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Neuropharmacology 53 (2007) 563e573 www.elsevier.com/locate/neuropharm VRX-03011, a novel 5-HT4 agonist, enhances memory and hippocampal acetylcholine efflux Eric G. Mohler a,b,1, Sharon Shacham c, Silvia Noiman c, Frank Lezoualc’h d,e, Sylvain Robert d,e, Monique Gastineau d,e, Joseph Rutkowski c, Yael Marantz c, Aline Dumuis f, Joel Bockaert f, Paul E. Gold b,g, Michael E. Ragozzino a,h,* a Department of Psychology, University of Illinois at Chicago, Chicago, IL 60607, USA Department of Psychology, University of Illinois UrbanaeChampaign, Champaign, IL 68210, USA c Epix Pharmaceuticals, Lexington, MA 02421, USA d INSERM U769, IFR-141, Faculté de Pharmacie, F-92296 Châtenay-Malabry, France e Université Paris-Sud, IFR-141, Faculté de Pharmacie, F-92296 Châtenay-Malabry, France f Institut de Génomique Fonctionnelle, Université de Montpellier, CNRS UMR5203; INSERM U661, F-34094 Montpellier, France g Neuroscience Program, University of Illinois UrbanaeChampaign, Champaign, IL 68210, USA h Laboratory of Integrative Neuroscience, University of Illinois at Chicago, Chicago, IL 60607, USA b Received 27 March 2007; received in revised form 11 June 2007; accepted 20 June 2007 Abstract Recent evidence suggests that 5-hydroxytryptamine (5-HT)4 receptor activity enhances cognition and provides neuroprotection. Here we report the effects of VRX-03011, a novel partial 5-HT4 agonist, that is both potent (Ki w 30 nM) and highly selective (Ki > 5 mM for all other 5-HT receptors tested). In separate experiments, rats received VRX-03011 (0.1e10 mg/kg i.p.) 30 min prior to spontaneous alternation testing in a no-delay or a 30-s delay condition. VRX-03011 (1, 5 and 10 mg/kg, but not 0.1 mg/kg) significantly enhanced delayed spontaneous alternation performance while none of the doses enhanced performance in the no-delay test. VRX-03011 (1 and 5 mg/kg) concomitantly enhanced hippocampal acetylcholine output and delayed spontaneous alternation scores compared to that of vehicle controls, but had no effect on hippocampal acetylcholine release under a resting condition. Moreover, suboptimal doses of VRX-03011 and the acetylcholinesterase inhibitor galanthamine combined to enhance memory. VRX-03011 also regulated amyloid precursor protein (APP) metabolism by inducing a concentration-dependent increase in the non-amyloidogenic soluble form of APP (sAPPa) with an EC50 w 1–10 nM. VRX-03011 had no effect on contractile properties in guinea pig ileum or colon preparations with an EC50 > 10 mM and did not alter rat intestinal transit at doses up to 10 mg/kg. These findings suggest that VRX-03011 may represent a novel treatment for Alzheimer’s disease that reduces cognitive impairments and provides neuroprotection without gastrointestinal side effects. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Serotonin; Acetylcholine; Hippocampus; Memory; 5-HT4; Alzheimer’s disease; Microdialysis; Behavior 1. Introduction * Corresponding author. Department of Psychology, University of Illinois Chicago, 1007 West Harrison Street MC 285, Chicago, IL 60607, USA. Tel.: þ1 312 413 2630; fax: þ1 312 413 4122. E-mail address: [email protected] (M.E. Ragozzino). 1 Present address: Abbott Laboratories, Abbott Park, IL 60064, USA. 0028-3908/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2007.06.016 Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that results in severe cognitive impairment (Ho et al., 2002; Hodges et al., 1992). Dysfunction and death of cholinergic neurons comprise the central neuropathology observed in AD. Reductions in cholinergic markers are commonly observed in hippocampal and cortical areas, as well as being 564 E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573 correlated with the severity of dementia (Hyman et al., 1984; Wilcock et al., 1982). Accordingly, the predominant treatment for AD focuses on increasing cholinergic tone through the use of acetylcholinesterase inhibitors (AChE-I) (Aruglia et al., 2004; Venneri et al., 2005). Treatment with AChE-I in AD has led to modest effects in cognition and sometimes unwanted side effects, e.g. depression (Frankfort et al., 2006). Another potential treatment approach is to target other neurotransmitter system ligands that modulate acetylcholine (ACh) release and may be used alone or in combination with AChE-I. One potential neurotransmitter to target is 5-HT. There is some evidence that 5-HT neurotransmission is altered in AD (Garcia-Alloza et al., 2005; Meltzer et al., 1998). More specifically, post-mortem binding studies in AD reported decreased density of 5-HT4 receptors in the hippocampus and neocortex (Reynolds et al., 1995; Wong et al., 1996, but see Lai et al., 2003). Furthermore, an increasing body of evidence indicates that the 5-HT4 receptor modulates ACh release, and is a promising target for the development of novel therapies for AD (Bockaert et al., 2004). The 5-HT4 receptor was first cloned from rat (Gerald et al., 1995). In human, eight splice variants have been identified (a, b, c, d, e, f, g, and n). With few exceptions, the pharmacology of all of the splice variants is nearly identical (reviewed in Bockaert et al., 2004), though the level of constitutive activity of the receptor has been found to differ between isoforms, with longer C-terminals generally associated with diminished constitutive activity (Claeysen et al., 1999). 5-HT4 receptors are localized in the heart (Kaumann, 1990), intestine (Craig and Clarke, 1990), adrenal cortex (Idres et al., 1991), bladder (Tonini and Candura, 1996), and the central nervous system (Patel et al., 1995). Within the brain, highest expression is found in the hippocampus, basal ganglia, and amygdala (Medhurst et al., 2001), regions associated with learning and memory. Developing pharmacological agents that target the 5-HT4 receptor may also be valuable because 5-HT4 receptor activation produces secretion of the soluble amyloid precursor protein (sAPPa) (Maillet et al., 2004; Robert et al., 2001). sAPPa may have neuroprotective effects by potentially blocking the formation and the toxic effect of b-amyloid peptide (Ab), a major component of the senile plaques associated with AD (Lezoualc’h and Robert, 2003). Cho and Hu (2006) demonstrated that a partial 5-HT4 agonist can inhibit the formation of Ab in primary cortical cultures from Tg2576 mice. Moreover, stimulation of central 5-HT4 receptors increases sAPPa in the mouse hippocampus and cortex (Chacard-Castel et al., 2007). The importance of 5-HT4 receptors in mnemonic processes is further supported by several studies in rodents which found that partial 5-HT4 agonists enhance learning and memory, either alone (Lamirault and Simon, 2001; Lelong et al., 2003; Matsumoto et al., 2001; Moser et al., 2002) or in combination with an AChE-I (Lamirault et al., 2003; Moser et al., 2002). Although there is some evidence suggesting that 5-HT4 receptor activity modulates ACh release (see Bockaert et al., 2004 for a review), some partial 5-HT4 receptor agonists when administered centrally have been found to increase brain ACh levels under non-mnemonic conditions (Consolo et al., 1994; Matsumoto et al., 2001). Thus, these drugs may indiscriminately enhance cholinergic tone. Related to this issue, there is no evidence that a partial 5-HT4 receptor agonist enhances ACh output under conditions that require learning and memory. This limitation is compounded by some previously developed 5-HT4 receptor agonists producing adverse drug reactions in gastrointestinal functions (Briejer et al., 2001; Wooltorton, 2004). Thus, a partial 5-HT4 agonist that more selectively modulates ACh output during mnemonic conditions without peripheral side effects could prove to be of significant therapeutic value in dementia. The findings described above suggest that partial 5-HT4 agonists may provide effective and novel treatments to ameliorate cognitive and neuropathological deficits in conditions associated with memory impairments and neurodegeneration. The present experiments examined the profile of VRX-03011, a novel partial 5-HT4 receptor agonist (see Fig. 1). Because previous experiments did not investigate whether a partial 5HT4 agonist enhances hippocampal ACh output during a memory test, VRX-03011 was tested in a spontaneous alternation task in which rodents choose the least recently visited arm in a maze and thus must remember recent arm selections. Previous experiments demonstrated that hippocampal ACh efflux is important for spontaneous alternation performance and memory-enhancing agents concomitantly augment spontaneous alternation performance and hippocampal ACh output (Ragozzino et al., 1996, 1998). Because earlier investigations showed synergistic effects with partial 5-HT4 agonists and AChE-Is (Lamireault et al, 2003; Moser et al., 2002), subefficacious doses of VRX-03011 were examined in combination with the AChE-I, galanthamine. To investigate whether VRX-03011 modified sAPPa levels, the effects of VRX03011 on the induction of sAPPa secretion were tested in vitro. Finally, to determine whether VRX-03011 affected gastric motility, VRX-03011 was studied in gastrointestinal models. 2. Methods All studies were carried out in accordance with the Declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals adopted by the U.S. National Institutes of Health. 2.1. Radioligand binding studies All radioligand binding studies were carried out by CEREP (Paris, France) under contract with Predix Pharmaceuticals Inc. (now EPIX Pharmaceuticals K+ O- O N H S N N O Fig. 1. VRX-03011, Potassium salt of 6,7-dihydro-4-hydroxy-7-isopropyl-6oxo-N-(3-(piperidin-1-yl)propyl)thieno[2,3-b]pyridine-5-carboxamide. E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573 Inc.). Binding was initially tested using 1 mM VRX-03011. A specific Ki was determined in all cases where greater than 50% displacement was observed at 1 mM. The binding affinities for other 5-HT receptors as well as 55 other proteins, including other G-protein coupled receptors, ion channels and transporters, were evaluated. When available, the experiments were carried out using recombinant human receptors cloned into various cell types as shown in Table 1. 2.2. Cell culture and transfection for splice variants of the 5-HT4 receptor The cDNAs coding for the 5-HT4(a), 5-HT4(b) and/or 5-HT4(e) receptors were subcloned into the pRK5 vector and, were introduced into COS-7 cells by transfection as previously described (Claeysen et al., 1999). IMR32 cells and Chinese hamster ovary (CHO) cells stably expressing the h5-HT4(d) or the h5-HT4(e) receptor isoform were cultured as previously described (Mialet et al., 2000; Robert et al., 2001). For transient transfection experiments, CHO cells stably expressing the h5-HT4(d) receptor isoform were transfected with the cDNA encoding the human APP695 using jetPEIÔ (Polyplus-transfection, Illkirch, France) according to the manufacturer instructions. 2.3. Membrane preparation and radioligand binding assay for splice variants of the 5-HT4 receptor In competitive binding analyses against 5-HT, the binding affinities of VRX03011 to 5-HT4 receptor isoforms were tested using membranes prepared from COS-7 cells transiently transfected with the recombinant 5-HT4(a) receptor splice variants or 5-HT4(e) receptor isoforms. Membranes were prepared from transiently transfected cells plated on 15 cm dishes and grown in DMEM with 10% FBSd for 6 h and 20 h in DMEM without FBSd. Radioligand binding studies with [3H]GR 113808 were performed on membrane preparation of guinea pig striatum that express the endogenous 5-HT4 receptors as previously described (Ansanay et al., 1996). Protein concentration in the samples was determined with the Bio-Rad protein assay. Competition and saturation experiments were analyzed by non-linear regression using the computer program LIGAND (Murson and Rodbard, 1980). 2.4. cAMP production in transfected cells To test 5-HT4 receptor variants, COS-7 cells were transiently transfected with 5-HT4(a), 5-HT4(b) or 5-HT4(e) receptor isoforms. COS-7 cells were transfected with the appropriate cDNA and plated into 24-well clusters (70,000 cells/well). Twenty-four hours post-transfection, cAMP production levels were determined as follows. Cells were stimulated for 5 min with the 565 appropriate concentrations of 5-HT, 0.1 mM L-ascorbic acid and 0.1 mM RO-20-1724, a phosphodiesterase inhibitor, in 250 ml of HEPES-buffered saline (20 mM HEPES, 150 mM NaCl, 4.2 mM KCl, 0.9 mM CaCl2, 0.5 mM MgCl2, 0.1% glucose, 0.1% BSA) at 37 C. The same volume of Triton X-100 (0.1%) was added to stop the reaction, and then the cells were incubated 30 min at 37 C. Quantification of cAMP production was performed by Homogenous Time Resolved FluorescenceÔ (HTRF) using the cAMP Dynamic kit (CIS Bio International, Bagnols sur Cèze, France) according to the manufacturer’s instructions. 2.5. Behavioral pharmacology experiments 2.5.1. Animals Male LongeEvans rats (Charles River Laboratories, Indianapolis, IN) weighing approximately 350 g were used for all experiments. Subjects were individually housed in plastic cages in a temperature controlled room with a 12 h light/dark cycle (lights on at 07:00 h). Rats had free access to food and water except for the 24 h preceding testing, at which time they were restricted to approximately 12 g of food. 2.5.2. Maze apparatus A four-arm cross-maze made of 0.6 cm thick black plastic was used for all behavioral testing. The maze was placed on a table that was 72 cm above the floor. Each arm was 55 cm long 10 cm wide. The height of the arm walls was 15.0 cm. Each arm contained a food well (3.2 cm diameter 1.6 cm high) that was 3 cm away from the end wall. Each food-well hole was 2.3 cm in diameter and 1.6 cm deep. 2.5.3. Spontaneous alternation (no-delay) and drug injection procedures In the spontaneous alternation test, rats were placed in the testing room 5 min before testing. Rats were allowed to freely explore the maze for 12 min. The number and sequence of arm entries was recorded. An arm entry was recorded when all four paws entered an arm. An alternation was defined as entry into four different arms on overlapping quadruple sets of arm entries; e.g., a quadruple set consisting of arm choices A,D,C,B was an alternation but a quadruple set consisting of arm choices A,D,A,C was not. The percent alternation score is equal to the ratio of (actual alternations/possible alternations) multiplied by 100; chance performance on this task is 22.2%. Rats that made less than 15 arm entries in 12 min were excluded from the analysis. This criterion is similar to that used in a previous experiment (Ragozzino et al., 1996). Thirty minutes before testing, each rat received either VRX-03011 (0.1, 1, 5, or 10 mg/kg, i.p.) (Epix Pharmaceuticals Inc., Lexington, MA) or vehicle (5% DMSO/sterile water). All doses were based on the weight of the salt. Each rat received a total injection volume of 5 ml/kg. The number of rats in Table 1 Serotonin receptor binding profile for VRX-03011 Receptor Ligand Ligand concentration (nM) Cells/Tissue Affinity for VRX-03011 (nM) 5-HT4a 5-HT4e 5-HT4 5-HT1A 5-HT1B 5-HT1D 5-HT2A 5-HT2B 5-HT2C 5-HT3 5-HT5A 5-HT6 5-HT7 [3H]GR 113808 [3H]GR 113808 [3H]GR 113808 [3H]8-OH-DPAT [125I]Cyanopindolol (CYP) [3H]Serotonin [3H]Ketanserin [3H]LSD [3H]Mesulergine [3H]BRL 43694 [3H]LSD [3H]LSD [3H]LSD 0.42 0.42 0.1 0.5 0.1 2 0.5 1.2 1.0 0.5 1.0 2.0 4.0 COS-7 cells expressing human cDNA COS-7 cells expressing human cDNA Guinea pig striatum HEK293 cells expressing human cDNA Rat cerebral cortex Bovine caudate HEK293 cells expressing human cDNA CHO cells expressing human cDNA CHO cells expressing human cDNA HEK293 cells expressing human cDNA HEK293 cells expressing human cDNA HEK293 cells expressing human cDNA CHO cells expressing human cDNA 31 17 36 >5 mMa >5 mMa >5 mMa >5 mMa >5 mMa >5 mMa >5 mMa >5 mMa >5 mMa >5 mMa All binding studies were carried out by CEREP except for 5-HT4a and 5-HT4e receptor. LSD, lysergic acid diethylamide. a Binding affinity estimated based on % displacement at 1 mM VRX-03011. 566 E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573 each group was 6, except the vehicle group (n ¼ 10) and the VRX-03011 (5 mg/kg) group (n ¼ 9). 2.5.4. Spontaneous alternation (30-s delay) and drug injection procedures A different set of rats was used in this experiment. In the delayed version, each rat was allowed to freely choose an arm, but after making a choice, a rat was blocked into that arm for 30 s. The block was a 21.5 cm 12 cm piece of plastic. After the 30-s delay, the block was removed and a rat was allowed to enter another arm. The test session lasted 15 min. Rats that made fewer than 11 arm entries in 15 min were excluded from the analysis. Each rat received an intraperitoneal injection 30e32 min before testing. Three separate experiments were carried out using spontaneous alternation with a 30-s delay. A separate set of rats was used for each experiment. In the first experiment, a rat received one of the following treatments: vehicle; VRX-03011 (0.1, 1, 5, or 10 mg/kg); GR 125487 1 mg/kg (Tocris, Ellisville, MO) or a combination of GR 125487 (1 mg/kg) and VRX-03011 (1 mg/kg). In the second experiment, delayed spontaneous alternation testing was combined with in vivo microdialysis using the following treatments: vehicle or VRX-03011 (1 or 5 mg/kg). In the third experiment the interaction between VRX-03011 and the AChE-I, galanthamine was investigated. This experiment had the following treatment groups: vehicle; VRX-03011 (0.03 mg/kg); galanthamine (0.3 or 2.5 mg/kg) (Tocris); a combination of VRX-03011 and galanthamine (0.3 mg/kg). All doses were based on the weight of the salt. Rats received a total injection volume of 5 ml/kg. In the experiment testing VRX-03011 in 30-s delayed spontaneous alternation, the number of rats in each group was 6. In the combined in vivo microdialysis and delayed spontaneous alternation experiment the number of rats in each group ranged from 7 to 9. In the experiment examining VRX-03011 and galanthamine, the number of rats in each group ranged from 6 to 8. 2.5.5. Surgery Each rat for the in vivo microdialysis experiment received stereotaxic surgery to implant cannulae into the hippocampus. Rats were injected with atropine sulfate (0.2 ml; of a 250 mg/ml solution, i.p.) and the anesthetic sodium pentobarbital (50 mg/kg, i.p.). The rat was then placed in the stereotaxic frame and 10-mm guide cannulae (CMA microdialysis) were implanted bilaterally at stereotaxic coordinates 5.2 posterior to bregma, 5.2 lateral to the midline, and 3.7 ventral to the surface of the skull. The cannulae were secured in place with skull screws and dental acrylic. Rats were allowed to recover for 1 week after surgery. 2.5.6. Microdialysis procedures The day before testing, each rat was placed in a large plastic bowl and a microdialysis probe was inserted into the cannula for 3 min. The purpose of this was to preclude the possibility that levels of ACh at the time of testing were abnormal as a result of damage from initial probe insertion (Westerink, 1995). On the day of testing, a 3-mm dialysis probe (CMA, North Chelmsford, MA) was inserted through the guide cannula into the hippocampus. The dialysis probe was connected to polyethylene tubing (CMA), which was connected to a quartz swivel (Instech, Plymouth Meeting, PA), an additional length of tubing, and then to an infusion pump (Harvard Apparatus, Holliston, MA). The probe was perfused continuously at a rate of 1.5 ml/min with artificial cerebrospinal fluid, which consisted of 128 mM NaCl, 2.5 mM KCl, 1.3 mM CaCl2, 2.1 mM MgCl2, 0.9 mM NaH2PO4, 2.0 mM Na2HPO4, 1.0 mM dextrose and adjusted to pH 7.4 by NaOH. To reliably detect ACh levels in the dialysate, the reversible AChE-I, neostigmine bromide (0.1 mM) (Sigmae Aldrich, St. Louis, MO) was added to the artificial cerebrospinal fluid. A previous study demonstrated that 0.1 mM concentration of neostigmine in the artificial cerebrospinal fluid is a minimal amount needed to reliably detect behaviorally-induced changes in brain ACh efflux (Chang et al., 2006). 2.5.7. Drug injection and microdialysis sample collection On the day of testing, a microdialysis probe was inserted into the hippocampus and the rat was placed in a large plastic bowl. The first 60 min of perfusate was not analyzed to allow for equilibration between the brain tissue and perfusion solution before testing. Subsequently, 12-ml samples were collected at 8-min intervals. Three baseline samples were collected, and then rats were injected i.p. with VRX-03011 (1 or 5 mg/kg) or vehicle (5% DMSO/sterile water) with an injected volume of 5 ml/kg. Four additional baseline samples were collected followed by the placing of the rat in the maze to begin delayed spontaneous alternation testing as described above. Delayed spontaneous alternation testing lasted 16 min. Thus, the testing session begin 32 min after receiving the drug or vehicle. After testing was over, each rat was returned to the plastic bowl and two posttest samples were collected. The sample size in each group ranged from 7 to 9. 2.5.8. Drug injection and resting condition procedure Resting condition measurements were made in rats that had completed delay spontaneous alternation task with in vivo microdialysis collection. There was a washout period of at least 1 week in between sessions. Microdialysis samples were collected from the hemisphere opposite that used during maze testing to ensure that tissue damage from the previous dialysis session did not affect ACh output. Previous observations revealed diminishing ACh levels from the same site with repeated dialysis sessions in the hippocampus (Gold and Ragozzino, unpublished observations). The microdialysis procedure was identical to the behavioral testing procedure, except that a rat was not placed in the maze. Each rat received a different treatment in the resting condition than in the behavioral testing condition. For this experiment the sample size in each group was 6. 2.5.9. ACh assay Samples (10 ml) were assayed for ACh using high-pressure liquid chromatography with electrochemical detection (Bioanalytical Systems, West Lafayette, IN). Samples were loaded on a microbore analytical column for separation of ACh and choline. Following separation, an enzymatic post-column reactor containing acetylcholinesterase and choline oxidase converted ACh to choline and acetate and choline to betaine and hydrogen peroxide. ACh and choline were further broken down into stoichiometric quantities of hydrogen peroxide. Hydrogen peroxide was broken down and detected by a glassy carbon wired electrode coated with horseradish peroxidase at þ100 mV versus and Ag/AgCl reference electrode. The mobile phase consisting of 50 mM Na2HPO4, 0.3 mM EDTA and 0.005% ProClin was delivered at a rate of 100 mL/min by a solvent delivery system. 2.5.10. Histology After completion of behavioral testing, each rat received a lethal dose of sodium pentobarbital. A probe dipped in 2.5% Chicago blue stain was inserted in each guide cannula to highlight the location of the probe. All rats were perfused intracardially with 0.9% phosphate-buffered saline, followed by a 4% formaldehyde solution. Brains were removed and stored in a 4% formaldehyde solution. The brains were frozen and cut in coronal sections (40 mm) on a cryostat. The brain sections were mounted on slides, dried, and stained with cresyl violet to assess the location of the cannula tips or probes. Rats with placements extending beyond the hippocampus were excluded from all analyses. 2.5.11. Statistical analysis For all behavioral experiments, the threshold for statistical significance was set at P < 0.05. In each version of the spontaneous alternation task, a one-way analysis of variance (ANOVA) was used to identify differences in percent alternation scores and number of arm entries across groups. A Fisher Least Significant Difference post-hoc test was used to compare treatment and control group measures. For ACh measurements, data was converted to a percentage of the baseline measurement. Specifically, the first three baseline data points were averaged, and all subsequent data points were expressed as a percentage of that average. Changes in ACh output were expressed as a percentage of baseline output. For the measurement of hippocampal ACh efflux during spontaneous alternation testing and the resting condition, an ANOVA with repeated measures was used to identify differences in ACh efflux across the different test samples (seven baseline periods, two behavioral testing periods and two post-test periods). A Dunnett post-hoc test was used to determine whether ACh output differed in the testing periods compared to that of baseline for the different groups. E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573 2.6. Measurement of sAPPa CHO cells stably expressing the h5-HT4(e) receptor and APP695 and IMR32 cells were cultured overnight in 5% dialyzed FCS-containing medium, followed by a serum free medium for a period of 4 h. Then, 5-HT4 ligands were added for 30 min in CHO cells or 4 h in IMR32 cells. After this period of incubation, conditioned supernatants were collected and sAPPa was determined as previously described (Robert et al., 2001). 2.7. Guinea pig colon functional assay The activity of VRX-03011 at the 5-HT4 receptor in the guinea pig colon bioassay was determined as previously described (Gale et al., 1994). Briefly, segments of guinea pig distal colon were suspended between two stainless-steel hooks in 20-ml organ baths. The baths containing an oxygenated (95% O2 and 5% CO2) and pre-warmed (37 C) physiological salt solution of 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 1.2 mM KH2PO4, 25 mM NaHCO3 and 11 mM glucose, pH 7.4. Pyrilamine (1 mM), methysergide (1 mM) and ondansetron (10 mM) were also present throughout the experiments to block the histamine H1, 5-HT2 and 5-HT3 receptors, respectively. The tissues were stretched to a resting tension of 1 g then allowed to equilibrate for 60 min during which time they were washed repeatedly and the tension readjusted. To test for agonist activity the tissues were exposed to increasing concentrations of VRX-03011. To test for antagonist activity, tissues were exposed to increasing concentrations of VRX-03011 or the reference antagonist GR 113808, 30 min before exposure to a single effective concentration of the reference agonist 5-HT (0.3 mM). GR113808 was generously donated by Glaxo (Ware, Herts, UK). 2.8. Intestinal transit in rats The effects of VRX-03011 on gastrointestinal motility were determined in male OFA-SD IGS rats weighing 144 to 208 g (n ¼ 8 per treatment group). Rats were fasted for 24 h prior to i.v. administration of either vehicle (12.5% DMSO in saline) or VRX-03011 (3 or 10 mg/kg i.v., expressed as base) on the test day. Forty minutes later, a pulp consisting of 10% vegetable charcoal in 2% Arabic gum was given by gavage (10 ml/kg body weight). Twenty minutes after charcoal meal, animals were euthanized by CO2 inhalation. The whole gut (from stomach to cecum) was removed as rapidly as possible and laid out on a centimeter measuring stick. The whole gut was examined in coded order and the length of the intestine from the pyloric sphincter to the ileocecal junction as well as the distance traveled by the charcoal front were both measured and recorded. For each animal, the intestinal transit was determined as a percentage of the distance traveled by the charcoal front, relative to the total length of the small intestine. Treatment effects relative to controls were analyzed by one-way ANOVA followed by a pairwise multiple comparisons (Dunnett’s method) where necessary. Differences between groups were considered significant at P < 0.05. 3. Results 3.1. Binding affinity and specificity for the 5-HT4 receptor VRX-03011 demonstrated high affinity for the two recombinant 5-HT4 receptor isoforms, 5-HT4 (a) and (e) (Kis ¼ 31 and 17 nM, respectively), and for the striatal 5HT4 receptor (Ki ¼ 36 nM). Experiments testing competitive displacement by VRX-03011 using several proteins revealed considerable 5-HT4 receptor selectivity, with a Ki > 5 mM for all other 5-HT receptors (Table 1). Furthermore, VRX03011 exhibited no binding activity for 55 other proteins tested (Kis > 1 mM), including other G-protein coupled 567 receptors, ion channels and transporters (See supplementary information at end of manuscript). VRX-03011 was only active at s1 and s2 receptors (Kis ¼ 79 and 40 nM, respectively). In COS-7 cells transfected with 5-HT4 (a), (b) and (e) variants, VRX-03011 increased cAMP formation in a concentration-dependent manner with high potency (EC50 of 15e93 nM) and intrinsic agonist activity between 41% and 61% relative to 5-HT (Table 2). Taken together these results demonstrate that VRX-03011 is a selective partial agonist for the 5-HT4 receptor. 3.2. Spontaneous alternation testing In the no-delay spontaneous alternation task, an ANOVA revealed there was not a significant treatment effect, F(4,32) ¼ 2.36, P > 0.05. Although the effect was not significant, only the 5 mg/kg of VRX-03011 showed a trend for improved alternation scores of w12% (see Fig. 2a). The mean number of arm entries among the groups was comparable, suggesting that general activity levels are not affected by the drug, F(4,32) ¼ 0.48, P > 0.05 (see Fig. 2b). In this experiment, 13 rats were excluded from the analyses because an insufficient number of arm entries were completed during the behavioral test. The distribution of these rats among the treatment groups was as follows: vehicle (n ¼ 2); VRX03011 0.1 mg/kg (n ¼ 1); VRX-03011 1 mg/kg (n ¼ 2); VRX-03011 5 mg/kg (n ¼ 3); VRX-03011 10 mg/kg (n ¼ 5). The results from the 30-s delay spontaneous alternation test are illustrated in Fig. 3. In the delayed spontaneous alternation task, VRX-03011 treatment significantly enhanced alternations in a dose-dependent manner, F(6,35) ¼ 4.01, P < 0.005. A Fisher LSD post-hoc test indicated that VRX-03011 significantly improved delayed alternation scores at the 1, 5 and 10 mg/kg doses; P’s < 0.05 vs. vehicle controls. When rats received the 5-HT4 receptor antagonist GR 125487 (1 mg/kg) co-administered with VRX-03011 (1 mg/kg), the antagonist reduced the alternation scores back down to control levels (P > 0.05 vs. vehicle controls). GR 125487 (1 mg/kg) alone was without effect. The mean number of arm entries among all groups was comparable, F(6,35) ¼ 0.70, P > 0.05, suggesting that VRX-03011 alone or in combination with GR 125487 did not affect general activity. In this experiment, six rats were excluded from the analyses because an insufficient number of arm entries were completed during the behavioral test. The distribution of these rats among the treatment groups was as follows: vehicle (n ¼ 4) and VRX-03011 0.1 mg/kg (n ¼ 2). Table 2 Stimulation of cAMP production by VRX-03011 in COS-7 cells expressing recombinant 5-HT4 receptors Receptor VRX-03011 EC50 (nM) VRX-03011 Emax (%) 5-HT4(a) 5-HT4(e) 5-HT4(b) 15e93 17e52 18e42 41e53 30e61 39e59 E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573 70 60 (a) 70 * 60 Percent Alternation (mean ± SEM) (a) Percent Alternation (mean ± SEM) 568 50 40 30 20 10 0 Veh 0.1 1.0 5.0 50 40 † 30 20 0 Vehicle 40 0.1 mg/kg 1 mg/kg 5 mg/kg VRX-03011 35 30 25 (b) 20 15 10 GR 125487 VRX-03011 mg/kg 1 mg/kg 1 mg/kg + GR 125487 1 mg/kg 30 25 10 5 0 Veh 0.1 1.0 5.0 10 VRX-03011 Fig. 2. Effect of VRX-03011 treatment on standard spontaneous alternation performance in a four arm cross-maze. (a) Effect of VRX-03011 (0.1e 10 mg/kg) treatment on spontaneous alternation performance in a four arm cross-maze. VRX-03011 did not significantly improve performance compared to that of vehicle controls. The dashed line denotes chance performance (approximately 22%) (b) Mean number of arm entries in the delayed spontaneous alternation test. All groups made comparable number of arm entries in the test session. 3.3. Hippocampal ACh efflux and spontaneous alternation testing To determine whether enhancement of memory by VRX03011 is accompanied by modulation of brain ACh output, the effects of VRX-03011 (1 mg/kg and 5 mg/kg) on delayed spontaneous alternation performance and hippocampal ACh efflux were assessed simultaneously (Fig. 4a). Probe placements for rats included in the analyses were concentrated in the CA3 and CA2 regions of the hippocampal formation at the level of the splenium of the corpus callosum along the anterioreposterior plane. Three rats were excluded from the analyses because cannulae were outside of the hippocampal formation. These probe placements were found in the fimbria and/or external capsule. Comparable to the results described above, VRX-03011 (1 and 5 mg/kg) significantly enhanced delayed spontaneous alternation scores to 48.6 6.3% and 44.9 4.2% (S.E.M.), respectively, compared to that of vehicle control-treated rats which had a mean score of 16.6 3.9%, F(2,20) ¼ 12.30, P < 0.05. Again, the memory enhancement with VRX03011 treatment occurred without altering the number of arm entries, F(2,20) ¼ 0.14, P > 0.05. The mean number of arm entries for the 1 mg/kg, 5 mg/kg and vehicle control groups was 14.4 1.4, 15 1.2 and 14 1.5, respectively. Number of Entries (mean ± SEM) Numberof Entries (mean ± SEM) * 10 10 VRX-03011 (b) * 20 15 10 5 0 Vehicle 0.1 mg/kg 1 mg/kg 5 mg/kg VRX-03011 10 GR 125487 VRX-03011 mg/kg 1 mg/kg 1 mg/kg + GR 125487 1 mg/kg Fig. 3. Effect of VRX-03011 treatment on delayed spontaneous alternation performance in a four-arm cross-maze. (a) Effect of VRX-03011 (0.1e 10 mg/kg) treatment on delayed spontaneous alternation performance in a four arm cross-maze. VRX-03011 (1 mg/kg) significantly enhanced performance compared to that of vehicle controls. *P < 0.05 vs. vehicle controls. y P < 0.05 vs. VRX-03011 (1, 5 and 10 mg/kg). n ¼ 6 for each group. The dashed line denotes chance performance (approximately 22%) (b) Mean number of arm entries in the delayed spontaneous alternation test. All groups made comparable number of arm entries in the test session. For those rats who had an accurate cannula placement, seven were excluded from the analyses because of an insufficient number of arm entries during the behavioral test. The distribution of these rats among the treatment groups was as follows: vehicle (n ¼ 1); VRX-03011 1 mg/kg (n ¼ 3) and VRX-03011 5 mg/kg (n ¼ 3). The average absolute value of hippocampal ACh in baseline samples was 889.4 126.8 fmol per 10 ml sample for the vehicle group, 745.6 20.9 fmol per 10 ml sample for the VRX-03011 1 mg/kg group, and 601.6 72.5 fmol per 10 ml sample for the VRX-03011 5 mg/kg group; this difference was not significant: F(2,21) ¼ 1.61, P > 0.05. An ANOVA with repeated measures was employed to determine whether hippocampal ACh efflux was different among the groups across the different test samples. The analysis revealed there was a significant group effect, F(2,21) ¼ 4.95, P < 0.05, a significant effect of test, F(10,210) ¼ 64.47, P < 0.05, as well as a significant group test interaction, F(20,210) ¼ 2.03, P < 0.05. A E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573 225 200 Vehicle VRX-03011 1 mg/kg VRX-03011 5 mg/kg 175 150 125 100 75 8 16 24 Bselinea 32 40 48 56 Pre-Testing Drug Injection Percent of Baseline Hippocampal ACh Efflux (Mean ± s.e.m.) (b) 3.4. Combined treatment with VRX-03011 and the AChE-I galanthamine * 250 64 72 80 88 Testing Post-Testing Time (Min) 250 225 200 Vehicle VRX-03011 1 mg/kg VRX-03011 5 mg/kg 175 150 The interaction between suboptimal doses of VRX-03011 and the AChE-I galanthamine was tested in the delayed spontaneous alternation task and compared with a known effective dose of galanthamine (2.5 mg/kg). An ANOVA revealed a significant treatment effect on alternation scores, F(4,27) ¼ 7.82, P < 0.05 (see Fig. 5a). A post-hoc analyses revealed that the combination of VRX-03011 (0.03 mg/kg) and galanthamine (0.3 mg/kg), that were individually ineffective for enhancing memory, resulted in a significant increase in delayed alternation scores compared to that of vehicle controls (P < 0.05). Galanthamine (2.5 mg/kg) also significantly improved alternation scores compared to that of vehicle controls (P < 0.05). The enhancement observed with the combination of VRX03011 and galanthamine was similar in magnitude to that of (a) 125 70 60 100 75 8 16 24 Baseline 32 40 48 56 64 72 Resting Condition 80 88 Drug Injection Time (Min) Fig. 4. Effect of VRX-03011 treatment on hippocampal ACh efflux during delayed spontaneous alternation testing and resting conditions (a) Effect of VRX-03011 (1 and 5 mg/kg) treatment on ACh release during delayed spontaneous alternation testing. VRX-03011 at 1 and 5 significantly enhanced efflux compared to that of vehicle controls during maze testing, *P < 0.05 vs. vehicle controls. No significant differences were found during baseline, pre-test or post-test periods. (b) ACh efflux during resting condition. VRX03011 treatment did not affect ACh efflux at any point. Percent Alternation (mean ± SEM) Percent of Baseline Hippocampal ACh Efflux (Mean ± s.e.m.) (a) 569 * 40 30 20 10 0 Vehicle (b) VRX-03011 Galanthamine VRX-03011 Galanthamine 0.03 mg/kg 0.3 mg/kg 0.03 mg/kg 2.5 mg/kg + Galanthamine 0.3 mg/kg 30 25 Number of Entries (mean ± SEM) Dunnett test revealed that vehicle controls, as well as both drug groups exhibited a significant increase in hippocampal ACh efflux during the behavioral test periods compared to their respective baseline levels (P’s < 0.05). To better understand the interaction, the groups differences in ACh output were analyzed in the different conditions. A Bonferroni t-test revealed that ACh output among the groups was comparable during the baseline periods (P’s > 0.05). In contrast, VRX-03011 at 1 and 5 mg/kg significantly increased ACh output during both test samples compared to that of vehicle controls (P’s < 0.05). In the first post-test period, ACh output remained significantly elevated in the VRX-03011 5 mg/kg compared to that of vehicle controls (P < 0.05). In the final post-test sample, ACh output among the groups was comparable (P’s > 0.05). To determine the specificity of VRX-03011 treatment potentiating hippocampal ACh output during memory testing, the effects of VRX-03011 (1 and 5 mg/kg) on hippocampal ACh output was further examined while a rat remained in a holding chamber (Fig. 4b). In this non-mnemonic condition, ACh efflux in the hippocampus was no different in rats receiving vehicle or VRX-03011 (1 and 5 mg/kg) at any time point, F(2,15) ¼ 0.32, P > 0.05. * 50 * * 20 * 15 10 5 0 Vehicle VRX-03011 Galanthamine VRX-03011 Galanthamine 0.03 mg/kg 0.3 mg/kg 0.03 mg/kg 2.5 mg/kg + Galanthamine 0.3 mg/kg Fig. 5. Effects of treatment with VRX-03011 (0.03 mg/kg), galanthamine (0.3 and 2.5 mg/kg), and combination of the galanthamine (0.3 mg/kg) and VRX03011 (0.03 mg/kg) on delayed spontaneous alternation performance in a four arm cross-maze. (a) VRX-03011 at 0.03 mg/kg or galanthamine 0.3 mg/kg alone did not significantly enhance performance compared to that of vehicle controls. In contrast, VRX-03011 0.03 mg/kg and galanthamine 0.3 mg/kg significantly enhanced performance in a manner similar to that of galanthamine 2.5 mg/kg, *P < 0.05 vs. vehicle. (b) Mean number of arm entries in the delayed spontaneous alternation test. Galanthamine 2.5 mg/kg, VRX03011 0.03 mg/kg, and the combination of galanthamine 0.3 mg/kg and VRX-03011 0.03 significantly increased arm entries, *P < 0.05 vs. vehicle. E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573 570 galanthamine (2.5 mg/kg) alone, suggesting a synergistic effect of the two drugs. The number of arm entries for this experiment is illustrated in Fig. 5b. There was also a significant overall effect on arm entries, F(4,27) ¼ 9.94, P < 0.05. A post-hoc test found that galanthamine (2.5 mg/kg), VRX-03011 (0.03 mg/kg), and the combination of galanthamine (0.3 mg/kg) and VRX-03011 (0.03 mg/kg) resulted in significantly more entries than vehicle alone (P’s < 0.05). VRX-03011 resulted in a concentration-dependent weak inhibition of the 5-HT-induced contraction, consistent with an antagonist activity (IC50 ¼ 2.2 mM in ileum and IC50 ¼ 280 nM in colon). When VRX-03011 was administered to rats, doses up to 10 mg/kg had no effect on the intestinal transit compared with that of controls (62.2 1.0% of total intestinal length for 10 mg/kg VRX-03011-treated vs. 60.2 2.2% for control). 3.5. sAPPa secretion The present findings indicate that VRX-03011 is a highly selective and potent partial 5-HT4 receptor agonist with adequate brain penetration (brain [ng/g] to serum [g/ml] ratio of 0.93) in rats following intravenous administration of 1 mg/ kg. VRX-03011 did not affect performance in the no-delay condition, but produced a robust enhancement in the delayed spontaneous alternation task. Furthermore, the VRX-03011induced enhancement occurred in a dose-dependent manner. Although VRX-03011 also binds to s1 and s2 receptors, as do the 5-HT4 agonists BIMU1 and RS67333 (Lelong et al., 2003), the 5-HT4 receptor antagonist GR125487 reversed the VRX-03011-induced memory enhancement, consistent with the idea that these effects are a result of 5-HT4 receptor activation. VRX-03011 enhancement of memory performance is comparable to past findings showing that other partial 5-HT4 receptor agonists enhance short-term memory (Lamirault and Simon, 2001; Marchetti et al., 2000, 2004; Marchetti-Gauthier et al., 1997; Moser et al., 2002) and reverse memory deficits in aged rats (Moser et al., 2002). Taken together, the results suggest that activation of central 5-HT4 receptors enhances memory. One issue about the interpretation of the present results related to memory is whether VRX-03011-induced improvement in delayed spontaneous alternation is principally due to affecting mnemonic processes. The spontaneous alternation test takes advantage of rodents’ natural tendency to alternate among spatial locations (Dember and Fowler, 1958). In order for a rat to alternate in this test, a rat must always enter a different arm in consecutive sets of four arm entries. This alternation of arm entries requires a memory for recent arm choices. Consistent with the idea that this task has a mnemonic component, inserting a delay between arm choices decreases alternation scores as observed in a previous study (Ragozzino et al., 1996). Furthermore, increasing the number of arms used and thus to remember decreases alternation scores (Ragozzino et al., 1996; Walker et al., 1991). Moreover, alternations did not occur because rats exhibit a fixed response pattern, whether it be a simple response sequence, e.g. always turning right or a more complex response sequence, e.g. go straight, turn right, turn left, go straight. If this was the case, then alternation scores would be perfect or near perfect. However, in the delay task controls performed near chance performance (22.2%). In addition, even under conditions in which VRX030111 enhanced performance, alternation scores did not approach 100% nor lead rats to produce a fixed response pattern. Finally, removal of extramaze cues decreases alternation performance indicating that rats are using extramaze spatial The effect of VRX-03011 on sAPPa secretion was tested in CHO cells co-expressing the human APP695 and the human 5HT4(e) receptor (h5-HT4(e)), a neuronal h5-HT4 isoform which is one of the most highly expressed isoform in the human brain (Medhurst et al., 2001). VRX-03011 induced a concentrationdependent increase in sAPPa levels (Fig. 6a), with an EC50 w 1e10 nM. Moreover, VRX-03011 had a comparable effect on sAPPa to the full 5-HT4 receptor agonist, prucalopride, which was used at 10 mM as a positive control (Fig. 6a). A dose-dependent study has previously shown that prucalopride used at this concentration of 10 mM induced a maximal effect on sAPPa secretion in CHO cells expressing the 5-HT4(e) receptor (Lezoualc’h and Robert, 2003). In addition, VRX03011 strongly increased the level of extracellular sAPPa at native h5-HT4 receptors in IMR32 human neuroblastoma cells (Fig. 6b). 3.6. Gastrointestinal functioning To explore possible side effects related to the gastrointestinal systems, the activity of VRX-03011 in guinea pig colon and ileum as well as rat intestine was investigated. VRX03011 had no contractile effect on guinea pig ileum or colon preparations (EC50 > 10 mM). However, preincubation with (a) Prucalo pride HCl salt of VRX-03011 (M) CT 10-10 10-9 10-8 10-7 10-6 10-5 10-5 4.4 4.0 9.0 9.4 7.1 4.1 Secreted sAPPα Relative Secretion 1.0 1.4 (b) Prucalo pride HCl salt of VRX-03011 (M) CT 10-10 10-9 10-8 10-7 10-6 10-5 2.7 3.1 3.9 1.2 10-5 Secreted sAPPα Relative Secretion 1.0 1.0 1.4 2.5 Fig. 6. Effects of treatment with VRX-03011 on secretion levels of non-amyloidogenic sAPPa and Ab. Representative immunoblot showing the effects of increasing concentrations of VRX-03011 on the cellular release of sAPPa in (a) CHO cells stably expressing the h5-HT4(e) receptor isoform and (b) IMR32 neuroblastoma cells. 4. Discussion E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573 cues to guide their arm entries as opposed to using some consistent response sequence (Ragozzino and Gold, unpublished observations). These findings suggest that the spontaneous alternation task requires the use of spatial working memory. The present findings demonstrating that VRX-03011 enhances delayed spontaneous alternation may have implications for the development of novel treatments in AD. Recent reports suggest that working memory deficits are among the first cognitive impairments manifested in AD or its precursor, Mild Cognitive Impairment (MCI) (Bäckman et al., 2005). The memory test used in the behavioral experiments is analogous to some of the memory tests employed for testing AD and MCI patients, e.g. Corsi block-tapping test to assess visuospatial working memory (Gron et al., 2006; Pasquier et al., 2001). One possibility is that activation of central 5-HT4 receptors may facilitate working memory in MCI and AD. Clearly, more studies are needed to examine the effects of VRX03011 in a broader array of animal models, as well as learning and memory tests to more completely characterize the potential benefits of this novel drug in alleviating cognitive deficits. VRX-03011 treatment not only enhanced delayed spontaneous alternation performance, but also concomitantly potentiated an increase in hippocampal ACh output. Previous studies found that partial 5-HT4 agonists enhanced brain ACh output under baseline conditions (Consolo et al., 1994; Matsumoto et al., 2003). However, previous experiments did not demonstrate whether partial 5-HT4 agonists modulate brain ACh output under mnemonic demands. An important aspect concerning the pattern of the present findings is that activation of 5-HT4 receptors improved spontaneous alternation performance and increased hippocampal ACh output only under the delayed spontaneous condition but did not significantly modify ACh output in the resting condition. The lack of effect observed in the resting condition is comparable to findings showing that intracerebroventricular injections of BIMU 1 or BIMU 8 do not affect hippocampal ACh output (Consolo et al., 1994). This selective modulation of hippocampal ACh output by VRX-03011 may allow an increase in cholinergic tone that corresponds to specific cognitive demands (improving signal-to-noise), rather than a global increase in ACh induced with AChE-Is (generally increasing ‘‘gain’’ in the system) (Sarter et al., 2005). One issue related to VRX-03011 potentiating hippocampal ACh output during spontaneous alternation testing is whether the drug modulates ACh output only under mnemonic conditions that engage the hippocampal cholinergic system or also affect hippocampal ACh levels when increased pharmacologically, e.g. AChE-I treatment. The latter situation is unlikely because the perfusion solution used in the in vivo microdialysis experiment contained the AChE-I, neostigmine. Despite this, in the resting condition VRX-03011 did not increase hippocampal ACh output compared to pre-drug baseline levels. Because a single concentration of neostigmine was used in the perfusion solution, it is possible that a different concentration of neostigmine would have led to VRX-03011 potentiating hippocampal ACh efflux in a resting condition. Because neostigmine was also in the perfusion solution for the combined 571 spontaneous alternation and in vivo microdialysis test, another possibility is that the combination of an AChE-I with a memory test that activates the hippocampal cholinergic system led to a partial 5-HT4 agonist augmenting ACh efflux. However, the present results are consistent with previous results demonstrating that agents which concomitantly enhance hippocampal ACh output and spontaneous alternation performance do not affect hippocampal ACh output in a resting condition even when using a range of different concentrations of neostigmine in the perfusion solution (Ragozzino et al., 1996, 1998). Thus, a parsimonious interpretation of the results is that VRX-03011 administration potentiated hippocampal ACh output during a memory test that engages the hippocampal cholinergic system. In the future, this question can be further addressed by examining systemic injections of an AChE-I and VRX-03011 on hippocampal ACh output in a resting condition without using a AChE-I in the perfusion solution. At a behavioral level, the present experiment did observe an interaction between an AChE-I and a partial 5-HT4 agonist. More specifically, suboptimal doses of VRX-03011 and galanthamine co-administered enhanced delayed spontaneous alternation performance. At present, AChE-Is are the most common treatment for dementia. This treatment approach has limited effects in reducing the cognitive deficits associated with AD (Winblad and Jelic, 2004). One possible reason for this may be that AChE-Is cause a global increase in ACh release (generally increasing ‘‘gain’’ in the system) rather than specifically improving the signal-to-noise ratio (Sarter et al., 2005). There is clearly a need for novel approaches in alleviating cognitive impairments in dementia. Because activation of 5-HT4 receptors modulates ACh output and may have neuroprotective effects, treatments that target central 5-HT4 receptors may prove beneficial in alleviating the severe mnemonic deficits found in AD and other types of dementia. The selective enhancement of ACh output during a mnemonic condition suggests that VRX-03011 activation of 5-HT4 receptors may be an efficacious strategy for improving cholinergic function. In addition, combining low doses of 5-HT4 agonists with low doses of AChE-Is may allow greater flexibility in the treatment of AD. Titrating doses of VRX-03011 and galanthamine may allow adjustments of both signal-to-noise ratios through 5-HT4 receptor activation and increasing gain through AChE-Is, maximizing therapeutic effectiveness in AD patients. Furthermore, AChE-Is induce secondary peripheral effects, such as nausea, vomiting, and muscle cramping (Cummings, 2003). It is possible that a low dose of VRX-03011 combined with a low dose of an AChE-I may result in less serious side effects than a therapeutic dose of either drug alone, although this was not examined in the present study. In the experiment investigating the combination of VRX03011 and galanthamine, some groups exhibited an increase in the number of arm entries during spontaneous alternation testing. Unclear is why a low dose of VRX-03011 enhanced arm entries in this study. Importantly, there is not a relationship in arm entries and spontaneous alternation performance as some treatments did not enhance memory but increased arm entries while other treatments enhanced memory and also 572 E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573 increased arm entries. This can even be observed with the same drug treatment. For example, in the case of VRX-03011 0.03 mg treatment, this dose enhanced arm entries when administered alone or in combination with galanthamine 0.3 mg. When VRX-03011 0.3 mg was administered alone it had no effect on alternation scores and when administered with galanthamine 0.3 mg it enhanced alternation scores. Moreover, this lack of relationship between alternation performance and number of arm entries is consistent with a large series of experiments that examined a wide variety of drugs on spontaneous alternation (Ragozzino and Gold, 1991; Walker et al., 1991). Another important finding supporting the potential therapeutic utility of VRX-03011 is that the drug exhibited possible neuroprotective activity by increasing secretion of the non-amyloidogenic sAPPa at native and recombinant 5-HT4 receptors. VRX-03011 had no effect on sAPPa in CHO cells expressing only the human APP695 receptor (data not shown). However, in CHO cells co-expressing the human APP695 and the human 5-HT4(e) receptor, VRX03011 induced a dose-dependent increase in sAPPa. This suggests that reduction of sAPPa by VRX-03011 is mediated by the 5-HT4(e) receptor. Non-amyloidogenic secreted sAPPa has potent neuroprotective functions against neurotoxic insults such as glutamate and Ab. Furthermore, sAPPa promotes neurite outgrowth, regulates neuronal excitability and has memory-enhancing effects in animal models of dementia (Bour et al., 2004; Lezoualc’h and Robert, 2003). These possible neuroprotective actions suggest that VRX-03011 may be a useful new treatment for AD. As a next step, it will be interesting to test whether VRX-03011 may slow down disease progression in transgenic animal models of AD. Interestingly, it has been recently shown that 5-HT4 may increase sAPPa in a transgenic mouse line over-expressing the ‘‘London’’ mutant of human APP (Cachard-Chastel et al., 2007). One last important consideration for potential AD treatments is the absence of adverse peripheral actions. The human 5-HT4 receptor is expressed in the brain as well as the gastrointestinal system. Therefore, potential therapeutic limitations of 5-HT4 receptor agonists come from possible adverse drug reactions involving gastrointestinal organs (Briejer et al., 2001). VRX-03011 had no agonist effects on the activity of the guinea pig colon and ileum or rat intestine. These data suggest VRX-03011 is a highly selective partial 5-HT4 receptor agonist that does not affect gastrointestinal functions. Overall, the findings suggest that VRX-03011 has a unique set of properties that could provide both symptomatic relief of cognitive impairments as well as possible neuroprotective therapeutic effects in patients with AD in the absence of adverse gastrointestinal actions. Acknowledgments This work was supported by Epix Pharmaceuticals, Inc. S.S., S.N., J.R., and Y.M. are employees of Epix Pharmaceuticals, which conducted tests of gastrointestinal functioning. CEREP (Paris, France) was contracted by Predix Pharmaceuticals (Currently Epix Pharmaceuticals) for radioligand binding assays. Work examining splice variants of the 5HT4 Receptor was completed by A.D. and J.B. Spontaneous alternation testing and in vivo microdialysis studies were completed by E.G.M., P.E.G., and M.E.R. Measurement of sAPPa was completed by F.L., S.R., and M.G. E.G.M. is currently employed by Abbott Laboratories. Abbott Laboratories is not affiliated with this work in any way. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neuropharm.2007.06.016. References Ansanay, H., Sebben, M., Bockaert, J., Dumuis, A., 1996. Pharmacological comparison between [3H]GR 113808 binding sites and functional 5-HT4 receptors in neurons. Eur. J. Pharmacol. 298, 165e174. Bäckman, L., Jones, S., Berger, A.-K., Laukka, E.J., Small, B.J., 2005. Cognitive impairment in preclinical Alzheimer’s disease: a meta-analysis. Neuropsychology 19, 520e531. Bockaert, J., Claeysen, S., Compan, V., Dumuis, A., 2004. 5-HT4 receptors. Curr. Drug Targets 3, 39e51. Bour, A., Little, S., Dodart, J.C., Kelche, C., Mathis, C., 2004. A secreted form of the beta-amyloid precursor protein (sAPP695) improves spatial recognition memory in OF1 mice. Neurobiol. Learn. Mem. 81, 27e38. Briejer, M.R., Prins, N.H., Schuurkes, J.A., 2001. Effects of the enterokinetic prucalopride (R093877) on colonic motility in fasted dogs. Neurogastroenterol. Motil. 13, 465e472. Cachard-Chastel, M., Lezoualc’h, F., Dewachter, I., Croes, S., Devijver, H., Langlois, M., Van Leuven, F., Sicsic, S., Gardier, A.M., 2007. 5-HT4 receptor agonists increase sAPPa levels in the cortex and hippocampus of male C57BL/6j mice. Br. J. Pharmacol. 150, 883e892. Chang, Q., Savage, L.M., Gold, P.E., 2006. Microdialysis measures of functional increases in ACh release inthe hippocampus with and without inclusion of acetylcholinesterase inhibitors in the perfusate. J. Neurochem. 97, 697e706. Cho, S., Hu, Y., 2006. Activation of 5-HT4 receptors inhibits secretion of b-amyloid peptides and increases neuronal survival. Exp. Neurol. 203, 274e278. Claeysen, S., Sebben, M., Bécamel, C., Bockaert, J., Dumuis, A., 1999. Novel brain-specific 5-HT4 receptor splice variants show marked constitutive activity: Role of the C-terminal intracellular domain. Mol. Pharmacol. 55, 910e920. Consolo, S., Arnaboldi, S., Giorgi, S., Russi, G., Ladinsky, H., 1994. 5-HT4 receptor stimulation facilitates ACh release in rat frontal cortex. Neuroreport 5, 1230e1232. Cummings, J.L., 2003. Use of AChE-Is in clinical practice: evidence-based recommendations. Am. J. Geriatr. Psychiatry 11, 131e145. Dember, W.N., Fowler, H., 1958. Spontaneous alternation behavior. Psych. Bull. 55, 412e428. Frankfort, S.V., Appels, B.A., De Boer, A., Tulner, L.R., Van Campen, J.P., Koks, C.H., Beijnen, J.H., 2006. Treatment effects of rivastigmine on cognition, performance of daily living activities and behaviour in Alzheimer’s disease in an outpatient geriatric setting. Int. J. Clin. Pract. 60, 646e654. Gale, J.D., Grossman, C.J., Whitehead, J.W., Oxford, A.W., Bunce, K.T., Humphrey, P.P., 1994. GR113808: a novel, selective antagonist with high affinity at the 5-HT4 receptor. Br. J. Pharmacol. 111, 332e338. Garcia-Alloza, M., Gil-Bea, F.J., Diez-Ariza, M., Chen, C.P., Francis, P.T., Lasheras, B., Ramirez, M.J., 2005. Cholinergic-serotonergic imbalance contributes to cognitive and behavioral symptoms in Alzheimer’s disease. Neuropsychologia 43, 442e449. E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573 Gron, G., Brandenburg, I., Wunderlich, A.P., Riepe, M.W., 2006. Inhibition of hippocampal function in mild cognitive impairment: targeting the cholinergic hypothesis. Neurobiol. Aging. 27, 78e87. Ho, G.J., Hansen, L.A., Alford, M.F., Foster, K., Salmon, D.P., Galasko, D., Thal, L.J., Masliah, E., 2002. Age at onset is associated with disease severity in Lewy body variant and Alzheimer’s disease. Neuroreport 13, 1825e1828. Hodges, J.R., Salmon, D.P., Butters, N., 1992. Semantic memory impairment in Alzheimer’s disease: Failure of access or degraded knowledge? Neuropsychologia 30, 301e314. Hyman, B.T., Van Horsen, G.W., Damasio, A.R., Barnes, C.L., 1984. Alzheimer’s disease: cell-specific pathology isolates the hippocampal formation. Science 225, 1168e1170. Lai, M.K., Tsang, S.W., Francis, P.T., Esiri, M.M., Hope, T., Lai, O.F., Spence, I., Chen, C.P., 2003. [3H]GR113808 binding to serotonin 5HT(4) receptors in the postmortem neocortex of Alzheimer disease: a clinicopathological study. J. Neural Transm. 110, 779e788. Lamirault, L., Simon, H., 2001. Enhancement of place and object recognition memory in young adult and old rats by RS 67333, a partial agonist of 5-HT4 receptors. Neuropharmacology 41, 844e853. Lamirault, L., Guillou, C., Thal, C., Simon, H., 2003. Combined treatment with galanthaminium bromide, a new cholinesterase inhibitor, and RS 67333, a partial agonist of 5-HT4 receptors, enhances place and object recognition in young adult and old rats. Prog. Neuropsychopharmacol. Biol. Psychiatry 21, 185e195. Lelong, V., Lhonneur, L., Dauphin, F., Boulouard, M., 2003. BIMU 1 and RS 7333, two 5-HT4 receptor agonists, modulate spontaneous alternation deficits induced by scopolamine in the mouse. Nauyn-Schmiedebergs Arch. Pharmacol. 367, 621e628. Lezoualc’h, F., Robert, S.J., 2003. The serotonin 5-HT4 receptor and the amyloid precursor protein processing. Exp. Gerontol. 38, 159e166. Maillet, M., Robert, S.J., Lezoualc’h, F., 2004. New insights into serotonin 5-HT4 receptors: a novel therapeutic target for Alzheimer’s disease? Curr. Alzheimer Res. 1, 79e86. Marchetti-Gauthier, E., Roman, F.S., Dumuis, A., Bockhaert, J., SoumireuMourat, B., 1997. BIMU 1 increases associative memory in rats by activating 5-HT4 receptors. Neuropharmacology 36, 697e706. Marchetti, E., Dumuis, A., Bockaert, J., Soumireu-Mourat, B., Roman, F.S., 2000. Differential modulation of the 5-HT4 receptor agonists and antagonists on rat learning and memory. Neuropharmacology 39, 2017e2027. Marchetti, E., Chaillan, F.A., Dumuis, A., Bockaert, J., Soumireu-Mourat, B., Roman, F.S., 2004. Modulation of memory processes and cellular excitability in the dentate gyrus of freely moving rats by a 5-HT4 receptors partial agonist, and an antagonist. Neuropharmacology 47, 1021e1035. Matsumoto, M., Togashi, H., Mori, K., Ueno, K., Ohashi, S., Kojima, T., Yoshioka, M., 2001. Evidence for involvement of central 5-HT(4) receptors in cholinergic function associated with cognitive processes: behavioral, electrophysiological, and neurochemical studies. J. Pharmacol. Exp. Ther. 296, 676e682. Medhurst, A.D., Lezoualc’h, F., Fischmeister, R., Middlemiss, D.N., Sanger, G.J., 2001. Quantitative mRNA analysis of five C-terminal splice-variants of the human 5-HT4 receptor in the central nervous system by TaqMan real time RT-PCR. Mol. Brain Res. 90, 125e134. Meltzer, C.C., Smith, G., DeKosky, S.T., Pollock, B.G., Mathis, C.A., Moore, R.Y., Kupfer, D.J., Reynolds 3rd, C.F., 1998. Serotonin in aging, 573 late-life depression, and Alzheimer’s disease: the emerging role of functional imaging. Neuropsychopharmacology 18, 407e430. Mialet, J., Berque-Bestel, I., Sicsic, S., Langlois, M., Fischmeister, R., Lezoualc’h, F., 2000. Pharmacological characterization of the human 5HT4(d) receptor splice variant stably expressed in chinese hamster ovary cells. Br. J. Pharmacol. 131, 827e835. Moser, P.C., Bergis, O.E., Jegham, S., Lochead, A., Duconseille, E., Terranova, J.P., Caille, D., Berque-Bestel, I., Lezoualc’h, F., Fischmeister, R., Dumuis, A., Bockaert, J., George, P., Soubrie, P., Scatton, B., 2002. SL65.0155, a novel 5hydroxytryptamine4 receptor partial agonist with potent cognition-enhancing properties. J. Pharmacol. Exp. Ther. 302, 731e741. Pasquier, F., Grymonprez, L., Lebert, F., van der Linden, M., 2001. Memory impairment differs in frontotemporal dementia and Alzheimer’s disease. Neurocase 7, 161e171. Ragozzino, M.E., Gold, P.E., 1991. Glucose effects on mecamylamine-induced memory deficits and decreases in locomotor activity in mice. Behav. Neural Biol. 56, 271e282. Ragozzino, M.E., Unick, K., Gold, P.E., 1996. Hippocampal acetylcholine release during memory testing: Augmentation by glucose. Proc. Natl. Acad. Sci. USA 93, 4693e4698. Ragozzino, M.E., Pal, S.N., Unick, K., Stefani, M.R., Gold, P.E., 1998. Modulation of hippocampal acetylcholine release and of memory by intrahippocampal glucose injections. J. Neurosci. 18, 1595e1601. Reynolds, G.P., Mason, S.L., Meldrum, A., De Keczer, S., Parnes, H., Eglen, R.M., Wong, E.H., 1995. 5-Hydroxytryptamine (5-HT)4 receptors in post-mortem human brain tissue: distribution, pharmacology and effects of neurodegenerative diseases. Br. J. Pharmacol. 114, 993e998. Robert, S.J., Zugaza, J.L., Fischmeister, R., Gardier, A.M., Lezoualc’h, F., 2001. The human serotonin 5-HT4 receptor regulates secretion of nonamyloidogenic precursor protein. J. Biol. Chem. 276, 44881e44888. Sarter, M., Hasselmo, M.E., Bruno, J.P., Givens, B., 2005. Unraveling the attentional functions of cortical cholinergic inputs: interactions between signal-driven and cognitive modulation of signal detection. Brain Res. Rev. 48, 98e111. Venneri, A., McGeown, W.J., Shanks, M.F., 2005. Empirical evidence of neuroprotection by dual cholinesterase inhibition in Alzheimer’s disease. Neuroreport 16, 107e110. Walker, D.L., McGlynn, T., Grey, C., Ragozzino, M., Gold, P.E., 1991. Naloxone modulates the behavioral effects of cholinergic agonists and antagonists. Psychopharmacology 105, 57e62. Westerink, B.H.C., 1995. Brain microdialysis and its application for the study of animal behaviour. Behav. Brain Res. 70, 103e124. Wilcock, G.K., Esiri, M.M., Bowen, D.M., Smith, C.C.T., 1982. Alzheimer’s disease: correlation of cortical choline acetyltransferase activity with the severity of dementia and histological abnormalities. J. Neurol. Sci. 57, 407e417. Winblad, B., Jelic, V., 2004. Long-term treatment of Alzheimer disease: efficacy and safety of acetylAChE-Is. Alzheimer Dis. Assoc. Disord. 18 (Suppl. 1), S2eS8. Wong, E.H.F., Reynolds, G.P., Bonhaus, D.W., Hsu, S., Eglen, R.M., 1996. Characterization of [3H]GR 113808 binding to 5-HT 4 receptors in brain tissues from patients with neurodegenerative disorders. Behav. Brain Res. 73, 249e252. Wooltorton, E., 2004. Tegaserod (Zelnorm) for irritable bowel syndrome: reports of serious diarrhea and intestinal ischemia. Can. Med. Assoc. J. 170, 1908.