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NEUROPHARMACOLOGY

The Serotonin_1A Receptor Partial Agonist S15535 [4-(Benzodioxan-5-yl)1-(indan-2-yl)piperazine] Enhances Cholinergic Transmission and Cognitive Function in Rodents: A Combined Neurochemical and Behavioral Analysis

Departments of Psychopharmacology (M.J.M., A.G., B.D.C., J.M.R., A.D.) and Cerebral Pathology (P.L.), Institut de Recherches Servier, Paris, France; Porsolt & Partners Pharmacology (S.R., R.P.), Boulogne-Billancourt, France; Pharmacobiol (A.M.), Centro de Investigación y Estudios Avanzados, Mexico City, Mexico; Istituto di Ricerche Farmacologiche "Mario Negri" (M.C.), Milan, Italy; Université de Bordeaux 1-Centre National de la Recherche Scientifique-Unité de Recherche Associée 339 (R.J.), Laboratoire de Neurosciences Comportementales et Cognitives, Talence cedex, France; Institut de Recherches Internationales Servier (E.M.), Courbevoie cedex, France; and Institut de Recherches Servier (J.L.P.), Suresnes, France

Received April 7, 2004; accepted May 13, 2004.

	Abstract

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These studies examined the influence of the selective 5-hydroxytryptamine (serotonin) (5-HT)_1A receptor partial agonist S15535 [4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine] upon cholinergic transmission and cognitive function in rodents. In the absence of acetylcholinesterase inhibitors, S15535 dose-dependently (0.04–5.0 mg/kg s.c.) elevated dialysis levels of acetylcholine in the frontal cortex and dorsal hippocampus of freely moving rats. In the cortex, the selective 5-HT_1A receptor antagonist WAY100,635 [(N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclo-hexanecarboxamide) fumarate] dose-dependently (0.0025–0.63) blocked this action of S15535. By contrast, in dorsal hippocampus, WAY100,635 mimicked the induction of acetylcholine release by S15535. In a social recognition paradigm, S15535 dose-dependently (0.16–10.0) improved retention, an action blocked by WAY100,635 (0.16), which was ineffective alone. Furthermore, S15535 dose-dependently (0.04–2.5) and WAY100,635 reversibly abolished amnesic properties of the muscarinic antagonist scopolamine (0.63) in this procedure. Cognitive deficits provoked by scopolamine in autoshaping and Morris water-maze procedures were likewise blocked by S15535 at doses of 0.63 to 10.0 and 0.16 to 2.5, respectively. In a two-platform spatial discrimination task, in which S15535 similarly abrogates cognitive deficits elicited by scopolamine, injection of S15535 (1.0 and 10.0 µg) into dorsal hippocampus blocked amnesic effects of the 5-HT_1A agonist 8-hydroxy-2-dipropylaminotetralin (0.5 µg). Finally, S15535 (0.16–0.63) improved performance in a spatial, delayed nonmatching to sample model in mice, and in an operant delayed nonmatching to sample model in old rats, S15535 (1.25–5.0 mg/kg p.o.) increased response accuracy and reduced latency to respond. In conclusion, S15535 reinforces frontocortical and hippocampal release of acetylcholine and displays a broad-based pattern of procognitive properties. Its actions involve both blockade of postsynaptic 5-HT_1A receptors and engagement of 5-HT_1A autoreceptors.

However, of particular importance to the influence of 5-HT_1A receptors upon learning and memory is their complex interrelationship with cholinergic networks (Cassel and Jeltsch, 1995; Steckler and Sahgal, 1995), which fulfill a crucial role in cognitive-attentional processes (Sarter and Bruno, 1997; Terry and Buccafusco, 2003). At the postsynaptic level, stimulation of 5-HT_1A receptors on pyramidal cells in the hippocampus interferes with the excitatory actions of colocalized muscarinic receptors (Claustre et al., 1988). Furthermore, inhibitory postsynaptic 5-HT_1A receptors are localized on cholinergic cell bodies in the medial septum and diagonal band of Broca, which innervate the hippocampus, and on cholinergic perikarya of the nucleus basalis of Meynert, which project to the FCX (Van den Hooff and Galvan, 1992; Kia et al., 1996). Concerning 5-HT_1A autoreceptors, their activation may indirectly enhance corticolimbic release of acetylcholine (ACh) via relief of a tonic, inhibitory influence of serotonergic pathways upon cholinergic projections (Steckler and Sahgal, 1995). Nevertheless, the precise influence of 5-HT_1A receptor ligands upon cholinergic transmission in vivo remains unclear, not least because neurochemical studies have generally been performed in the presence of acetylcholinesterase inhibitors (AChEIs) that modify drug actions (Fujii et al., 1997; Bruno et al., 1999; Ichikawa et al., 2002; Gobert et al., 2003; Herzog et al., 2003).

Behavioral studies provide complementary evidence for a complex role of 5-HT_1A receptors, in interaction with cholinergic pathways, in the control of memory and attention. For example, under certain conditions high-efficacy 5-HT_1A receptor agonists mimic and reinforce the negative influence of muscarinic antagonists upon learning and memory (Steckler and Sahgal, 1995; Meneses, 1999; Winsauer et al., 1999), actions reflecting stimulation of postsynaptic 5-HT_1A receptors in hippocampus (Carli et al., 1995a; Ohno and Watanabe, 1996), FCX (Edagawara et al., 1998), septum (Bertrand et al., 2000), and amygdala (Liang, 1999). Evidence has also been acquired that engagement of hippocampal 5-HT_1A receptors compromises cognitive performance in humans (Yasuno et al., 2003). In contrast, blockade of postsynaptic 5-HT_1A receptors can relieve learning and retention deficits elicited by muscarinic antagonists (Carli et al., 1995a, 1999; Misane and Ogren, 2003; Pitsikas et al., 2003). Although the role of presynaptic 5-HT_1A receptors remains comparatively unclear, under certain conditions their stimulation may favor memory formation (Cole et al., 1994; Carli et al., 2000; see Discussion).

The above-mentioned observations encourage particular interest in low-efficacy 5-HT_1A receptor agonists, which, reflecting differential sensitivity, activate and block pre- and postsynaptic 5-HT_1A receptors, respectively (Millan et al., 2000). Correspondingly, the aim of the present work was to characterize the influence of S15535, a selective partial agonist at 5-HT_1A receptors (Millan et al., 1994, 1997a,b; Newman-Tancredi et al., 1999, 2003), upon cholinergic transmission and cognitive-attentional function in rodents. First, using a dialysis procedure not requiring the use of AChEIs (Ichikawa et al., 2002; Gobert et al., 2003), we evaluated the influence of S15535 upon extracellular levels of ACh in the FCX and dorsal hippocampus (DH). Second, we examined the influence of S15535 upon cognitive function in a broad array of behavioral models (Table 1). Third, in certain procedures, we examined the actions of WAY100,635 that share antagonist properties with S15535 at postsynaptic 5-HT_1A receptors but that block its agonist actions at 5-HT_1A autoreceptors. Antagonism of the effects of S15535 would suggest a role of presynaptic 5-HT_1A receptors in its effects. Furthermore, in one study, S15535 was microinjected into the DH in association with the 5-HT_1A agonist 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT), to directly evaluate a potential postsynaptic locus of activity. Finally, because S15535 blocks the amnesic actions of scopolamine in a two-platform spatial discrimination (TPSD) task in rats (Carli et al., 1999), in several other paradigms we likewise determined the influence of S15535 upon the amnesic properties of scopolamine.

	Materials and Methods

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Animals. The experiments were conducted on male rats or mice kept under standard laboratory conditions. They are housed in sawdust-lined cages with, unless otherwise specified below, unrestricted access to standard chow and water. Laboratory temperature was 21 ± 1°C and humidity was 60 ± 5%. There was a 12-h light/dark cycle (lights on at 7:00–8:00 AM). All animal use procedures conformed to international European ethical standards and to (86/609-EEC) decrees of the French National Committee (décret 87/848), the U.S. National Research Council, and the National Institutes of Health for the care and use of laboratory animals.

Dialysis and Chromatographic Procedures. Experiments were carried out on Wistar rats (Iffa Credo, l'Arbresle, France) weighing 225 to 250 g at the time of surgery, which was performed under pentobarbital anesthesia (60 mg/kg i.p.). As described previously (Gobert et al., 2003), rats were mounted in a Kopf stereotaxic frame, and a single guide cannulae (CMA/11) was implanted in the FCX or DH with coordinates for FCX being AP, +2.2; L, 0.6; and DV, –0.2 and for DH being AP, –3.8; L, 2.0; and DV, –2.0. Rats were singly housed and allowed to recover for 5 days before dialysis. On the day of dialysis, a cuprophan CMA/11 probe (4 mm in length for the FCX and 2 mm in length for the DH, 0.24-mm o.d.) was slowly lowered into position. It was perfused at 1 µl/min with a phosphate-buffered solution of 147.2 mM NaCl, 4 mM KCl, and 2.3 mM CaCl₂ at pH 7.3. Two hours after implantation, 20-min dialysate samples were collected for 3 h. Three basal samples were collected before drug administration. In the antagonist studies, WAY100,635 or vehicle was injected 20 min before S15535 or vehicle. Furthermore, the actions of the muscarinic antagonist scopolamine were examined and, in one study, S15535 was administered before scopolamine. ACh was quantified in the absence of AChEIs, essentially as described by Gobert et al. (2003). Twenty-microliter dialysate samples were collected on 10 µl of acetic acid (0.01%). Twenty-microliter aliquots were then analyzed by high-performance liquid chromatography. The mobile phase was composed of 50 mM Na₂HPO₄ and 0.5% proclin (BAS, Congleton, UK), adjusted to pH 8.2 with H₃PO₄. The stationary phase was comprised of a cation ion exchanger (Sepstik, 530 x 1 mm, particle size 10 µm; BAS), a precolumn (preimmobilized enzyme reactor, 55 x 1 mm) of choline oxydase/catalase (BAS), and a postcolumn (postimmobilized enzyme reactor, 50 x 1 mm) of choline oxydase/ACh esterase (BAS) maintained at 35°C. An amperometric detector (BAS LC-4B) was used for quantification. The electrode was set at +100 mV versus Ag/AgCl. The glassy carbon electrode (MF2098; BAS) was coated with a peroxidase-redox polymer. The mobile phase was delivered at a flow rate of 0.14 ml/min. The sensitivity of the assay for ACh was 0.1 pg (= 0.55 fmol) (injected in a volume of 20 µl). The influence of drugs and vehicle was expressed relative to basal values (defined as 100%). Data were analyzed by analysis of variance (ANOVA) with sampling time as the repeated within-subject factor.

Social Recognition Test. The test was adapted from the procedure described by Perio et al. (1989). Experiments were carried out on Wistar rats (Elevage Janvier, le Genest-Saint-Isle, France) weighing 240 to 260 g. Juvenile Wistar rats, 25 to 30 days old, were used as a social stimulus. Adult rats were individually housed for 2 days before testing. On the test day, each adult was placed in its home cage on the observation table. After 5-min habituation to the new environment, a juvenile was introduced into the home cage for a first 5-min session. In the "time-induced retention deficit" variant of the procedure, a second 5-min session was conducted 120 min later with introduction of the same juvenile (for evaluation of the promnesic influence of S15535) or a different juvenile (to control for the specificity of its actions). S15535 was administered 1 min after the end of the first 5-min session. In the antagonist study, WAY100,635 or vehicle was administered immediately before S15535 or vehicle. In the "scopolamine-induced amnesia" procedure, the second 5-min session immediately followed the first one and likewise with the same juvenile. S15535, WAY100,635, or vehicle was administered 45 min before testing, and scopolamine (0.63 mg/kg s.c.) or vehicle was administered 30 min before testing. The influence of WAY100,635 upon the interaction between S15535 and scopolamine was studied with administration of the drugs (or vehicle) 60, 45, and 30 min before testing, respectively. In all procedures, an observer unaware of drug treatment recorded the total duration of social behavior (i.e., the time spent by the adult rat primarily in sniffing, but also in after biting, jumping, or crawling over or under the juvenile) during the first (T1) and second (T2) sessions. Adults displaying T1 values of <60 s were not examined further. The data analyzed were the difference in active social behavior between the two sessions (T2–T1). The dose-response curve for S15535 was analyzed by one-way ANOVA followed by Dunnett's test. In antagonism studies, and in the "time-induced retention deficit" procedure, data were analyzed by two-way ANOVA followed by Newman-Keuls test.

Scopolamine-Induced Amnesia in the Morris Water-Maze Test. Wistar rats (Elevage Janvier), weighing 180 to 220 g, were used for these experiments. They were housed in groups of five or six. The method used follows that described by Porsolt et al. (1995). The Morris water-maze consisted of a circular water tank (150 cm in diameter) filled with water (27°C) and with an escape platform (15 cm in diameter) located 18 cm from the perimeter, always in the same position 2 cm beneath the surface of the water. Water was made opaque by addition of milk powder, thereby rendering the platform invisible. The animals were given a single training session that consisted of four consecutive trials in the water-maze separated by 1 min. For each trial, the animal was placed in the maze at one of two different starting points equidistant from the escape platform and allowed to find the escape platform where it was left for 60 s before starting a new trial. If the escape platform was not found within 120 s, the rat was placed on it for 60 s by the experimenter. S15535 or vehicle was given 60 min before the session. Scopolamine (0.5 mg/kg i.p.) or vehicle was administered 30 min before the session. The time (in seconds) required to find the escape platform (escape latency) for each trial was measured. Data were analyzed by comparing treated groups with appropriate control groups using unpaired t tests.

Two-Platform Discrimination Task in a Water-Maze. Experiments were performed on singly housed, Sprague-Dawley rats (CD-COBS; Charles River, Italica, Calco, Italy) of 200- to 250-g weight. As described previously (Carli et al., 1999), animals were anesthetized with equithesin (3 ml/kg), and cannulae were implanted into the CA1 region of the DH at AP, +5.2; L, ±2.0; and DV, +7.3 mm relative to the interaural zero point. Guide cannulae of 23-gauge stainless steel were bilaterally placed 2 mm above the injection site coordinates, and, for testing, bilateral injection units were inserted (30-gauge stainless steel tubing), which terminated 2 mm below the tip of the guide cannulae. Drugs or vehicle was delivered by a CMA/100 perfusion pump (CMA Microdialysis, Stockholm, Sweden) connected via PP10 tubing to the cannula. The flow rate was 0.5 µl/min and the volume infused was 1 µl. For behavioral testing, a circular pool of 1.5-m diameter and 0.5-m height was used. It was filled to a depth of 0.29 m with water (26°C) rendered opaque by addition of a food dye. It was located in the middle of a room containing several visual cues on the walls. Two visible square (11 x 11 cm) platforms projected 2.0 cm above the water surface of the pool. Both platforms were made of gray Perspex. One was fixed; the other (sinkable) was filled with expanded polystyrene and anchored by a thread to a solid movable base on the bottom of the pool. Rats were trained for 8 days (10 trials a day) to swim to the fixed (correct) escape platform. The floating (incorrect) platform was positioned across successive trials in a random sequence of eight locations around the pool, whereas the fixed (correct) platform was always at the center of one of the eight quadrants. At the beginning of the trial, rats were placed in the pool at one of eight possible randomized starting locations. The trial terminated when the rat escaped onto the rigid platform. Intertrial intervals were 2 to 4 min, and daily sessions lasted approximately 30 min. A correct trial was when the rat escaped onto the rigid platform without touching the floating platform. If the rat did not choose either the correct or incorrect platform within 60 s, the trial was stopped and an omission error recorded. The first choice in each trial was recorded as correct or incorrect. Testing took place at the end of the 8-day water-maze training period. S15535 (1.0 or 10.0 µg) or vehicle, and 8-OH-DPAT (5.0 µg) or vehicle, were infused 15 and 10 min, respectively, before testing. Choice accuracy of spatial discrimination was measured as the proportion of correct choices (total correct choices/total correct choices + total incorrect choices). Significant differences in the proportions of correct choices among various treatments were examined by two-way ANOVA followed by Tukey's test.

Operant, Delayed Nonmatching to Sample Procedure. Wistar rats (Elevage Janvier), 19 months old (627–660 g), were housed two per cage and maintained with restricted access to food (15 g of UAR 113 per day) during the course of the experiments. Young controls were 2 months old (mean weight 264 g) at the beginning of the experiment. As described previously (Porsolt et al., 1995), the experiments were conducted in standard, soundproof operant chambers for rats (30 x 25 x 30 cm; Coulbourn Instruments, Lehigh Valley, PA) fitted with a white house light, one or two retractable levers, and a food pellet (45-mg) dispenser. In the one-lever configuration, the lever was located centrally above the food tray. In the two-lever configuration, the two levers were located at the same level on either side of the food tray. The chambers were connected to a programming system (MED.PC; MED Associates, East Fairfield, VT) that controlled the experiments and collected the data automatically. The animals were initially submitted to 13 lever-pressing acquisition sessions (one per day), according to a fixed ratio 1 schedule of reinforcement, first using the central lever (seven sessions), and second, with the right or left lever presented in a pseudorandom order (six sessions). Then, the animals were submitted to 10 delayed nonmatching to sample (DNMTS) sessions over two consecutive weeks. During this phase, the chambers were fitted with the two-lever configuration, and each session consisted of 35 successive trials separated by a 10-s intertrial interval. A trial started with the presentation of the left or right lever. A response on this lever resulted in the retraction of the lever, the delivery of a food pellet and the onset of a 5-s delay. If the animal did not press the lever within 20 s (one-lever omission), the lever was retracted and a new lever presentation occurred 5 s later. After expiration of the 5-s delay, the two levers were inserted into the chamber. The animals received a food pellet only if they pressed the lever not previously presented 5 s (DNMTS). Incorrect responses (presses on the same lever) were not reinforced. Both correct and incorrect responses were followed by retraction of the two levers and onset of the next intertrial interval (10 s). If the animal did not press a lever within 20 s (two-lever omission), the levers were retracted and the next intertrial started. Sessions continued until 35 choice responses between the two levers were completed or 30 min had elapsed. S15535 (aged rats) or vehicle (aged and young rats) was administered p.o. 60 min before each session. Two additional administrations were performed on the weekend between the 2 weeks of DNMTS sessions. Data analyzed were the percentage of correct responses (response accuracy) and choice latency to respond. They were pooled week by week. Dose effects were analyzed for each week by ANOVA followed by Dunnett's test.

Spatial, Delayed Nonmatching-to-Sample Test in Mice. C57BL/6 mice of approximately 30 g were housed in individual cages 2 weeks before the study. Access to food was restricted to maintain body weight at 88% of free-feeding values. As described previously (Guillou et al., 1993), the apparatus consisted of an eight-arm radial maze comprising a central platform (diameter 30 cm) from which radiated eight arms (50 x 11 cm). Access to each arm was controlled by a gate. After a 2-day (days 1 and 2) adaptation period to the apparatus, mice were tested daily during 16-day chronic treatment (s.c.) with S15535 or vehicle administered 30 min before each session. From day 3 to day 9, animals learnt the basic "simple rule" of the task during one daily session of eight trials separated by 1-min intervals. At each trial, the mouse was presented two arms, for example, 1 and 6, by the successive opening of the corresponding gates. Subsequently, each of these two arms was again presented but with the simultaneous opening of an adjacent, nonpreviously visited gate; for example, 1 and 2 and then 5 and 6. Each time, the animal had to select the arm that had not been visited previously: in this case 2 and then 6. For each session (eight trials), there were 16 possible correct responses. Mice were required to respond correctly at a level of at least 13/16 over two consecutive sessions to progress to the next phase. In the "sequenced" procedure of training, three additional arms were interposed between presentation and recognition of the arm to memorize. For example, the presentation and visit of arm 8 was followed by arms 4, 1, and 6 before choosing between arms 7 and 8. During the first 3 days, mice were submitted to eight trials of this type with, in addition, four trials where only one arm was interposed (simple rule procedure). Thereafter, for three more days, the same procedure was applied, but five arms were interposed between the presentation of the arm to memorize and its recognition. Finally, in the "mixed" procedure of testing (over 3 days), animals were confronted with a random sequence of trials whereby either one, three, or five arms were interposed between the initial arm shown and the final choice. There were eight trials per day. Data were analyzed for all the periods of the study, and the percentage of correct choices was calculated from the total number of choices. For the simple rule, acquisition period, or the number of sessions needed to reach criterion (13/16 correct choices on two consecutive sessions), was determined. The actions of S15535 were evaluated in the mixed procedure by ANOVA followed by the Fisher protected least significant difference test.

Autoshaping Learning Task. The experiments were performed on 12-week-old, group-housed Wistar rats (200–250 g) derived from an internal breeding colony (Centro de Investigación y Estudios Avanzados, Mexico City, Mexico). After a 1-week habituation period, the amount of food was gradually decreased, and body weight was maintained at 85% of free-feeding weight throughout the study. The protocol used was described in detail previously (Meneses, 2003). Briefly, standard, soundproof operant chambers for rats (25 x 29 x 25 cm; Coulbourn Instruments) were used. Cages were constantly illuminated during training and test sessions. In the middle of one wall, 4 cm above the floor, was a clear, retractable lever with a built-in light. A food tray was located 5 cm to the right of the lever. Solid-state programming equipment was used for coordination and recording (Coulbourn Instruments). On the training day, each rat was placed in the chamber with 50 food pellets (each of 45 mg) available in the food tray. The training, autoshaping session began once the rat had eaten all the pellets. This session consisted of 10 trials, each involving presentation of an illuminated, retractable lever for 8 s (conditioned stimulus) followed by delivery of one food pellet (unconditioned stimulus). The lever was then retracted and the light turned off for 60 s before starting a new trial. If the animal pressed the lever (conditioned response), the trial was immediately stopped (with delivery of the food pellet, lever withdrawal, and extinction of the light). The test session occurred 24 h later and consisted of 20 trials. Scopolamine (0.17 mg/kg i.p.) or vehicle was given immediately after the training session, and S15535 or vehicle was administered 10 min after scopolamine. Data were the number of conditioned responses during the test session (i.e., lever press responses during conditioned stimulus/20 trials) and were analyzed by ANOVA followed by Dunnett's test.

Chemicals and Drugs. All drug doses are in terms of the base. For systemic administration, drugs were dissolved in sterile water, if necessary, plus a few drops of lactic acid, and pH was adjusted to as close to neutrality (>5.0) as possible. Drugs were administered s.c. with injection volumes of 1 ml/kg (rats) or 10 ml/kg (mice), unless specified below. For intracerebral infusions, drugs were dissolved in saline or 1% ascorbic acid solution; the volume infused was 1 µl per injection site. In the operant DNMTS study, S15535 was administered orally (injection volume 10 ml/kg). S15535 (4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine) methane sulfonate and WAY100,635 [(N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclo-hexanecarboxamide) fumarate] were synthesized by J. L. Péglion (Servier). Scopolamine HCl and 8-OH-DPAT HBr were purchased from Sigma (Chesnes, France).

	Results

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Influence of S15535 upon Dialysis Levels of ACh in the FCX of Freely Moving Rats (Fig. 1). As quantified in the absence of AChEIs, basal dialysate levels of ACh in the FCX were 3.76 ± 0.13 pg/20 µl (26.0 ± 0.9 fmol/20 µl). A single injection of vehicle induced a modest and transient increase in extracellular levels of ACh, although the influence of two consecutive vehicle injections was more prolonged. Compared with vehicle, S15535 evoked a marked, sustained, and dose-dependent (0.04–5.0 mg/kg s.c.) increase in cortical levels of ACh. The maximal effect was attained at a dose of 5.0 mg/kg: 236 ± 20% versus basal values (defined as 100%). Upon a further increase in the dose of S15535 to 10.0 mg/kg, there was no additional effect (not shown). Indeed, at a dose of 10.0 mg/kg, although statistically significant (P < 0.01 versus vehicle), the elevation in extracellular levels of ACh (176 ± 15%) elicited by S15535 was less pronounced than for the dose of 5.0. The selective 5-HT_1A receptor antagonist WAY100,635 (0.01–0.63 mg/kg s.c.) did not significantly modify levels of ACh itself. In its presence, the facilitatory influence of S15535 upon ACh release was dose-dependently (0.0025–0.63 mg/kg s.c.) abrogated.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Influence of S15535 upon dialysis levels of ACh in the frontal cortex of freely moving rats: dose-response relationship and interaction with WAY100,635. A, dose-response relationship for S15535. B, dose-response relationship for WAY100,635. C, dose-dependent blockade of the effect of S15535 by WAY100,635 (0.0025–0.63 mg/kg) (area under the curve analysis). Dialysate levels are expressed as a percentage of basal preinjection values that were defined as 100%. These were 3.76 ± 0.13 pg/20 µl of dialysate. Data are means ± S.E.M. n 5/value. ANOVA with dose as between factor and time as within factor was performed over the 2 h after drug administration. A, S15535 (0.04), F(1,10) = 0.6, P > 0.05; S15535 (0.63), F(1,10) = 17.2, P < 0.01; and S15535 (5.0), F(1,11) = 41.3, P < 0.01. B, WAY100,635 (0.01), F(1,10) = 0.1, P > 0.05; WAY100,635 (0.16), F(1,9) = 0.6, P > 0.05; and WAY100,635 (0.63), F(1,9) = 0.1, P > 0.05. C, influence of S15535 (5.0), F(1,19) = 19.6, P < 0.01; interaction of S15535 with WAY100,635 (0.0025), F(1,16) = 0.1, P > 0.05; interaction of S15535 with WAY100,635 (0.04), F(1,17) = 5.8, P < 0.05; and interaction of S15535 with WAY100,635 (0.63), F(1,16) = 21.0, P < 0.01. In A and B, asterisks indicate significance of drug-treated groups versus vehicle-treated group after ANOVA, and in C, the "sharp" indicates the significance of vehicle plus S15535 versus vehicle plus vehicle values, and asterisks indicate the significance of WAY100,635 plus S15535 versus vehicle plus S15535 values after ANOVA. *, P < 0.05.

Influence of S15535 upon Dialysis Levels of ACh in the DH of Freely Moving Rats (Fig. 2). Basal levels of ACh in the DH, determined in the absence of AChEIs, were 1.13 ± 0.07 pg/20 µl (7.8 ± 0.5 fmol/20 µl). Administration of vehicle provoked a modest and transient elevation in extracellular levels of ACh. Dialysis levels of ACh were robustly and dose-dependently enhanced by S15535: maximal effect at a dose of 5.0 mg/kg, 224 ± 22% versus basal values (defined as 100%). By analogy to the FCX, compared with the dose of 5.0 mg/kg, a further increase in the dose of S15535 to 10.0 mg/kg s.c. did not achieve any additional elevation in ACh levels: 160 ± 13%, P < 0.05 to vehicle (not shown). However, in contrast to the FCX, WAY100,635 elicited a dose-dependent (0.01–0.63) elevation in extracellular levels of ACh in DH. That is, it mimicked the action of S15535. Accordingly, WAY100,635 (0.63) did not attenuate the increase in ACh levels provoked by S15535 (5.0) (not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Influence of S15535 and WAY100,635 upon dialysis levels of ACh in the dorsal hippocampus of freely moving rats. A, dose-response relationship for S15535. B, dose-response relationship for WAY100,635. Dialysate levels are expressed as a percentage of basal preinjection values that were defined as 100%. These were 1.13 ± 0.07 pg/20 µl of dialysate. Data are means ± S.E.M. n 5/value. ANOVA with dose as between factor and time as within factor was performed over the 2 h after drug administration. A, S15535 (0.63), F(1,11) = 0.1, P > 0.05; S15535 (2.5), F(1,13) = 5.8, P < 0.05; and S15535 (5.0), F(1,12) = 23.6, P < 0.01. B, WAY100,635 (0.01), F(1,12) = 0.3, P > 0.05; WAY100,635 (0.16), F(1,12) = 8.8, P < 0.05; and WAY100,635 (0.63), F(1,13) = 27.9, P < 0.01. Asterisks indicate significance of drug-treated groups versus vehicle-treated group. *, P < 0.05.

Influence of S15535 upon the Increase in Dialysis Levels of ACh Elicited by Scopolamine (Fig. 3). Scopolamine elicited a robust, dose-dependent (0.04–2.5), and sustained elevation in extracellular levels of ACh both in the FCX and in the DH. These effects were expressed across similar dose ranges. However, the magnitude of the influence of scopolamine upon ACh levels was more pronounced in the DH compared with the FCX. In the presence of a "modest" dose of S15535 (0.63 mg/kg), which elicited only a slight elevation in ACh levels, the facilitatory influence of a modest dose of scopolamine (0.63 mg/kg) upon ACh levels in FCX was greater compared with pretreatment with vehicle. In DH, a dose of 0.08 mg/kg scopolamine was selected for the interaction study. In the presence of a modest dose of S15535 (0.63), the influence of scopolamine was greater compared with vehicle pretreatment.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Influence of scopolamine alone and after pretreatment with S15535 upon extracellular levels of ACh in the frontal cortex and dorsal hippocampus of freely moving rats. A and B, dose-response relationship for increases in ACh levels elicited by scopolamine in the frontal cortex and dorsal hippocampus, respectively. C and D, influence of scopolamine after pretreatment with S15535 upon ACh levels in the frontal cortex and dorsal hippocampus, respectively. Data are means ± S.E.M. n 5/value. ANOVA results follow. A, scopolamine (0.04), F(1,9) = 4.4, P > 0.05; scopolamine (0.63), F(1,9) = 14.9, P < 0.01; and scopolamine (2.5), F(1,10) = 23.2, P < 0.01. B, scopolamine (0.04), F(1,11) = 1.4, P > 0.05; scopolamine (0.16), F(1,12) = 11.1, P < 0.01; scopolamine (0.63), F(1,12) = 15.6, P < 0.01; and scopolamine (2.5), F(1,11) = 34.3, P < 0.01. C, influence of S15535, F(1,13) = 6.2, P < 0.05; influence of scopolamine, F(1,13) = 3.9, P < 0.05; and interaction, F(1,12) = 5.4, P < 0.05. D, influence of S15535, F(1,11) = 3.2, P > 0.05; influence of scopolamine, F(1,14) = 17.5, P < 0.01; and interaction, F(1,14) = 6.9, P < 0.05. In A and B, asterisks indicate significance of scopolamine versus vehicle values after ANOVA, and in C and D, asterisks indicate the significance of S15535 plus scopolamine versus vehicle plus scopolamine values after ANOVA. *, P < 0.05.

Actions of S15535 Alone in the Social Recognition Test (Fig. 4). Adult, vehicle-treated animals rats spent equivalent time in both sessions investigating a juvenile animal, suggesting that, with this prolonged intersession interval (120 min), there was an absence of retention of the juvenile. Administered immediately after the first session, S15535 dose-dependently and significantly reduced the time of investigation of the juvenile rat during the second session, suggesting improved retention. Furthermore, at the highest dose tested (10 mg/kg), S15535 did not modify the time devoted (second session) to interaction with a juvenile different from that presented during the first session. This result supports a specific effect of S15535 upon memory processes. At a dose of 0.16 mg/kg, WAY100,635 was inactive in this procedure (same juvenile in the first and second sessions), suggesting a lack of influence upon retention. Furthermore, in the presence of WAY100,635, the influence of S15535 (10.0 mg/kg) upon interaction time was abolished.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Influence of S15535 upon cognitive function in the social recognition test in rats: dose-response relationship and influence of pretreatment with WAY100,635. Top, dose-response curve for the improvement of retention by S15535 (same juvenile) and control for the specificity of its actions (different juvenile). VEH, vehicle. Data are means ± S.E.M. n = 9 to 13/value. One-way ANOVA for the dose-response of S15535: F(4,44) = 7.3, P < 0.01. Closed asterisks indicate significance of differences between S15535 and vehicle values in Dunnett's test. Two-way ANOVA for specificity of the action of S15535; influence of juvenile, F(1,35) = 22.9, P < 0.01; S15535, F(1,35) = 18.0, P < 0.01; and interaction, F(1,35) = 15.5, P < 0.01. The open asterisk indicates the significance of the difference in Newman-Keuls test between values for the same juvenile/S15535 (10.0) versus a different juvenile/S15535 (10.0). Bottom, effect of S15535 (10.0 mg/kg s.c.) after pretreatment with WAY100,635 (0.16 mg/kg s.c.). Data are means ± S.E.M. n = 10 to 11/value. Two-way ANOVA results follow. Influence of WAY100,635, F(1,38) = 6.1, P < 0.05; influence of S15535, F(1,38) = 13.3, P < 0.01; and interaction, F(1,38) = 7.1, P < 0.05. The closed asterisk indicates the significance of the difference between vehicle/S15535 and vehicle/vehicle values, and the open asterisk indicates the significance of differences between WAY100,635/S15535 and vehicle/S15535 values in Newman-Keuls test. *, P < 0.05.

Influence of S15535 upon Scopolamine-Induced Amnesia in the Social Recognition Test (Fig. 5). When the second session took place immediately after the first session, adult, vehicle-treated animals spent substantially less time in social contact with the same juvenile compared with the first session ("retention"). In contrast, scopolamine-treated animals spent the same amount of time in social contact in the second compared with the first session (absence of retention). This scopolamine-induced, short-term retention deficit was dose-dependently reversed by S15535, which did not itself modify performance in the absence of scopolamine. In contrast to S15535, WAY100,635 (0.16 mg/kg) did not modify the effect of scopolamine. In its presence, the ability of S15535 to abrogate the actions of scopolamine was attenuated.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Influence of S15535 compared with WAY100,635 upon scopolamine-induced short-term memory deficits in the social recognition test in rats. A, blockade of scopolamine (0.63 mg/kg s.c.)-induced amnesia by S15535. B, lack of blockade of scopolamine-induced amnesia by WAY100,635 (0.16 mg/kg s.c.). C, reversal of blockade of scopolamine-induced amnesia by S15535 (0.63 mg/kg s.c.) in the presence of WAY100,635. SCOPO, scopolamine; VEH, vehicle. Data are means ± S.E.M. n = 5 to 10/value. ANOVA results follow. A, influence of S15535 versus scopolamine, F(4,23) = 11.0, P < 0.01; and effect of S15535 alone, F(4,30) = 1.2, P > 0.05. B, influence of WAY100,635, F(1,31) = 1.1, P > 0.05; influence of scopolamine, F(1,31) = 62.1, P < 0.001; and interaction, F(1,31) = 0.6, P > 0.05. In A and B, closed asterisks indicate the significance of differences between vehicle/vehicle and vehicle/scopolamine values in unpaired t tests, and open asterisks indicate the significance of differences between S15535/scopolamine and vehicle/scopolamine values in Dunnett's test. C, in animals receiving vehicle, influence of S15535, F(1,18) = 0.4, P > 0.05; influence of WAY100,635, F(1,18) = 0.9, P > 0.05; and interaction, F(1,18) = 0.1, P > 0.05. In animals receiving scopolamine, influence of S15535, F(1,20) = 26.2, P < 0.001; influence of WAY100,635, F(1,20) = 5.5, P < 0.05; and interaction, F(1,20) = 6.7, P < 0.05. Asterisks indicate the significance of differences (in rats treated with scopolamine) between vehicle/S15535 and vehicle/vehicle or WAY100,635/S15535 values in Newman-Keuls test. *, P < 0.05.

Influence of S15535 upon Scopolamine-Induced Amnesia in the Morris Water-Maze Test (Fig. 6). Control (vehicle/vehicle) animals showed progressively diminished escape latencies over the four consecutive trials in the Morris water-maze. Scopolamine-treated rats did not, however, display a significant reduction in escape latencies over the four consecutive trials, suggesting a learning deficit. That is, scopolamine abolished the decrease in escape latency from trial to trial. The difference between vehicle/vehicle and vehicle/scopolamine groups was significant for the last two trials. S15535 markedly decreased the escape latencies of scopolamine-injected animals at doses of 0.16, 0.63, and 2.5 mg/kg. Significant differences versus scopolamine were obtained for trial 4 at the dose of 0.16 mg/kg, and for trials 3 and 4 at doses of 0.63 and 2.5 mg/kg. However, at the highest dose examined (10.0 mg/kg), S15535 did not modify the action of scopolamine.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Influence of S15535 upon scopolamine-induced learning deficits in the Morris water-maze task. Data are means ± S.E.M. n = 12/value. Asterisks indicate significance of differences versus vehicle/scopolamine values in unpaired t tests. *, P < 0.05.

Influence of S15535 upon 8-OH-DPAT-Induced Amnesia in a Two-Platform Discrimination Task (Fig. 7). Bilateral administration of 8-OH-DPAT into the CA1 region of the DH (5 µg/µl/injection site) resulted in a significant decrease in the percentage of correct choices in the TPSD model, suggesting reduced retention. In contrast, administration of S15535 (1.0 and 10.0 µg/µl bilaterally) into this structure did not significantly decrease correct choices. Indeed, at the higher dose tested, there was a clear tendency toward an increase in correct choices, although this effect did not obtain statistical significance. Both doses of S15535 significantly attenuated the effects of 8-OH-DPAT. At the higher dose, the effects of 8-OH-DPAT were abolished.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Influence of bilateral, intracerebral infusions of S15535 (1.0 or 10.0 µg) and 8-OH-DPAT (5.0 µg) into the dorsal hippocampus upon the number of correct choices emitted by pretrained rats in the water-maze test. Data are means ± S.E.M.. n = 7 to 11/value. VEH, vehicle. For the dose of 1.0 µg of S15535, ANOVA results follow. Effect of 8-OH-DPAT, F(1,42) = 19.1, P < 0.01; effect of S15535, F(1,42) = 7.1, P < 0.05; and interaction, F(1,42) = 2.3, P > 0.05. Dose of 10.0 µg of S15535: effect of 8-OH-DPAT, F(1,21) = 11.0, P < 0.01; effect of S15535, F(1,21) = 12.1, P < 0.01; and interaction, F(1,21) = 0.5, P > 0.05. Closed asterisks indicate the significance of differences between vehicle/8-OH-DPAT and vehicle/vehicle values, and open asterisks indicate significance of differences between S15535/8-OH-DPAT and vehicle/8-OH-DPAT values in Tukey's test. *, P < 0.05.

Influence of S15535 upon the Acquisition of an Operant, Delayed Nonmatching to Sample Task in Aged Rats (Fig. 8). Vehicle-treated aged rats displayed impaired acquisition in comparison with young controls as shown by the absence of an increase in correct responding during the second week of acquisition. Although not significant during the first week of testing, this acquisition deficit attained statistical significance during the second week. At this time, the intermediate dose of S15535 (5.0 mg/kg) significantly reversed the deficit associated with age. Aged rats showed a significant decrease in latency to respond during both weeks of evaluation. During the first test week, S15535 induced a marked reduction in latency to respond at 5.0 mg/kg, and in the second week, latencies to respond were decreased by doses of both 1.25 and 5.0 mg/kg. In contrast, the highest dose of S15535 (20.0) failed to modify latencies to respond relative to vehicle-treated control animals.

View larger version (28K): [in this window] [in a new window]   Fig. 8. Influence of S15535 upon the acquisition of an operant, delayed nonmatching to sample task in the aged rat. VEH, vehicle. Data are means ± S.E.M. n = 10 to 11/value. ANOVA results for dose effect in aged rats follow. For percentage of correct responses (top), week 1, F(3,40) = 2.4, P < 0.05; and week 2, (3,40) = 4.3, P < 0.05. For latency to respond (bottom), week 1, F(3,40) = 4.3, P < 0.05; and week 2, F(3,40) = 3.7, P < 0.05. Closed asterisks indicate significance of differences between vehicle/young and vehicle/aged rat values. Open asterisks indicate significance of differences between S15535 and vehicle values in the aged group. *, P < 0.05.

Action of S15535 in a Spatial, Delayed Nonmatching to Sample Paradigm in Mice (Fig. 9). Administered throughout the training period, S15535 did not modify the number of sessions required by mice to achieve criterion (13/16 correct choices on two consecutive sessions) during the simple rule phase of acquisition: vehicle, 3.9 ± 1 sessions; S15535, 0.16 mg/kg s.c., 4.9 ± 0.9; S15535, 0.63 mg/kg s.c., 4.0 ± 1; S15535, 2.5 mg/kg s.c., 3.6 ± 0.7; and S15535, 10.0 mg/kg s.c., 4.3 ± 0.6; n = 7/value, F(4,30) = 0.3, P > 0.05. However, in a mixed testing paradigm, S15535 (0.16 and 0.63 mg/kg s.c.) significantly increased the number of correct choices emitted by mice at the low level (one arm interposed visit) of difficulty. At higher doses (2.5 and 10.0 mg/kg), S15535 was not significantly effective. A similar pattern of data were seen for the two higher levels of difficulty (three and five arms interposed) with significant drug effects in both cases (see Fig. 9 legend), although post hoc analyses of individual doses versus vehicle just failed to reach statistical significance. Finally, using a combined analysis across all levels of difficulty, the global increase in correct responses elicited by S15535 (0.16 and 0.63) was marked and significant.

View larger version (32K): [in this window] [in a new window]   Fig. 9. Influence of S15535 upon performance in a mixed procedure test of a spatial, delayed nonmatching to sample paradigm in mice. Data are means ± S.E.M. n = 7/value. One, 3, and 5 refer to varying levels of difficulty (see text), and combined refers to the collapsed, global values pooled across the three levels. For one arm, F(4,30) = 6.3, P < 0.01; for three arms, F(4,30) = 4.3, P < 0.01; for five arms, F(4,30) = 3.2, P < 0.05; and for combined, F(4,30) = 7.2, P < 0.001. Asterisks denote significance of S15535 versus vehicle differences in the Fisher protected least significant difference test. *, P < 0.05.

Actions of S15535 in an Autoshaping Learning Task (Fig. 10). In an initial study, administration of scopolamine (0.17 mg/kg i.p.) immediately after the training phase resulted in a pronounced reduction in conditioned responses during the test session 24 h later compared with vehicle-injected animals, suggesting impairment of consolidation. S15535 (1.0 mg/kg i.p.) did not modify the number of conditioned responses alone but blocked their reduction by scopolamine. Percentages of conditioned responses were as follows: vehicle/vehicle (11 ± 1%, n = 8); vehicle/scopolamine (2 ± 1%, n = 8); S15535/vehicle (11 ± 3%, n = 8), and S15535/scopolamine (14 ± 3, n = 8). Two-way ANOVA results follow: effect of scopolamine, F(1,28) = 2.2, P < 0.05; effect of S15535, F(1,28) = 6.5, P < 0.05; and interaction, F(1,28) = 6.5, P < 0.05. Differences between vehicle/vehicle and vehicle/scopolamine and between vehicle/scopolamine and S15535/scopolamine groups were significant in Newman-Keuls test (P < 0.05). In an additional study, the dose-response relationship for the influence of S15535 upon the action of scopolamine was examined (Fig. 10), and it was found to be active at doses of 0.63 and higher, although not at the lowest dose (0.16) evaluated.

View larger version (32K): [in this window] [in a new window]   Fig. 10. Influence of S15535 upon scopolamine-induced amnesia in an autoshaping learning task. Data are means ± S.E.M. n = 8/value. VEH, vehicle. One-way ANOVA results for dose-effect of S15535 versus scopolamine are F(3,28) = 6.5, P < 0.01. The closed asterisk indicates the significance of differences between vehicle and scopolamine values. Open asterisks indicate the significance of differences between S15535/scopolamine and vehicle/scopolamine values in Dunnett's test. *, P < 0.05.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Influence upon Frontocortical versus Hippocampal Cholinergic Transmission. Using a highly sensitive dialysis system not necessitating use of AChEIs, resting levels of ACh in both FCX and DH were substantially lower than acquired in the presence of AChEIs to improve detection limits (Ichikawa et al., 2002; Gobert et al., 2003). Vehicle administration elicited transient elevations in ACh levels in line with previous studies of handling, the effects of which are blunted by high concentrations of AChEIs (Giovannini et al., 1998; Ichikawa et al., 2002; Gobert et al., 2003). AChEIs also modify the influence of 5-HT_1A receptor ligands upon ACh levels (Fujii et al., 1997; Ichikawa et al., 2002). It is thus of importance that the dose-dependent and robust elevation in FCX and DH levels of ACh elicited by S15535 was seen in the absence of AChEIs. This action of S15535 accords with work suggesting a facilitatory influence of 5-HT_1A agonists upon corticolimbic ACh release (Consolo et al., 1996; Somboonthum et al., 1997; Nakai et al., 1998) and the question arises as to underlying mechanisms. Although this issue was not directly addressed herein, parallel studies with the antagonist WAY100,635 afford several insights into this issue.

Extending single-dose studies with the prototypical 5-HT_1A agonist 8-OH-DPAT (Ichikawa et al., 2002), and confirming the implication of 5-HT_1A sites, WAY100,635 dose-dependently abolished the S15535-induced increase in ACh release in FCX. It is unlikely that S15535 activates 5-HT_1A sites in the FCX itself because local administration of 5-HT_1A agonists does not enhance cortical release of ACh (Giovannini et al., 1998). Furthermore, S15535 behaves as an antagonist at cortical populations of (postsynaptic) 5-HT_1A receptors (Newman-Tancredi et al., 1999, 2003). Although lesions of serotonergic pathways do not invariably modify the influence of 5-HT_1A agonists upon cortical levels of ACh (Consolo et al., 1996; Somboonthum et al., 1997), it is likely that activation of 5-HT_1A autoreceptors by S15535 (Millan et al., 1994, 1997a,b) indirectly disinhibits ACh release in FCX (Cassel and Jeltsch, 1995; Steckler and Sahgal, 1995). On the other hand, the inflection of the dose-response for S15535 at high doses might be explained by the onset of weak agonist actions at inhibitory, postsynaptic 5-HT_1A receptors localized on cholinergic cell bodies (Van den Hooff and Galvan, 1992; Kia et al., 1996).

Interestingly, induction of hippocampal ACh release by S15535 was mimicked by WAY100,635, indicative of a role for postsynaptic 5-HT_1A receptor blockade. This finding may seem surprising inasmuch as Izumi et al. (1994) and Nakai et al. (1998) reported that stimulation of hippocampal 5-HT_1A sites by local administration of 8-OH-DPAT increased ACh release. However, Fujii et al. (1997) showed that 8-OH-DPAT is inactive when AChEIs are not added to the perfusate to artificially boost baseline levels, conditions corresponding to the present work. Moreover, in in vitro studies of the hippocampus, 5-HT_1A agonists did not enhance ACh release (Steckler and Sahgal, 1995). Rather than blockade of hippocampal 5-HT_1A sites, S15535 and WAY100,635 may, in fact, enhance hippocampal ACh release by blocking tonically active, inhibitory 5-HT_1A sites on cholinergic cell bodies. This would be consistent with findings that lesions of serotonergic neurons increase extracellular levels of ACh in the DH (Steckler and Sahgal, 1995; Birthelmer et al., 2002).

Social Memory: Recognition of a Young Conspecific. Social memory is integrated in the olfactory system, inter-connected cortical structures, and the amygdala, although the role of the hippocampus remains controversial. Indeed, the social recognition model differs markedly from other procedures exploited herein (Perio et al., 1989; Bannerman et al., 2002; Ferguson et al., 2002). Curiously, actions of 5-HT_1A receptor ligands have not, to date, been documented in this paradigm, so the robust, dose-dependent effects of S15535 are of particular interest. Furthermore, interaction with a novel juvenile was not affected by S15535, underpinning its specificity of actions and consistent with its lack of influence upon motor function (Millan et al., 1994, 1997a,b). Reflecting the role of cholinergic mechanisms in social memory, performance is perturbed by lesions of cholinergic pathways (Perio et al., 1989; Winters et al., 2000). Correspondingly, when the intertest interval was reduced to preclude a spontaneous retention deficit, scopolamine disrupted performance, an action abolished by S15535. Inasmuch as WAY100,635 blocked the actions of S15535 in this model, a predominant role of 5-HT_1A autoreceptors is indicated, although this awaits direct confirmation.

Hippocampally Integrated Spatial Memory: Morris Water-Maze and TPSD. Hippocampal cholinergic pathways play a major role in spatial memory and, correspondingly, performance in the water-maze is severely disrupted by scopolamine (Porsolt et al., 1995). The reversal of its effects by S15535 resembles findings of a previous study with MDL73005 which likewise acts as an agonist and antagonist at pre- and postsynaptic 5-HT_1A receptors, respectively, although its D₂ antagonist properties complicate interpretation of its actions (Bertrand et al., 2001). Furthermore, in contrast to that single (low)-dose study of MDL73005(8-[2-{1,4-benzodioxan-2-ylmethylamino) ethyl]-8-azaspiro[4.5]decane-7,9-dione), evaluation of the dose-response relationship for S15535 revealed that the highest dose was inactive, mimicking its biphasic influence upon performance in the spatial DNMTS procedure (see below) and upon ACh release (see above). The reasons underlying the biphasic dose-response curve for S15535 in the water-maze are unclear, although, as mentioned above, high doses may recruit 5-HT_1A sites inhibitory to cholinergic cell bodies. Inasmuch as the negative influence of scopolamine upon spatial memory can be abrogated either by blockade of postsynaptic 5-HT_1A receptors (Carli et al., 1995a,b; Ohno and Watanabe, 1996) or by stimulation of 5-HT_1A autoreceptors (Cole et al., 1994; Carli et al., 2000), their relative contribution to attenuation of scopolamine-induced amnesia by low doses of S15535 remains to be established. Nevertheless, supporting a role of presynaptic sites, their engagement by S15535 and other agonists (Carli et al., 2000) abolishes amnesic properties of scopolamine in a related model of spatial learning, the TPSD (Carli et al., 1999). In this model, blockade of postsynaptic 5-HT_1A receptors by WAY100,635 alleviates amnesic effects of intrahippocampal 8-OH-DPAT, although it is inactive alone (Carli et al., 1995b). By analogy, S15535 did not exert procognitive actions in the TPSD but abolished the influence of intrahippocampal 8-OH-DPAT. This finding suggests that, under certain conditions, antagonism of postsynaptic 5-HT_1A receptors by S15535 may enhance spatial memory.

Spatial Working Memory: Delayed Nonmatching to Sample Models. "Delayed nonmatching to position" models of short-term, spatial working memory, in which rats choose between spatially separated, retractable levers, are dependent upon the operation of cholinergic mechanisms in the hippocampus and FCX (McAlonan et al., 1995; Herremans et al., 1996). Although it has been questioned whether mnemonic (compared with motor) factors underlie the perturbation of performance in this model provoked by activation of hippocampal 5-HT_1A receptors, stimulation of 5-HT_1A autoreceptors specifically improves cognitive function (Cole et al., 1994; Porsolt et al., 1995; Stanhope et al., 1995; Warburton et al., 1997; Pache et al., 2003). Accordingly, S15535 enhanced performance of mice in the present spatial DNMTS (place) variant of this paradigm at autoreceptor-activating doses that exert little influence upon motor function (Millan et al., 1994, 1997a,b). Procognitive actions of S15535 were lost at higher doses, perhaps reflecting the onset of postsynaptic 5-HT_1A receptor activation (see above). A biphasic dose-response curve also was seen in a related procedure of operant, working memory, an operant DNMTS task in aged rats, in which the influence of drugs upon memory (response accuracy) and attention (latency to respond) can be differentiated (Porsolt et al., 1995). Interestingly, S15535 preferentially improved the compromised attentional function displayed by aged rats. Although the positive influence of S15535 upon response accuracy was less marked, the lack of deterioration is in itself important because it refutes the contention that, via recruitment of 5-HT_1A receptors in the dorsal raphe nucleus, S15535 may trigger excessive vigilance and impulsive (premature and inaccurate) responding (Steckler and Sahgal, 1995; Bizot and Thiébot, 1996).

Nonspatial Reference Memory: Autoshaping Task. Cholinergic mechanism in the hippocampus have been implicated in the integration of autoshaping cognitive tasks (Meneses, 1999). Furthermore, indicative of a modulatory role of 5-HT_1A receptors in autoshaping, it is accompanied by alterations in levels of postsynaptic (decreased) and presynaptic (increased) 5-HT_1A receptors (Tomie et al., 2003), whereas mice genetically deprived of 5-HT_1A receptors reach criteria more rapidly than their wild-type counterparts (Pattij et al., 2003). Depletion of endogenous pools of 5-HT impaired the enhancement of consolidation by 8-OH-DPAT, implicating a role for 5-HT_1A autoreceptors in its actions (Meneses, 2003). Inasmuch as S15535 exerts procognitive properties via engagement of presynaptic 5-HT_1A receptors in other procedures examined herein, its inactivity alone was unexpected. Although S15535 mimicked the ability of 8-OH-DPAT (Meneses, 2003) to prevent induction of retroactive amnesia by scopolamine, similar procognitive actions have been reported for WAY100,635 in this model (Meneses, 2003). Thus, whether the attenuation of scopolamine-amnesia by S15535 reflects engagement of presynaptic 5-HT_1A autoreceptors (like 8-OH-DPAT) or blockade of postsynaptic sites (like WAY100,635) will require further study.

Mechanism(s) of Procognitive Actions of S15535: Role of ACh and Interaction with Scopolamine. These data with S15535 underpin a role of 5-HT_1A sites in the modulation of ACh release and mnemonic function. However, the relationship between these actions remains unclear, and there was not an absolute correspondence between the influence of S15535 upon ACh levels compared with its actions in the behavioral models. For example, S15535 was active in the social interaction and DNMTP paradigms at doses lower (0.16 mg/kg) than those elevating extracellular levels of ACh. This difference may reflect the limited sensitivity of dialysis techniques that measure increases in transmitter levels in the extracellular space but not in the synaptic cleft itself. On the other hand, S15535 showed procognitive actions at doses of up to 10 mg/kg in the autoshaping model, whereas its influence upon ACh levels peaked at a dose of 5 mg/kg. This difference may reflect the difficulty of comparing doses obtained in behavioral versus neurochemical procedures. A further explanation may be that noncholinergic mechanisms recruited by high doses of S15535 contribute to its influence upon cognition (see below).

The question as to how S15535 and other 5-HT_1A receptor ligands relieve the amnesic effects of scopolamine requires elucidation. One possibility is that increases in ACh release elicited by S15535 and scopolamine (which blocks inhibitory M₂ autoreceptors) collectively overcome the latter's antagonism of postsynaptic muscarinic receptors and concurrently stimulate nicotinic receptors. This possibility is supported by the greater increase in FCX and DH release of ACh evoked by scopolamine in the presence of S15535. In addition, blockade of inhibitory 5-HT_1A receptors colocalized with excitatory muscarinic receptors in the DH may alleviate the amnesic effects of scopolamine independently of ACh outflow (Claustre et al., 1988; Fujii et al., 1997; Misane and Ogren, 2003). On the other hand, Carli et al. (1999) proposed that activation of 5-HT_1A autoreceptors blocks scopolamine-induced amnesia via polysynaptic, cortico-hippocampal loops operating independently of ACh release. Moreover, reflecting agonist properties at 5-HT_1A autoreceptors, S15535 enhances dopamine release in FCX (Millan et al., 1997a,b) wherein activation of D₁ receptors facilitate cognitive function and enhances ACh release (Consolo et al., 1996; Arnsten, 1997). The increase in noradrenaline release elicited by S15535 in FCX and hippocampus may also improve cognitive-attentional function (Arnsten, 1997; Millan et al., 1997a,b). Finally, antagonist properties of S15535 at postsynaptic 5-HT_1A receptors may reinforce glutamatergic mechanisms of memory (Schechter et al., 2002). These comments emphasize the broad and complex role of 5-HT_1A receptors in the modulation of mnemonic function and the difficulty of defining the role of (multiple) mechanisms implicated in the influence of S15535 and other 5-HT_1A receptor ligands upon cognitive performance.

Conclusions. Over similar dose ranges, S15535 enhances cortico-hippocampal ACh release and facilitates cognitive function in a broad array of behavioral models. Its actions involve both engagement of 5-HT_1A autoreceptors and blockade of postsynaptic 5-HT_1A receptors, though their respective contributions require further elucidation. An improvement of cognitive function would offer advantages relative to benzodiazepines in the management of anxiolytic states. However, the precise therapeutic utility of low-efficacy 5-HT_1A receptor ligands in the treatment of cognitive symptoms associated with psychiatric and neurological disorder awaits further experimental and clinical evaluation.

	Acknowledgements

 
We thank Laetitia Cistarelli, Rodolphe Billiras, Dorothée Sicard, and Roberto González for excellent technical assistance.

	Footnotes

 
A.M. was partially supported by Consejo Nacional de Ciencia y Tecnologia Grant 39534.

doi:10.1124/jpet.104.069625.

ABBREVIATIONS: FCX, frontal cortex; 5-HT, 5-hydroxytryptamine (serotonin); ACh, acetylcholine; AChEI, acetylcholinesterase inhibitor; DH, dorsal hippocampus; 8-OH-DPAT, 8-hydroxy-2-dipropylaminotetralin; TPSD, two-platform spatial discrimination; ANOVA, analysis of variance; DNMTS, delayed nonmatching to sample; S15535, 4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine; WAY100,635, (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclo-hexanecarboxamide fumarate.

Address correspondence to: Dr. Mark J. Millan, Institut de Recherches Servier, Centre de Recherches de Croissy, 125 chemin de Ronde 78290 Croissy/Seine, France. E-mail: mark.millan{at}fr.netgrs.com

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