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NEUROPHARMACOLOGY

Comparison of Galantamine and Donepezil for Effects on Nerve Growth Factor, Cholinergic Markers, and Memory Performance in Aged Rats

Program in Clinical and Experimental Therapeutics, University of Georgia College of Pharmacy, Medical College of Georgia, Augusta, Georgia (C.M.H., D.A.G., E.J.H., L.W.D., M.L.M., S.P.W., A.V.T.); Small Animal Behavior Core, Medical College of Georgia, Augusta, Georgia (A.V.T.); Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia (A.V.T.); Department of Psychology, University of Michigan, Ann Arbor, Michigan (V.P.); and Department of Biostatistics, Medical College of Georgia Augusta, Georgia (J.L.W.)

Received July 21, 2005; accepted October 6, 2005.

	Abstract

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This study was designed to determine 1) whether repeated exposures to the acetylcholinesterase inhibitors (AChEIs) galantamine (GAL) or donepezil (DON) resulted in positive effects on nerve growth factor (NGF) and its receptors, cholinergic proteins, and cognitive function in the aged rat, and 2) whether GAL had any advantages over DON given its allosteric potentiating ligand (APL) activity at nicotinic acetylcholine receptors. Behavioral tests (i.e., water maze and light/dark box) were conducted in aged Fisher 344 rats during 15 days of repeated (subcutaneous) exposure to either GAL (3.0 or 6.0 mg/kg/day) or DON (0.375 or 0.75 mg/kg/day). Forty-eight hours after the last drug injection, cholinergic receptors were measured by [¹²⁵I]-(±)-exo-2-(2-iodo-5-pyridyl)-7-azabicyclo[2.2.1]heptane ([¹²⁵I]IPH; epibatidine analog), ¹²⁵I--bungarotoxin (¹²⁵I-BTX), [³H]pirenzepine ([³H]PRZ), and [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 ([³H]AFDX-384, or [³H]AFX) autoradiography. Immunochemical methods were used to measure NGF, high (TrkA and phospho-TrkA)- and low (p75 neurotrophin receptor)-affinity NGF receptors, choline acetyltransferase (ChAT), and the vesicular acetylcholine transporter (VAChT) in memory-related brain regions. Depending on dose, both GAL and DON enhanced spatial learning (without affecting anxiety levels) and increased [¹²⁵I]IPH, [³H]PRZ, and [³H]AFX (but decreased ¹²⁵I-BTX) binding in some cortical and hippocampal brain regions. Neither AChEI was associated with marked changes in NGF, NGF receptors, or VAChT, although DON did moderately increase ChAT in the basal forebrain and hippocampus. The results suggest that repeated exposures to either GAL or DON results in positive (and sustained) behavioral and cholinergic effects in the aged mammalian brain but that the APL activity of GAL may not afford any advantage over acetylcholinesterase inhibition alone.

It should be noted that whereas DON and GAL are categorized as AChEIs, they have important differences that may be of interest from a therapeutic perspective. DON is a potent (piperidine-based), noncompetitive, reversible AChEI with a long duration of action (for review, see Sugimoto et al., 2002). In contrast, GAL (a phenanthrene alkaloid) is a competitive, reversible AChEI with a considerably lower potency and shorter duration of action. GAL (also in contrast to DON) possesses allosteric potentiating ligand (APL) activity at nicotinic AChRs, which results in an amplification of the action of the acetylcholine in vitro (Albuquerque et al., 1997). It is unclear whether the latter action of GAL affects its clinical efficacy or whether such actions could result in neurotrophic and/or neuroprotective activity that has been observed in laboratory studies. For example, GAL protected human neuroblastoma cells from -amyloid and thapsigargin toxicity (Arias et al., 2004) and protected rat cortical neurons against glutamate toxicity (Takada et al., 2003). GAL preserved the viability of rat hippocampal slices exposed to oxygen and glucose deprivation (Sobrado et al., 2004) and restored cholinergic cells in antinerve growth factor (AD-11) mice (Capsoni et al., 2002). Furthermore, GAL's allosteric effects on nAChRs were associated with an up-regulation of the protective protein Bcl-2 (Arias et al., 2004). In a similar fashion, DON protected rat cortical neurons from glutamate neurotoxicity via ₄₂ and ₇ nicotinic receptors, prevented apoptotic neuronal death (Takada et al., 2003), and reversed the cholinergic and behavioral deficits in AD-11 mice (Capsoni et al., 2004).

We previously observed that repeated exposures to the prototypical nAChR agonist nicotine (in rats) resulted in an increase in high-affinity tropomyosin-receptor kinase A NGF receptors (TrkA) and their phosphorylation (Hernandez and Terry, 2005). From a potential therapeutic standpoint, such effects may be important since NGF is responsible for the maintenance, survival, and function of basal forebrain cholinergic neurons (for review, see Lad et al., 2003), cells that are severely damaged in advanced AD. Indeed, in our study cited above, nicotine was associated with increases in cholinergic proteins in memory-related brain regions (cortex, hippocampus), as well as enhanced performance of two spatial learning tasks. Such studies provide the impetus to investigate whether AChEIs, which have indirect effects on nAChRs, have similar (potentially neurotrophic) properties as nicotine and, furthermore, whether AChEIs that also possess APL activity at nAChRs (e.g., GAL) hold any therapeutic advantage.

The studies described in this report were thus designed to determine whether repeated exposures to GAL or DON resulted in positive effects on NGF and cholinergic proteins in the brain, as well as cognitive function in the aged rodent (a mammalian model that exhibits deficits in cholinergic markers and impaired memory performance; for review, see Zhang et al., 2002). Water maze testing was used to assess the effects of the AChEIs on spatial learning, after which sub-type-specific ligands and autoradiographic methods were used to measure the densities of the predominant nicotinic (₇ and ₄₂) and muscarinic (M₁ and M₂) receptor subtypes in the brain. Immunohistochemistry and/or enzyme-linked immunosorbent assays (ELISAs) were used to measure cholinergic and growth factor-related proteins including choline acetyltransferase (ChAT), the vesicular acetylcholine transporter (VAChT), TrkA, activated (i.e., phosphorylated) TrkA (phospho-TrkA), and low-affinity p75 neurotrophin receptors (p75^NTR), as well as the NGF peptide.

	Materials and Methods

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Drugs, Chemicals and Antibodies, and Commercial Sources
Drugs and Radioligands. The following drug sources are listed as follows: nicotine hydrogen tartrate and atropine sulfate (Sigma-Aldrich, St. Louis, MO); galantamine hydrobromide (Janssen Pharmaceuticals, Antwerp, Belgium); donepezil (A&A Pharmachem, Ottawa, ON, Canada); Ketaved and TranquiVed (Phoenix Scientific, St. Joseph, MO); and isoflurane (Abbott Laboratories, Abbott Park, IL). Radioligands were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA): [³H]choline chloride, [¹²⁵I]IPH, ¹²⁵I-BTX, [³H]PRZ, and [³H]AFX.

Chemicals. Cresyl violet acetate, hydrochloric acid (HCl), hydrogen peroxide (H₂O₂), sodium chloride (NaCl), sodium dodecyl sulfate (SDS), methanol (MeOH), and sucrose were obtained from Fisher Scientific Co. (Pittsburgh, PA). Kodak GBX Developer and GBX Fixer were made by Eastman Kodak (East Hampton, NY). Sigma-Aldrich was the source for 2-methylbutane, 5,5'-dithiobis-2-nitrobenzoic acid, acetone, acetylthiocholine iodide, glycerol, NP-40, paraformaldehyde, phenylmethanesulfonyl fluoride, Phosphatase Inhibitor Cocktail I and II, Protease Inhibitor Cocktail, potassium phosphate monobasic (KH₂PO₄), sodium carbonate (NaCO₃), sodium deoxycholate, sodium orthovanadate (Na₃VO₄), sodium phosphate dibasic (Na₂HPO₄), tetraisopropylpyrophosphoramide, Tris hydrochloride (Tris-HCl), and Tris base. Tissue-Tek OCT Compound was obtained from Sakura Finetek (Torrance, CA).

Antibodies and Substrates. ELISA methods. 1) Primary antibodies, see Table 1; 2) secondary antibodies horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). HRP-conjugated anti-phosphotyrosine (recombinant 4G10) was purchased from Upstate (Charlottesville, VA). 3) Substrate: 3,3',5,5'-tetramethylbenzidine (TMB) was from Kirkegaard and Perry Laboratories (Gaithersburg, MD).

Immunohistochemical (IHC) methods. Both mouse anti-ChAT and rabbit anti-mouse NGF were purchased from Chemicon International (Temecula, CA). Biotinylated secondaries and avidin-HRP complexes, were prepared from VECTASTAIN Elite ABC Kits, and 3,3-diaminobenzidine, the IHC substrate, and nickel chloride were purchased from Vector Laboratories (Burlingame, CA).

Study Subjects
Young (3-4 months old) and aged (22-24 months old) male Fisher 344 rats were obtained from the National Institute of Aging (Bethesda, MD) and housed in pairs in a temperature-controlled room (25°C) with free access to food (NIH-07 formula). Water was allowed ad libidum, and the animals were maintained on a 12-h light/dark cycle. 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 guidelines. Appropriate measures were taken to minimize pain or discomfort in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 80-23, revised 1996).

Study Protocol/Experimental Design
All animals were handled daily for several minutes for at least 1 week prior to experimentation. Saline or the test compounds (dissolved in saline) were subsequently administered twice daily (i.e., every 12 h) for 15 consecutive days. Tables 2 and 3 provide a summary of the subject numbers, the drug dosing protocol, experiments conducted, etc. All drugs were prepared and coded to blind the laboratory technicians to their identity.

GAL and DON Dosing. The doses of GAL and DON (see Table 2) were based on data obtained in rats in which brain levels of the compounds and the degree of AChE inhibition were compared across dose and time (Geerts et al., 2005) and on plasma AChE experiments conducted at the beginning of this study in our laboratory (see below). Our goal was to behaviorally test rats at a (daily) time point after injection when the acute (i.e., peak-related) drug effects would be minimized and levels of plasma AChE inhibition would be similar. Thus, behavioral testing took place between 12:00 PM and 4:00 PM each day. Furthermore, we selected doses of GAL that would be in the optimal range for allosteric potentiating ligand activity (range published at 1.5-5.0 mg/kg; see Geerts et al., 2005).

Plasma Acetylcholinesterase Activity. Blood samples were collected from three to four test subjects per group on day 13 of dosing and again at the time of sacrifice to assess plasma AChE activity. On day 13 of dosing, immediately after the water maze test session, rats were lightly anesthetized with ketamine, and approximately 300 µl of blood was collected into heparinized tubes from the tail vein. At the time of sacrifice (under ketamine/xylazine anesthesia), blood was withdrawn directly from the left ventricle immediately before perfusion. Plasma was separated from erythrocytes by centrifugation (2500g, 15 min, 4°C) and subsequently assayed spectrophotometrically using a modification of a method described in detail previously (Terry et al., 2003). Briefly, 100-µl plasma samples were added to 24-well plates containing the reaction mixture (7.5 nM acetylthiocholine iodide substrate and 6.9 mM 5,5'-dithiobis-2-nitrobenzoic acid in phosphate buffer at pH 7.9 at 37°C). Tetraisopropylpyrophosphoramide (100 µM) was also added to inhibit butyrylcholinesterase. Absorbance at 412 nm was recorded for 4 min with a µQuant Universal Microplate spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT). Data were expressed as micromoles substrate hydrolyzed per minute per milliliter of plasma, and the levels of enzyme activity for each drug dose relative to vehicle control levels were determined.

Behavioral Experiments
Locomotor Activity and the Light/Dark Preference Test. To assess the effects of GAL and DON 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 on day 12 of drug dosing (approximately 1 h after water maze testing; see below). This test is one of the most commonly used rodent models of anxiety (Holmes et al., 2001). Med Associates Inc. (St. Albans, VT) rat open field activity monitors (43.2 x 43.2 cm) were used for these experiments. They were fitted with dark box inserts (which are opaque to visible light) to cover 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 (lumen/m²) in the brightly lit zone, whereas the illumination level in the darkened zone was approximately 5 lux. The test was initiated by placing each subject into the lighted zone of the activity chamber. Activity (horizontal photobeam breaks) and the time spent in the light and dark zones of the apparatus was subsequently monitored and recorded continuously for 5 min.

Water Maze Testing. Testing apparatus. To determine the effects of age and the AChEIs on spatial learning, water maze experiments were performed in a circular pool (diameter, 180 cm; height, 76 cm) made of black plastic. The pool was filled to a depth of 35 cm of water (maintained at 25.0 ± 1.0°C). The pool was located in a large room with a number of extra-maze visual cues including geometric images (squares, triangles, circles etc.) hung on the wall, diffuse lighting, 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, and time and distance spent in each quadrant to a video tracking system (Actimetrics, Wilmette, IL).

Visible platform task. On the day prior to water maze hidden platform testing, a visible platform test was performed to assure 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 cm x 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. When a rat was successful (on its own accord without assistance), 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. Animals that were unable to locate the platform within the 90-s cutoff period for more than two of the four trials (after the initial successful trial) were eliminated from future water maze experiments.

Hidden platform task. For these experiments, an invisible (black) 10 x 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. Each rat was given two trials per day for 10 consecutive days to locate and climb on to the hidden platform. A trial was initiated by placing the rat in the water with its nose directly facing the pool wall (i.e., approximately 2 cm from the wall) in one of the four quadrants. The daily order of entry into individual quadrants was pseudo-randomized such that all four quadrants were used once every 2 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). Forty-eight hours following the last drug injection, a single probe trial was conducted for each study subject to measure spatial bias for the previous platform location. This was accomplished by removing the platform from the pool and measuring the time spent in the previous platform quadrant location.

Neurochemistry Experiments
Immediately after water maze probe trial testing, representative test subjects from each study group were sacrificed by rapid decapitation for autoradiographic and ELISA experiments and via perfusion (while under isoflurane anesthesia) for immunohistochemistry experiments.

Quantitative Receptor Autoradiography. Tissue preparation. After rapid decapitation, whole brains were extracted and flash frozen in dry ice-cooled 2-methylbutane, then stored at -70°C for at least 24 h prior to sectioning. Using a Microm HM cryostat (-18°C; Richard Alan Scientific, Kalamazoo, MI), the left hemisphere (n = 6 per group) of each brain was serially sectioned (16 µm) up to the midline onto chrome alum/gelatin-coated slides.

Preparation of standards. To define the response of the radiosensitive films to increasing amounts of radioactivity, standards containing increasing amounts of radioactivity were included on each film. For tritiated mAChR radioligands, tissue paste standards were prepared as described in Hernandez and Terry (2005) using homogenized whole rat brain tissue and [³H]choline chloride. The specific activity range of each set of tritiated tissue paste standards was 0.5 to 30.0 nCi/mg, as determined by a liquid scintillation counter. For iodinated nAChR radioligands, GE Healthcare iodinated Microscales were used with a specific activity range of 1.25 to 640.0 nCi/mg.

Radioligand binding to tissue sections. The densities of both central nAChRs and mAChRs were measured using the following radioligands: [¹²⁵I]IPH (1278.2 Ci/mmol), ¹²⁵I-BTX (118.5 Ci/mmol), [³H]PRZ (75.0 Ci/mmol) and [³H]AFX (120.0 Ci/mmol). For all autoradiographic experiments, slides were incubated with radioligands diluted in Kreb-Ringers-Hepes buffer (nAChR radioligands) or 50 mM Tris-HCl buffer (mAChR radioligands). For each radioligand, the receptor subtype target, incubation time, concentration, and duration of film exposure are listed in Table 4 (see Hernandez and Terry, 2005; Hohnadel et al., 2005, for further details). Nonspecific binding was determined by the addition of 300 µM or 1 mM nicotine hydrogen tartrate to the buffer prior to incubation with]¹²⁵I[IPH and ¹²⁵I-BTX, respectively. Nonspecific binding was determined by addition of 10 mM atropine sulfate to the buffer prior to the addition of [³H]PRZ or [³H]AFX.

Film exposure and development. After incubation with designated radioligands, slides were air-dried for at least 45 min and then stored overnight in a vacuum desiccator at room temperature. Slides were arranged in X-ray cassettes so that each film would include similar brain regions for a single rat from each group (i.e., a single youngsaline, aged-saline, low-dose DON, etc.), as well as standards. Autoradiograms were prepared by exposing the slides to Kodak Biomax MR film. All films were manually processed using GBX Developer (5 min), ultrapure water as the stop bath (30 s), and GBX Fixer (10 min) according to the manufacturer's instructions. Films were rinsed in running tap water for at least 10 min and hung to air-dry overnight. After films were developed, slides were stained with 0.5% cresyl violet (Paxinos and Watson, 1998) to better visualize and discriminate between structures and boundaries of an individual brain area.

Densitometry. Images of each section were captured from autoradiograms for the densitometry of individual brain regions using NIH Image Software (Bethesda, MD) and an imaging station [Macintosh PowerPC 8100/100I computer (Apple Computer, Cupertino, CA), QuickCapture imaging board (Data Translation, Inc., Marlboro, MA), CCD-300-RC Camera (Dage-MTI, Michigan City, IN), and a Northern Light Desktop Illuminator (Imaging Research, Inc., St. Catharines, ON, Canada). Quantification of receptor binding (as optical densities) was measured in a subset of forebrain areas important for learning and memory (see Tables 5, 6, 7, 8). Optical densities (OD) of the tissue paste standards or Microscales (with known nanocuries per milligram) were obtained, and a sigmoidal calibration curve (standard OD versus [nanocuries per milligram]) was generated using Table Curve 2D software (Systat Software, Point Richmond, CA). Once converted to units of nanocuries per milligram, all autoradiographic data were collated for each radioligand and entered into Microsoft Excel spreadsheets, then subsequently imported into (SAS 8.2; SAS Institute Inc., Cary, NC) for statistical analyses. All autoradiographic data sets are expressed as the mean nanocuries per milligram ± S.E.M.

View this table: [in this window] [in a new window]   TABLE 6 125I--Bungarotoxin autoradiography Areas with densitometry values that were significantly different from age-saline values are also designated with a bold font.

Immunohistochemistry. The IHC procedures used to localize NGF and ChAT in the hippocampal formation were based on modifications of the procedure of Parikh et al. (2004). Rats (n = 6/treatment group) were deeply anesthetized with a cocktail of ketamine/xylazine and then transcardially perfused with ice-cold PBS to remove any residual blood elements in the brain. Brains designated for NGF IHC were collected without fixation and flash frozen. Only rats designated for ChAT IHC were subsequently perfused with ice-cold 4% paraformaldehyde (in PBS), postfixed (2 h at 4°C), and cryoprotected by a 48-h incubation in 30% sucrose (in PBS) prior to being flash frozen. All brains were stored at -70°C for at least 24 h prior to cryosectioning.

Embedding and cryosectioning. Prior to cryosectioning, whole brains were embedded in OCT Compound. Cryosectioning for IHC was done using a Leica CM 3050S cryostat (Leica Microsystems, Deerfield, IL) at -20 ± 2°C. Coronal sections (NGF, 20 µm; ChAT, 40 µm) of whole brain were collected starting at the following stereotaxic coordinates: interaural, 4.84 mm; and bregma, -4.16 mm (Paxinos and Watson, 1998). The dorsal hippocampal formation, including the CA1 and CA3 region of the hippocampus proper, and the dentate gyrus (Paxinos and Watson, 1998) were analyzed. Only cryosections designated for NGF immunostaining were fixed in ice-cold acetone prior to blocking.

Rinsing and blocking. Cryosections were permeabilized prior to blocking with PBS/0.05% Tween 20 (PBS-T20) for 10 min at room temperature. PBS-T20 was also used to rinse all sections when necessary, as well as an antibody diluent for both staining methods. Nonspecific binding was blocked in sections with 10% normal goat (NGF) or horse (ChAT) serum diluted in PBS-T20 for 1 h at room temperature. Endogenous peroxidase was blocked for 30 min with 0.1% H₂O₂ and 100% methanol.

Immunostaining. Sections were incubated with rabbit anti-mouse NGF antibody (1:100) or mouse anti-ChAT antibody (10 µg/ml) overnight at 4°C. Sections were subsequently washed, then incubated for 2 h with biotinylated goat anti-rabbit IgG (1:50) or horse anti-mouse IgG (1:20) in 1.0% blocking buffer, respectively. After washing, sections were incubated for 1 h in avidin-biotin-HRP complex. HRP activity was detected and developed with and 3,3-diaminobenzidine in the presence of 0.02% H₂O₂ and nickel chloride.

Quantitative image analysis. The method for quantitation of NGF or ChAT immunoreactivity in hippocampal tissue sections from each treatment group (n = 6 for each treatment group) follows the methods described in Parikh et al. (2004). Images were viewed and digitally captured using a Zeiss Axioplan-2 Microscope equipped with CCD camera connected to a PC computer with Zeiss image analyses software (KS-300) by an experimenter blinded to treatment group (Carl Zeiss Inc., Thornwood, NY). For each tissue section, three nonoverlapping rectangular fields were captured and then digitized for the DG (582 x 455 µm²) and both the CA1 and CA3 regions (582 x 228 µm²).

Quantification of NGF immunostaining as optical density. To quantitate NGF immunoreactivity, three parameters were assessed using KS-300 software within each rectangular field: 1) the total number of stained cells, 2) the percentage of area occupied by stained cells, and 3) the OD of the stained cells. Positive staining was defined as any OD higher than a threshold OD (i.e., where only cell bodies but not processes were detectable). OD values were calculated by common logarithmic transformation of the ratio of incident to transmitted light. The whole OD range (0-2) was divided into 256 digitized values (0-255, corresponding to eight bits) to highlight variations in staining intensity. All the parameters described above were measured for the cells detected as positive ones. For each treatment group, NGF immunoreactivity was expressed as mean OD (MOD) of each treatment group (MOD_GROUP) ± S.E.M, which was based on the MOD of each subject per slice (i.e., MOD_slice = MOD of three analyzed fields/rectangles per slice - background OD_slice).

Quantification of ChAT immunostaining as fiber pixel density. Images from each rectangular field were converted to grayscale and adjusted to enhance the visibility of fibers using brightness, contrast, and masking functions in Adobe Photoshop 5.0 (Adobe Systems Inc., San Jose, CA). Quantitative data for ChAT immunoreactive fibers were expressed as fiber pixel density.

ELISA Experiments. Sample preparation for ELISA. Isofluraneanesthetized rats were decapitated, brains harvested, immediately frozen in dry ice-cooled 2-methylbutane, and then stored at -70°C. The Rat Brain Atlas by Paxinos and Watson (1998) was used as a guide for neuroanatomical landmarks for dissections of basal forebrain, hippocampal formation, cortex, and prefrontal cortex. Brain regions were homogenized in 10 volumes (e.g., 10 µl of homogenization buffer per mg of tissue) of ice-cold, modified RIPA buffer containing protease inhibitors and phosphatase inhibitors. Crude homogenates (on ice) were sonicated and then rocked at 4-5°C for 1 h on an orbital mixer. Crude pellet and supernatant fractions of the brain lysates were prepared by centrifugation at 14,000g for 30 min at 4-5°C. Each supernatant was clarified using 0.22-µm Durapore centrifugal filters (Millipore, Billerica, MA). The filtered supernatants (clarified brain lysates) were aliquoted into 0.5-ml tubes and stored at -20°C until used in the protein and ELISA analyses. Total protein in clarified brain lysates was determined using the Micro BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL).

ELISA methods. See Table 1 for details about the antibodies and incubation conditions used in indirect ELISAs for ChAT, VAChT, TrkA, and p75^NTR. The ELISA washing/rinsing buffer (PBST; pH 7.4) contained 1.7 mM KH₂PO₄, 5mMNa₂HPO₄, 150 mM NaCl, and 0.05% Triton X-100. The ELISA blocking buffer (M-PBST) contained 1% (w/v) nonfat dry milk (M) dissolved in PBST. Maxisorp 96-well ELISA plates from Nalge Nunc International (Rochester, NY) were used for all ELISA methods. During incubations, the ELISA plates were shaken—to gently mix contents of microwells— on a Jitterbug Microplate incubator/shaker (Boekel Scientific, Feasterville, MI) or on a platform orbital shaker. All PBST washes were done manually using an 8- or 12-channel multichannel pipettor. Brain lysates were diluted in 50 mM (pH 9.6) NaCO₃ buffer, and then 50 µl of the diluted brain lysate (0.3-0.5 µg/well) was added to designated wells of a microplate. The microplate was sealed with Parafilm, enclosed in a Ziploc bag that contained a wet paper towel, and slowly shaken overnight (16-20 h) at 4-5°C. The next morning, the contents of the lysate-coated microplate were discarded, the inverted plate was tapped sharply on a clean paper towel (to remove residual liquid), and the microwells were washed once with 200 µl of PBST (1 min on the Jitterbug, set at 5 1000 rpm). After every wash/rinse, residual liquid was removed by sharply tapping the inverted microplate onto a clean paper towel. The microwells were blocked with 1% (w/v) M-PBST (200 µl/well), sealing the wells with Parafilm, and slowing shaking for 1 to 4 h at room temperature. A 4-h block was required for the ChAT ELISA, because shorter blocking times resulted in higher backgrounds. Blocking was carried out for 1 h for the TrkA, p75^NTR, and VAChT ELISAs. After the blocking step, the microwells were washed/shaken once with 200 µl of PBST. Diluted primary antibody (50 µl/well) or 50 µl of M-PBST (negative control) was added to the brain lysate-coated/blocked microplate. The plate was sealed with Parafilm and slowly shaken for 2 h at room temperature. The microwells were washed/shaken five times with 200 µl of PBST after incubation with the primary (1°) antibody (antigen-specific antibody) and after incubation with the HRP-conjugated secondary (2°) antibody. The primary antibodies against VAChT, TrkA, and p75^NTR were detected using 50 µl/well of the 2° antibody: HRP-conjugated goat anti-rabbit IgG diluted 1:10,000 in M-PBST. The 1° antibody against ChAT was detected with 50 µl/well of HRP-conjugated goat anti-mouse IgG diluted 1:10,000 in M-PBST. The incubations with 2° antibodies were done in Parafilm-sealed plates, with slow shaking at room temperature for 1 h. TMB was used to detect the HRP-labeled secondary antibodies. TMB becomes blue (usually within 15-60 min) in the presence of HRP; color development was stopped (quenched) by adding 100 µl of 1 M HCl. Acidification changes the substrate color from blue to yellow, which was measured at 450 nm on a µQuant Microplate Spectrophotometer (Bio-Tek Instruments, Inc.; http://www.biotek.com).

Sandwich ELISA for phosphorylated-TrkA (Phospho-TrkA). A rabbit anti-TrkA antibody was used to capture (bind) the extracellular domain of the TrkA receptor. Theoretically, this capture antibody should orient the TrkA receptor (present in brain lysate) so that the intracellular domain of the receptor (containing the four tyrosinephosphorylation sites) is available to interact with the anti-phosphotyrosine antibody. Rabbit anti-TrkA (supplied at 1 µg/µl) was diluted to 1 µg/ml (1:1000) in 50 mM NaCO₃ (pH 9.6), and then 50 µl/well diluted anti-TrkA was added to microwells. The microplate was sealed with Parafilm, enclosed in a Ziploc bag containing a wet paper towel, and slowly shaken overnight (16-20 h) at 4-5°C. The next morning, the capture antibody was discarded, and the microwells were washed three times with 200 µl of PBST per well (using the same washing/shaking/tapping methods described in "ELISA Methods"). Microwells were blocked using 200 µl/well 1% (w/v) M-PBST; the Parafilm-sealed plate containing blocking agent was slowly shaken for 1 h at room temperature. The microwells were rinsed three times with 200 µl of PBST after the blocking step. Brain lysates were diluted to 1.4 µg of total protein/µl in 50 mM sodium carbonate buffer (pH 9.6), and then 50 µl of the diluted brain lysate was added to the blocked, anti-TrkA (capture antibody)-coated wells. The microplate was sealed with Parafilm and slowly shaken for 2 h at room temperature. The microwells were rinsed three times with 200 µl of PBST and then incubated with 50 µl of HRP-conjugated anti-phosphotyrosine diluted 1:500 in M-PBST. The plate was sealed with Parafilm and shaken for 2 h at room temperature. The microwells were rinsed three times with 200 µl of PBST, and then TMB was used to visualize the HRP as described above.

Statistical Analyses
All statistical analyses were performed using either SAS 8.2 (SAS Institute Inc., Cary, NC) or SigmaStat 2.03 (SPSS Inc., Chicago, IL). Statistical significance was assessed using an level of 0.05.

Behavioral Data, Plasma Acetylcholinesterase, Immunohistochemistry, and ELISA Results. A one- or two-way analysis of variance (ANOVA) (with repeated measures when indicated) was used for all age and treatment comparisons. In some cases, ranked data were used when the particular data set was non-normally distributed. A Tukey-Kramer multiple comparison procedure was used to examine post hoc differences when indicated.

Autoradiographic Data. Four different data sets were examined, [¹²⁵I]IPH, ¹²⁵I-BTX, [³H]PRZ, and [³H]AFX. All analyses were performed within each data set, since differences between data sets were not of interest. A repeated measures two-group ANOVA was used to examine differences in autoradiography measures between age (young and old) within areas of the brain. Animal was nested within age and was considered a random effect. Fixed effects were age and area of the brain. The two-factor interaction between age and area was included in the model. If the two-factor interaction was statistically significant, this indicated that the effect of age was different in the different areas of the brain. A Tukey-Kramer multiple comparison procedure was used to examine post hoc differences on the adjusted least square means of the two-factor interaction between ages within area of the brain. A repeated measures two-factor ANOVA was used to examine differences in autoradiographic measures between doses (high, low, or none) within areas of the brain for each drug type (DON, GAL). Animal was nested within dose and was considered a random effect. Fixed effects were dose and area of the brain. The two-factor interaction between dose and area was also included in the model. Post hoc differences on the adjusted least-square means of the two-factor interaction between doses within area of the brain were of interest.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Plasma AChE Activity
Age- and dose-related effects of DON and GAL on plasma AChE activity assayed on day 13 of dosing and 48 h after the last drug injection are illustrated in Table 9. As indicated, the two higher doses of the cholinesterase inhibitors (i.e., 0.75 mg/kg/day DON and 6.0 mg/kg/day GAL) were associated with approximately 18% inhibition when compared with aged saline control rats on day 13. This percentage probably reflects an actual (in vivo) level of inhibition approaching 80% due to the well known dilution effect observed with reversible AChEIs in vitro (Sweeney et al., 1989). There were no significant differences in AChE activity (compared with the aged saline control) in the younger rats or in the rats administered the lower doses of the AChEIs. After the 48-h washout (i.e., at the end of the study), AChE activity had fully recovered, and in fact, it seemed to be up-regulated in the animals previously treated with AChEIs.

Locomotor Activity and the Light/Dark Preference Test
Figure 1, A and B, illustrates the effects of age and the various drug treatments on horizontal activity and fear/anxiety-related behaviors, respectively. There was a significant age-related difference (p < 0.05) in ambulatory counts (i.e., horizontal photobeam breaks); specifically, older animals exhibited less locomotor activity than the younger subjects. There were no significant effects of the AChEIs on ambulatory counts in the aged animals. In addition, there were no significant age- or drug-related effects on the light/dark preference test, indicating that there were no effects on fear or anxiety-related behaviors that might confound interpretation of the results of water maze studies (see below).

View larger version (26K): [in this window] [in a new window]   Fig. 1. A, horizontal activity measured as the mean number of ambulatory counts (photobeam breaks/5 min). B, fear/anxiety related behavior (emergence neophobia) measured as the mean time spent ± S.E.M. in a brightly lit zone of the activity monitor. *, p < 0.05 versus aged saline (SAL) control. n = 14 to 22 rats per group.

Water Maze Testing
Visible Platform Test. The visible platform test was used as a method to insure that the test subjects were not impaired visually and did not exhibit other (nonmnemonic) behaviors that might have confounded the analyses. Thus, animals that were unable to locate the platform within the 90-s cutoff period for more than two of the four trials (after the initial successful trial) were eliminated from future water maze experiments, as were animals that demonstrated a lack of search behaviors, or thigmotaxis. The total number of animals eliminated from the study (all aged) was six: four that failed the visible platform test and two that became thigmotaxic (i.e., constantly circled around the perimeter of the pool) after passing the visible platform test, including one animal that developed a lesion on its tail. There was a significant group effect (F_5,99 = 5.9, p < 0.001) in the visible platform test; however, there was not a significant trial effect (F_3,15 = 0.42, p = 0.74) or group x trial interaction (F_297,419 = 1.3, p = 0.220) (data not shown). Post hoc analyses indicated that all of the groups of aged animals (either saline- or drug-treated) were somewhat impaired in this task compared with young saline treated animals. Visual inspection of the data indicated that performance was similar on trial one for all groups but separated in subsequent trials. This may indicate that visual ability was not significantly different but that more rigorous search behaviors and/or a learning effect may have contributed to the superior performance in the young animals.

View larger version (21K): [in this window] [in a new window]   Fig. 2. A to C, performance of a water maze hidden platform test (two trials/day) over 10 consecutive days of testing (mean latencies to find the platform). A, young versus aged rat comparison; B, DON dose comparison in aged rats; C, GAL dose comparison in aged rats. D, performance of water maze probe trials (mean percentage of the total time spent in the previous target quadrant ± S.E.M.) conducted 48 h after the last drug injection. *, p < 0.05 versus aged saline control; +, dose-related effect, p < 0.05. n = 14 to 22 rats. SAL, saline.

Swim Speeds. Swim speeds were also analyzed (data not shown) in an effort to further investigate treatment related differences in task performance. Average swim speeds ranged between approximately 15 to 20 cm/s across the groups for the 10 days of testing. Statistical analyses revealed that there was not an overall difference in the treatments (treatment effect, F_5,100 = 0.86, p = 0.51); however, there was a significant day effect (F_9,45 = 8.0, p < 0.001) and a significant group x trial interaction (F_900,1059 = 2.4, p < 0.001). Post hoc analyses indicated that all groups of animals swam somewhat more quickly in later trials (compared with earlier trials). Furthermore, early in testing (i.e., during days 1-3), the young animals swam slightly faster than all of the treated or untreated aged animals (i.e., p values were <0.05 or there was a strong trend, p < 0.1). There were no significant differences in swim speed after day 3.

Probe Trials. Figure 2D 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, F_5,95 = 7.3, p < 0.001). Post hoc analyses indicated that performance of the test was superior in the young animals as well as in the animals administered the higher doses of DON or GAL compared with aged saline controls.

Quantitative Receptor Autoradiography
Autoradiographic analyses of brain tissues (after memory testing) were conducted with subtype-specific cholinergic radioligands to nAChRs and 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). High-affinity (heteromeric / subunit complexes) and low-affinity (homomeric ₇) nAChRs were labeled with [¹²⁵I]IPH and ¹²⁵I-BTX, respectively. Of the high-affinity nAChRs, the ₄₂ subtype predominates in density and distribution in the mammalian central nervous system compared with other high-affinity subtypes such as ₆/₃₂ and ₃₄ (Perry et al., 2002). 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]PRZ and [³H]AFX, respectively. Representative images of the receptor autoradiographic experiments are provided in Fig. 3. Quantitative densitometric data are provided in Tables 5, 6, 7, 8.

View larger version (42K): [in this window] [in a new window]   Fig. 3. Representative autoradiograms illustrating receptors labeled by (top) [125I]IPH (high-affinity nAChRs) and 125I--bungarotoxin (low-affinity nAChRs); bottom, [3H]pirenzepine (M1 mAChRs) and [3H]AFDX 384 (M2 mAChRs) on 16-µM sagittal sections of brains from young and aged Fisher 344 rats treated with saline (SAL), DON, or GAL. Indicated areas are as follows: 1) entorhinal cortex, 2) presubiculum, 3) lamina I, 4) lamina II, 5) lamina III-VI, 6) lamina VI, 7) lamina I-IV, 8) prefrontal cortex, 9) hippocampal CA1 region, and 10) anterior olfactory nucleus.

[¹²⁵I]IPH. Across all study groups, there were highly significant regional binding differences (area effect, p < 0.0001) for [¹²⁵I]IPH. The highest binding was observed in thalamic and subicular areas, moderate binding was observed the cerebral cortex and individual cortical layers, and the lowest binding was observed in hippocampal regions. These findings are in agreement with the distributions observed in previous studies performed in rodents with epibatidine (Hernandez and Terry, 2005). In the group comparisons, there was a significant effect of age (p = 0.036) and a highly significant two-factor interaction between age and area (p = 0.0008). Inspection of Table 5 indicates that binding densities were lower in aged saline control rats compared with young controls in every brain region measured with the exception of the dentate gyrus. Post hoc analyses indicated, however, that differences met the required level of statistical significance (p < 0.05) only in the anterior thalamus and the presubiculum. In the treatment comparisons, neither the dose nor the dose x brain region interaction was significant (p > 0.05). Whereas, there were several areas in which binding was higher in both DON- and GAL-treated animals compared with age controls (e.g., cortical lamina), post hoc analyses indicated that differences met the required level of significance in the following cases: the lower and higher dose of DON in cingulate cortex (p = 0.0051 and p = 0.0008, respectively) and the post subiculum (p = 0.0045 and p < 0.0001, respectively) and the higher dose of GAL in the post subiculum (p < 0.0001).

¹²⁵I-BTX. Binding of ¹²⁵I-BTX was distributed across all areas of the brain that were analyzed, although highly significant regional binding differences (area effect p < 0.0001) were noted (Table 6). The highest ¹²⁵I-BTX binding densities were observed in the ventral and polymorphic regions of the dentate gyrus and the accessory olfactory bulbs, whereas slightly lower binding levels were observed across the remaining brain regions analyzed. This distribution is in general agreement with the findings of previous studies (Hernandez and Terry, 2005). In the group comparisons, there were no statistically significant age-related differences; however, there was a highly significant treatment x region interaction (p < 0.0001). Surprisingly, there were several cases where either DON or GAL was associated with decreased ¹²⁵I-BTX binding. Examples include olfactory areas with both compounds; GAL in the dentate gyrus, substantia innominata, and amygdala; and DON in the hippocampal (CA2) region. Conversely, DON (both lower and higher doses) was associated with an increase in ¹²⁵I-BTX binding sites in the mammillary nucleus.

[³H]PRZ. In all study groups, there were also highly significant regional differences (area effect, p < 0.0001) in binding. Binding was highest in the neocortex and hippocampal formation, whereas somewhat lower binding was observed in the subicular complex and basal ganglia (Table 7). Again, this distribution is in agreement with previous studies in rodents (Hernandez and Terry, 2005). In the group comparisons, there were no statistically significant age-related differences, whereas there was a nearly significant treatment x region interaction (p = 0.07). Interestingly, post hoc analyses indicated that both the higher and lower doses of DON and GAL were associated with a significant increase in [³H]PRZ binding sites in lamina II of cortex (medial region) and the olfactory bulbs.

[³H]AFX. Like [³H]PRZ binding, [³H]AFX binding (Table 8) was widely distributed in the cortex and hippocampal formation in all study groups and highly significant regional binding differences were noted (area effect, p < 0.0001). Unlike [³H]PRZ binding, [³H]AFX binding was also significant in the thalamus. This distribution of [³H]AFX sites is in general agreement with previous autoradiographic studies in rodents (Hernandez and Terry, 2005). In the group comparisons, there was a significant two-factor interaction between age and brain area (p = 0.03), although the Tukey multiple comparison procedure did not show any significant differences between young and old animals within any particular brain area. Inspection of Table 8 indicates that there were no particular age-related trends in the binding differences and both (subtle) increases and decreases in binding could be associated with age. In the treatment comparisons, there were no (statistically significant) overall treatment effects, treatment x region interactions or treatment x region x dose interactions with either DON or GAL. However, post hoc analyses indicated that the lower doses of DON and GAL were associated with increases in [³H]AFX binding in some cortical, olfactory, and hippocampal regions (significant with both the lower and higher doses of both AChEIs in the external plexiform layer of the olfactory bulbs).

Immunohistochemistry
NGF. Age and treatment comparisons were made by measuring the OD of immunostained cells and are provided in Table 10. As indicated, NGF immunoreactivity was highest in the DG; however, there were no statistically significant age- or treatment-related differences in NGF immunoreactivity in any of the hippocampal regions analyzed.

View larger version (112K): [in this window] [in a new window]   Fig. 4. Representative photomicrographs illustrating the effect of age and donepezil (0.75 mg/kg) on ChAT immunoreactivity in projections to the dentate gyrus and CA1 and CA3 regions of the hippocampus. Bars represent 80, 100, and 120 µm for the dentate gyrus, CA1, and CA3 regions, respectively.

View larger version (44K): [in this window] [in a new window]   Fig. 5. Brain levels of ChAT (A-C) and VAChT (D-F) proteins measured by indirect ELISA. For each ELISA (ChAT or VAChT), samples from one brain region for each treatment group (young, old, donepezil-treated, and galantamine-treated) were analyzed in the same 96-well ELISA plate, and equal amounts of total protein were analyzed across treatment groups. Data are expressed as relative levels (absorbance at 450 nm). Significant differences are indicated (*, p < 0.05; **, p < 0.01).

View larger version (40K): [in this window] [in a new window]   Fig. 6. Brain levels of TrkA receptor (A-C) and p75NTR (D-F) proteins measured by indirect ELISA. For each ELISA (TrkA or p75NTR), samples from one brain region for each treatment group (young, old, donepezil-treated, and galantamine-treated) were analyzed in the same 96-well ELISA plate, and equal amounts of total protein were analyzed across treatment groups. Data are expressed as relative levels (absorbance at 450 nm). Significant differences are indicated (*, p < 0.05; **, p < 0.01).

NGF and Phosphorylated TrkA (Phospho-TrkA). NGF levels were modestly increased in the cortex and prefrontal cortex (Fig. 7A) of aged rats (administered saline) compared with young rats, whereas phospho-TrkA was significantly decreased (Fig. 7D) in the prefrontal cortex of aged rats. DON had no measurable effect on levels of NGF (Fig. 7B) or phospho-TrkA (Fig. 7E) in any of the four brain regions evaluated. In contrast, GAL decreased NGF in the hippocampus (Fig. 7C). Similar to DON, GAL had no detectable effects on phospho-TrkA levels (Fig. 7F) in any of the four brain regions evaluated.

View larger version (40K): [in this window] [in a new window]   Fig. 7. Brain levels of NGF (A-C) and phosphorylated-TrkA (phospho-TrkA) receptor (D-F) proteins measured by sandwich ELISA. For each ELISA (NGF or phospho-TrkA), samples from one brain region for each treatment group (young, old, donepezil-treated, and galantamine-treated) were analyzed in the same 96-well ELISA plate, and equal amounts of total protein were analyzed across treatment groups. Data are expressed as relative levels (absorbance at 450 nm). Significant differences are indicated (*, p < 0.05; ***, p < 0.001).

	Discussion

Top Abstract Materials and Methods Results Discussion References

In the water maze task, the first observation was that aged vehicle-control rats were clearly impaired when compared with young controls. This was evident in both the hidden platform tasks and probe trials. Furthermore, depending on dose, aged rats repeatedly exposed to either AChEI (but particularly DON at the higher dose) performed better in the hidden platform test compared with aged (vehicle) controls. A positive effect of the higher doses of both GAL and DON was also observed in probe trials conducted 48 h after the last hidden platform trial (i.e., 48 h after the last drug injection). Therefore, both drugs seem to have positive effects on the acquisition (and potentially, sustained effects on the retention) of a spatial learning task. It should be noted, however, that since the aged control rats failed to fully reach an asymptomatic level of performance in the hidden platform trials, probe trial accuracy could have been biased in favor of the AChEIs. Interestingly, there is other evidence that repeated doses of GAL and DON can improve water maze performance in rodent models with relevance to AD. For example, daily doses of GAL (1.25 mg/kg) and DON (0.3 mg/kg) administered 30 min before testing improved deficits in water maze performance in amyloid-producing APP23 transgenic mice (Van Dam et al., 2005).

To evaluate the possibility that age or the AChEIs had effects on motor function or anxiety levels that might have influenced memory-related tests, light/dark preference experiments were performed. Heightened levels of anxiety have been documented to contribute to behavioral impairments in aged, memory-impaired rats (Rowe et al., 1998). Furthermore, at least theoretically, either AChEI could have anxiolytic actions as a result of the indirect effects of elevated synaptic acetylcholine on nAChRs; however, we were particularly interested in GAL since agonists at nAChRs have been observed to reduce anxiety in both humans and animals including rodents (Pomerleau, 1986; Brioni et al., 1993). The older animals in this study did exhibit reduced locomotor activity, although there were no significant drug effects on this phenomenon. Furthermore, there were no age- or drug-related differences in preference for the light or dark regions of the test apparatus, arguing against anxiety levels or anxiolytic drug actions as underlying group differences in water maze performance.

In autoradiographic studies, high-affinity nAChRs were modestly reduced across the brain in aged controls and notably lower in the anterior thalamus and subicular complex. Furthermore, both DON and GAL increased the expression of high-affinity nAChRs in some important memory-related brain areas (e.g., cingulate cortex, DON; postsubiculum, both DON and GAL). Similar findings regarding increased high-affinity nAChRs in memory areas and chronic GAL or DON treatment have been reported previously. For example, in homogenates of hippocampus and cortex of aged rats, an increase in [³H]nicotine binding was observed in animals that received GAL or DON for 33 days via osmotic minipumps compared with saline controls (Barnes et al., 2000). Similarly, a increase in [³H]epibatidine binding in the cortex was observed in aged rabbits that received GAL (3 mg/kg) for 3 weeks (Woodruff-Pak et al., 2001).

There were no marked age-related differences in ₇ nAChRs in the present study. Surprisingly, DON and GAL were associated with decreased ¹²⁵I-BTX binding in some brain regions (e.g., hippocampus), although DON was associated with increased BTX binding in the mammillary nucleus. There were also no significant age-related differences in M₁ mAChRs; however, both DON and GAL increased M₁ mAChR density in the lamina II layer of cortex (medial region) and the olfactory bulbs. The fact that DON was associated with some increases in [³H]PRZ binding is interesting, given reports of its nanomolar affinity (as an antagonist) at M₁ receptors (Snape et al., 1999). Although there is some evidence to suggest that GAL may bind mAChRs as well, concentrations up to 1000 µM did not significantly affect the activity of human M₁,M₂,M₃,M₄,orM₅ receptors expressed in Chinese hamster ovary cells in culture (Samochocki et al., 2003). There were no major age-related differences in the expression of M₂ mAChRs; however, both AChEIs did slightly increase binding in some cortical, olfactory, and hippocampal areas. Interestingly, in a study in 3-month-old transgenic mice overexpressing human AChE, GAL (4 mg/kg/day) administered for 10 days resulted in a down-regulation of M₁ and M₂ receptors compared with age-matched controls (Svedberg et al., 2004). A significant decrease in [³H]AFX binding but not in [³H]PRZ binding was observed in all of the brain regions analyzed in aged rats following chronic treatment with the AChEI tacrine (Zhang et al., 2002). Collectively, such results seem to suggest that the effect of AChEIs on cholinergic receptor densities are influenced by the subject's age, constitutive levels of AChE, the particular AChEI administered, and whether a drug-free washout period is imposed before neurochemical analyses are performed.

In the immunohistochemistry and ELISA studies, the effects of the AChEIs on NGF and its receptors, as well as the cholinergic markers ChAT and VAChT (i.e., proteins often described as essential for mnemonic processes; Gutierrez et al., 1997; Woolf et al., 2001), were assessed since they are often diminished in aged subjects and those with AD. NGF binding to its high-affinity receptor TrkA causes TrkA dimerization, followed by autophosphorylation of multiple tyrosine residues, which in turn initiates signal transduction events that generally promote the survival of cholinergic neurons. Conversely, signaling via the low-affinity p75^NTR typically (but not exclusively) activates pathways leading to cell death. These processes are currently among the potential therapeutic targets for neurodegenerative diseases (Lad et al., 2003). We found no significant age-related differences in NGF immunoreactivity in any of the hippocampal regions analyzed (see Table 10). This was further exemplified in ELISA experiments (in homogenates of whole hippocampus), although surprisingly, NGF was elevated in the cortex and prefrontal cortex of aged rats. The levels of ChAT and VAChT were decreased in the hippocampus of aged rats (in agreement with several previous studies), which may reflect a loss of cholinergic projections from basal forebrain nuclei to the hippocampus (Decker, 1987). In contrast to the hippocampus, aged rats had elevated ChAT and VAChT levels in the cortex. Although this observation was perplexing, equivocal results regarding cholinergic activity in the cortex of aged rodents have been observed previously. For example, both age-related decreases and no changes in cortical ChAT activity in rats (Sarter and Bruno, 1998) and increased (Sherman and Friedman, 1990) and no change (Bernstein et al., 1985) in cortical ChAT activity in mice have been reported. Furthermore, region-specific up-regulation of ChAT activity has been observed in the brains of humans previously diagnosed with mild cognitive impairment but not advanced AD (DeKosky et al., 2002), a finding that could reflect an early compensatory reaction to advancing cholinergic degeneration.

The drug effects on NGF and cholinergic proteins were also a bit difficult to interpret since increases, decreases, or no change in the proteins could be observed depending on the AChEI (and its dose) and the brain region studied. For example, a dose-dependent increase in basal forebrain ChAT was observed in aged rats treated with DON, whereas the lower dose of GAL decreased ChAT protein in the prefrontal cortex. ChAT protein levels were not affected by GAL in other brain regions, whereas ChAT immunoreactivity was increased in the CA3 hippocampal region of the aged subjects administered the higher dose of DON. Interestingly, DONrelated increases in ChAT activity have also been reported in cultured rat septal cholinergic neurons, and furthermore, DON restored water maze performance, ChAT activity, and NGF mRNA expression in experimental allergic encephalomyelitis in rats (D'Intino et al., 2005). Finally, neither DON nor GAL had significant effects on VAChT or phospho-TrkA levels, whereas both drugs were associated with decreases in both TrkA and p75^NTR in the prefrontal cortex.

In conclusion, the results of this study suggest that repeated exposures to either GAL or DON results in sustained (positive) behavioral effects and cholinergic changes in the aged mammalian brain without marked effects on NGF or its receptors. The lack of superiority of GAL over DON (at doses that produced similar degrees of AChE inhibition) may indicate that the APL activity of GAL does not afford any particular advantage over AChE inhibition alone. These studies do not exclude the possibility that AChEIs might have positive acute effects on growth factors that may be useful in the therapeutics of AD or that pure APLs (a current area of drug discovery research) could have therapeutic advantages over AChEIs.

	Footnotes

 
This work was supported in part by the Janssen Research Foundation and by the American Foundation for Pharmaceutical Education's Predoctoral Fellowship program.

doi:10.1124/jpet.105.093047.

ABBREVIATIONS: AD, Alzheimer's disease; AChE, acetylcholinesterase; AChEI, acetylcholinesterase inhibitor; DON, donepezil; GAL, galantamine; APL, allosteric potentiating ligand; nAChR, nicotinic acetylcholine receptor; NGF, nerve growth factor; TrkA, tropomyosin-receptor kinase A; ELISA, enzyme-linked immunosorbent assay; ChAT, choline acetyltransferase; VAChT, vesicular acetylcholine transporter; p75^NTR, p75 neurotrophin receptor; IPH, (±)-exo-2-(2-iodo-5-pyridyl)-7-azabicyclo[2.2.1]heptane; BTX, -bungarotoxin; PRZ, pirenzepine; AFX, 5,11-dihydro-11-[((2-(2-((dipropylamino)methyl)-1-piperidinyl)ethyl)amino)carbonyl]-6H-pyrido(2,3-b)(1,4)-benzodiazepin-6-one methanesulfonate (AFDX-384); HRP, horseradish peroxidase; TMB, 3,3',5,5'-tetramethylbenzidine; IHC, immunohistochemical; mAChR, muscarinic acetylcholine receptor; OD, optical density; PBS, phosphate-buffered saline; PBS-T20, PBS with 0.05% Tween 20; DG, dentate gyrus; MOD, mean OD; RIPA, radioimmunoprecipitation assay; M-PBST, 1% (w/v) nonfat dry milk in PBS with 0.05% (v/v) Triton X-100; ANOVA, analysis of variance; phospho-TrkA, phosphorylated TrkA.

Address correspondence to Dr. Alvin V. Terry, Jr., Professor, UGA College of Pharmacy, Director, Small Animal Behavior Core, CJ-1020, Medical College of Georgia, 1120 Fifteenth Street, Augusta, GA 30912-2450. E-mail: aterry{at}mail.mcg.edu