-impairing agents and their antagonism by reference antipsychotic and cholinergic agents. In the chronic neuroleptic studies, rats previously treated with HAL were impaired in water maze hidden platform tests and probe trials (i.e., spatial learning/acquisition and retention). ZIP-treated animals were also impaired during task acquisition, although the magnitude of the deficits was lower than with HAL. The absence of HAL- or ZIP-related effects on swim speeds, visible platform tests, or in light/dark box/activity monitor experiments, argues against the premise that residual drug effects on locomotor activity, visual acuity, or anxiety levels underlie the observed deficits in water maze performance. The final behavior experiments were conducted to assess the residual effects of the antipsychotics on PPI. Although a number of neuroleptic drugs (administered acutely) reverse or attenuate PPI deficits in pharmacological and neurodevelopmental models of schizophrenia (for review, see Geyer and Ellenbroek, 2003), the effects of chronic antipsychotic treatment (i.e., similar to the situation in schizophrenia) have not been evaluated. Several neurotransmitters, including dopamine, serotonin, and glutamate, are known to regulate PPI; however, observations that decreased cholinergic activity results in PPI disruption (Stanhope et al., 2001; Jones et al., 2005) were of particular interest to us in light of our previous findings of neuroleptic-associated decreases in ChAT (Terry et al., 2003). In the present study, neither HAL nor ZIP (i.e., in a normal, nonimpaired animal model) was associated with significant alterations in PPI or startle amplitude, even though these agents were associated with quite notable (negative) effects on cholinergic markers (see below).
The residual effects of chronic HAL and ZIP treatment on the neurotrophin NGF (and its receptors) and cholinergic proteins in memory-related brain areas were then assessed. NGF interacts with two plasma membrane receptors, the high-affinity TrkA receptor and the neurotrophin receptor p75NTR (p75) and provides the primary trophic support to the cholinergic basal forebrain and its projections to cortex and hippocampus. NGF binding to TrkA promotes TrkA autophosphorylation, which activates pathways that enhance cholinergic neuron survival, whereas NGF signaling via p75NTR typically (but not exclusively) activates pathways leading to cell death (see reviews, see Sofroniew et al., 2001; Counts and Mufson, 2005). ChAT and VAChT are commonly assessed as cholinergic markers since only neurons that release acetylcholine express these proteins (Wu and Hersh, 1994; Arvidsson et al., 1997). Although there were some regional differences in drug responses, in general, both HAL and ZIP tended to decrease the levels of cholinergic proteins (i.e., either ChAT, VAChT, or both) and decrease NGF (and/or its receptors). Surprisingly, in contrast to the immunohistochemical results described previously, we did not detect a decrease in ChAT protein by ELISAs performed with hippocampal lysates from HAL-treated rats. Although ChAT immunoreactivity in the hippocampus was clearly demarcated within the hippocampal subfields, the ELISAs measured ChAT protein in heterogynous tissue lysates that included the DG combined with the CA1 and CA3 subfields. Thus, the sensitivity to HAL-associated decreases in ChAT in the hippocampus may be lower when the tissues were combined. The differential finding could also be explained by the fact that ChAT immunoreactivity averaged across the hippocampal subfields decreased by ∼75% in ZIP-treated rats (i.e., at the 90-day time point), whereas the decrease was ∼55% in HAL-treated rats; therefore, the deficits may have simply been more easily detectable in the ZIP-treated animals by ELISA. An alternative explanation is that there are differential antipsychotic-related neurochemical responses to the 2-week washout.
The changes that were most striking and shared by both drugs in the ELISAs were decreases in NGF in the basal forebrain and prefrontal cortex and a decrease in phospho-TrKA in the hippocampus (i.e., brain areas known to affect acquisition and retention in a number of behavioral tasks; Kesner and Rogers, 2004). Surprisingly, p75NTR was decreased by both HAL and ZIP in the prefrontal cortex as well. Given the commonly described negative role of p75NTR on neurons, the finding was a bit perplexing, although in memory-related illnesses, such as Alzheimer's disease, TrkA depletion more reliably correlates with deteriorated cognitive ability than alterations in p75NTR (Counts et al., 2004), even though basal forebrain cholinergic neurons (commonly damaged in Alzheimer's disease) express both types of NGF receptors.
In the final portion of the study, we investigated whether chronic HAL or ZIP treatment resulted in persistent changes in cholinergic receptor densities. Given the aforementioned decreases in ChAT and VAChT (i.e., presynaptic proteins), we expected to observe reduced levels of presynaptic cholinergic receptors (i.e., nicotinic, and M2, muscarinic). Surprisingly, there were only a few minor changes (e.g., decreases in [3H]EPB binding in the cingulate cortex of rats administered HAL or ZIP). This observation seems to indicate that the antipsychotics affect proteins that are more directly influenced by NGF and involved in metabolic processes such as acetylcholine synthesis and storage (i.e., ChAT and VAChT) as opposed to cholinergic receptors.
In summary, the results of this study indicate that although ZIP (given chronically) seems somewhat superior to HAL due to less pronounced behavioral effects and a more delayed appearance of neurochemical deficits, both agents may be associated with deleterious time-dependent (and persistent) effects on the neurotrophin, NGF, and cholinergic neurons as well as spatial learning. Due to the fact that single doses of the antipsychotics were evaluated in this study, these data should be viewed with caution until more extensive dose-effect relationships for chronic treatment are established. Furthermore, it is not entirely clear whether the behavioral results observed in this study reflect some persistent neurochemical adaptation to chronic dosing or an antipsychotic withdrawal effect. Either scenario would likely have relevance to the therapeutics of schizophrenia given that chronic treatment periods are standard practice and that drug withdrawal periods are common (i.e., from poor compliance). It is also important to note that, similar to our water maze results, chronic treatment with FGAs such as HAL (Levin et al., 1987) as well as SGAs such as clozapine and risperidone (Rosengarten and Quartermain, 2002) has been associated with impaired acquisition in radial arm maze tasks as well. Collectively, these animal data suggest that there are potential limitations to extended therapy with both FGAs and SGAs, especially when cognitive function is considered.
Footnotes
-
This study was supported in part by Pfizer, Inc., and by the National Institute of Mental Health Grant MH 066233 (to A.V.T.).
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.105.099218.
-
ABBREVIATIONS: SGA, second generation antipsychotic; FGA, first generation antipsychotic; HAL, haloperidol; ChAT, choline acetyltransferase; NGF, nerve growth factor; ZIP, ziprasidone; 5HT, 5-hydroxytryptamine; PBS, phosphate-buffered saline; DG, dentate gyrus; PBST, phosphate-buffered saline/Tween 20; HRP, horseradish peroxidase; OD, optical density; AChEI, acetylcholinesterase inhibitor; mCPP, meta-chlorophenylpiperazine; PPI, prepulse inhibition; MK801, 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); ELISA, enzyme-linked immunosorbent assay; VAChT, vesicular acetylcholine transporter; p75NTR, p75 neurotrophin receptor; TrkA, tropomyosin receptor kinase A; P-TrkA, phosphotropomyosin receptor kinase A; SB, sample buffer; nAChR, nicotinic acetylcholine receptor; mAChR, muscarinic acetylcholine receptor; BTX, α-bungarotoxin; EPB, epibatidine; PRZ, pirenzepine; ANOVA, analysis of variance; DON, donepezil; SCOP, scopolamine; VEH, vehicle; AFDX 384, [3H]-5,11-dihydro-11-[((2-(2-dipropylamino)-methyl)-1-piperidinyl)ethyl)amino)carbonyl]-6H-pyrido(2,3-b)(1,4)-benzodiazepin-6-one methanesulfonate.
- Received November 29, 2005.
- Accepted May 11, 2006.
- The American Society for Pharmacology and Experimental Therapeutics