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P.1.c.040 Long-lasting cognitive enhancing effects of S 18986, a positive modulator of AMPA receptors, in the object recognition task in rats
P.1.c Basic neuroscience – Neuropharmacology to the end of the experiment and the concentration of 5-HT and dopamine levels were measured. The concentration values of extracellular dopamine and 5-HT levels were measured from the same dialysis samples. At the same time, PS and population EPSP in the responses to the single stimuli was measured. The extracellular DA and 5-HT levels didn’t change after the administration of aripiprasole 10, 20 and 40 mg/kg. And aripiprazole suppressed LTP as does dependent. These findings indicate that aripiprazole suppresses the excitatory synaptic transmission.
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to 6 hours post-dosing) cognitive enhancing activity in the object recognition task at 1 mg/kg. Furthermore, once a day administration seems to be more efficient than 3 administrations per day. References
P.1.c.040 Long-lasting cognitive enhancing effects of S 18986, a positive modulator of AMPA receptors, in the object recognition task in rats
[1] Bourasset F., Bernard K., Munoz C., Genissel P., Schermann J.M., 2005, Neuropharmacokinetics of a new alpha-amino-3-hydroxy-5-methyl-4isoxazole propionic acid (AMPA) modulator, S18986 [(S)-2,3-dihydro[3,4] cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide], in the rat, Drug Metab Dispos, 33, 1137–1143. [2] Lebrun C., Pilliere E., Lestage P., 2000, Effects of S 18986-1, A novel cognitive enhancer, on memory performances in an object recognition task in rats, Eur. J. Neurosci, 401, 205–212. [3] Rosi S., Giovannini M.G., Lestage P.J., Munoz C., Corte L.D., Pepeu G., 2004, S 18986, A positive modulator of AMPA receptors with cognition enhancing properties, increases ACh release in the hippocampus of young and aged rat, Neurosci. Lett., 361, 120–123.
V. Bertaina-Anglade1 ° , K. Bernard2 , C. Munoz2 , P. Morain2 , C. Drieu la Rochelle1 . 1 Biotrial, Preclinical Pharmacology, Rennes, France; 2 Institut de Recherches Internationales Servier, Neuro-Psychiatry, Courbevoie, France
P.1.c.041 Fluorescence in vitro studies of human adenosine A2A and serotonin 5HT1A receptors homodimerization
S 18986, a positive allosteric modulator of AMPA-type glutamate receptors, has been shown to possess cognitive enhancing properties in appropriated animal models such as the passive avoidance test (Rosi at al, 2004) and the object recognition task (Lebrun et al, 2000) among others. S 18986 pharmacokinetics showed a good blood–brain barrier permeability but a short plasma and brain terminal half-lives (approximately 1h) (Bourasset et al., 2005). The present study was designed to evaluate the optimal schedule of S 18986 administration. First, we assessed the maximal duration of action of S 18986 effects on memory performance after a single administration (1 mg/kg, once a day.) during 3 consecutive days. In addition, we studied the effect of a single administration (1 mg/kg, once a day.) versus the same daily dose given as three distinct administrations (0.3 mg/kg, ter in day). The memory performance was investigated by using the object recognition task in the Sprague-Dawley rat. In this memory task, considered as a model of episodic memory in rodents, two copies of the same object are presented to rats during a learning trial. In a second trial 24h later, one of the familiar object along with a novel object are presented to rats, recognition being assessed by the ability of rats to discriminate between both objects. Under these conditions of natural forgetting, control rats do not recognise the familiar object. In a first series of experiments, the duration of action of S 18986, orally administered at the active dose of 1 mg/kg (Lebrun et al., 2000) before each trial of the test, was investigated by increasing the delay between dosing and testing from 1 to 9 hours. An increase in memory performance, assessed by a recognition index (RI), was observed in S 18986-treated rats up to 6 hours after administration. This effect was maximal 3h following administration (recognition index was increased by 40% compared to vehicle-treated animals) and disappeared 9h following administration (−2% versus vehicle group). In a second series of experiments, S 18986 was orally administered 3 times per day at 0.3 mg/kg 9, 6 and 3 hours before each of the daily trial. Using this schedule of administration, RI increase (+16% versus vehicle group) was similar to that observed after a oncea-day administration of S 18986 at 1 mg/kg, 6h before each of the daily trial (+22% versus vehicle group). In both series of experiments, S 18986 did not altered exploratory behaviour or locomotor activity suggesting a direct effect on memory function and no unspecific activity. Taken together, results of the present study demonstrated that S 18986 displays a long-lasting (i.e. up
S. Lukasiewicz1 , E. Blasiak1 , A. Faron-Gorecka2 , Z. Wasylewski1 , M. Dziedzicka-Wasylewska2 ° . 1 Jagiellonian University, Department of Physical Biochemistry, Krakow, Poland; 2 Polish Academy of Sciences, Department of Pharmacology, Krakow, Poland Recently the view that G protein-coupled receptors (GPCRs) can function as monomeric polypeptides has been broadened by the data indicating the existence of GPCRs dimers or higherordered oligomers [Milligan & Bouvier, FEBS J, 272, 2914]. The ability to differentially epitope-tag GPCRs has been central to widespread use of coimmunoprecipitation following coexpression of two forms of the same GPCR in heterologous expression systems. However, such methodology might reflect non-specific aggregation following detergent extraction of proteins from cell membrane. The biophysical methods, using the F¨orster resonance energy transfer (FRET) phenomenon seem more appropriate for studies of GPCRs oligomerization. The present study addressed the question whether human serotonin 5-HT1A or adenosine A2A receptors can form homodimers. Each pair of receptors was tagged with Cyan Fluorescence Protein (CFP, fluorescence donor) or Yellow Fluorescence Protein (YFP, fluorescence acceptor). HEK 293 cell line was transiently transfected with the fusion proteins, i.e. with 5-HT1A-CFP and 5-HT1A-YFP or A2A-CFP and A2AYFP. As a control we used the HEK 293 cells transfected with the plasmid encoding CFP-YFP hybrid mutant, in which both CFP and YFP were linked together by a short 15 amino acid chain. The fluorescence intensity ratio, measured at maximum of fluorescence emission of about 475 nm and 528 nm for CFP and YFP proteins, respectively, was equal to ca. 0.38. We employed steady-state fluorescence spectroscopy (Fluorolog 3, Jobin Yvon) as well as time resolved fluorescence lifetime measurements (picosecond diode pulse laser coupled to the inverted fluorescence microscope, Olympus platform IX 71) and confocal microscopy (Leica TCS SP2). The first two approaches were applied for measurements in cell suspension; for confocal microscopy measurements the transfected cells were grown on coverslips, and were fixed with 3.5% paraformaldehyde for 15 min. The cells were harvested at 48 h after transfection; specific receptor ligands were added to the culture medium for 30 min or 2 h prior to harvesting. Using three independent approaches we have observed that both types of receptors exist as oligomeric complexes in
S245
to 6 hours post-dosing) cognitive enhancing activity in the object recognition task at 1 mg/kg. Furthermore, once a day administration seems to be more efficient than 3 administrations per day. References
P.1.c.040 Long-lasting cognitive enhancing effects of S 18986, a positive modulator of AMPA receptors, in the object recognition task in rats
[1] Bourasset F., Bernard K., Munoz C., Genissel P., Schermann J.M., 2005, Neuropharmacokinetics of a new alpha-amino-3-hydroxy-5-methyl-4isoxazole propionic acid (AMPA) modulator, S18986 [(S)-2,3-dihydro[3,4] cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide], in the rat, Drug Metab Dispos, 33, 1137–1143. [2] Lebrun C., Pilliere E., Lestage P., 2000, Effects of S 18986-1, A novel cognitive enhancer, on memory performances in an object recognition task in rats, Eur. J. Neurosci, 401, 205–212. [3] Rosi S., Giovannini M.G., Lestage P.J., Munoz C., Corte L.D., Pepeu G., 2004, S 18986, A positive modulator of AMPA receptors with cognition enhancing properties, increases ACh release in the hippocampus of young and aged rat, Neurosci. Lett., 361, 120–123.
V. Bertaina-Anglade1 ° , K. Bernard2 , C. Munoz2 , P. Morain2 , C. Drieu la Rochelle1 . 1 Biotrial, Preclinical Pharmacology, Rennes, France; 2 Institut de Recherches Internationales Servier, Neuro-Psychiatry, Courbevoie, France
P.1.c.041 Fluorescence in vitro studies of human adenosine A2A and serotonin 5HT1A receptors homodimerization
S 18986, a positive allosteric modulator of AMPA-type glutamate receptors, has been shown to possess cognitive enhancing properties in appropriated animal models such as the passive avoidance test (Rosi at al, 2004) and the object recognition task (Lebrun et al, 2000) among others. S 18986 pharmacokinetics showed a good blood–brain barrier permeability but a short plasma and brain terminal half-lives (approximately 1h) (Bourasset et al., 2005). The present study was designed to evaluate the optimal schedule of S 18986 administration. First, we assessed the maximal duration of action of S 18986 effects on memory performance after a single administration (1 mg/kg, once a day.) during 3 consecutive days. In addition, we studied the effect of a single administration (1 mg/kg, once a day.) versus the same daily dose given as three distinct administrations (0.3 mg/kg, ter in day). The memory performance was investigated by using the object recognition task in the Sprague-Dawley rat. In this memory task, considered as a model of episodic memory in rodents, two copies of the same object are presented to rats during a learning trial. In a second trial 24h later, one of the familiar object along with a novel object are presented to rats, recognition being assessed by the ability of rats to discriminate between both objects. Under these conditions of natural forgetting, control rats do not recognise the familiar object. In a first series of experiments, the duration of action of S 18986, orally administered at the active dose of 1 mg/kg (Lebrun et al., 2000) before each trial of the test, was investigated by increasing the delay between dosing and testing from 1 to 9 hours. An increase in memory performance, assessed by a recognition index (RI), was observed in S 18986-treated rats up to 6 hours after administration. This effect was maximal 3h following administration (recognition index was increased by 40% compared to vehicle-treated animals) and disappeared 9h following administration (−2% versus vehicle group). In a second series of experiments, S 18986 was orally administered 3 times per day at 0.3 mg/kg 9, 6 and 3 hours before each of the daily trial. Using this schedule of administration, RI increase (+16% versus vehicle group) was similar to that observed after a oncea-day administration of S 18986 at 1 mg/kg, 6h before each of the daily trial (+22% versus vehicle group). In both series of experiments, S 18986 did not altered exploratory behaviour or locomotor activity suggesting a direct effect on memory function and no unspecific activity. Taken together, results of the present study demonstrated that S 18986 displays a long-lasting (i.e. up
S. Lukasiewicz1 , E. Blasiak1 , A. Faron-Gorecka2 , Z. Wasylewski1 , M. Dziedzicka-Wasylewska2 ° . 1 Jagiellonian University, Department of Physical Biochemistry, Krakow, Poland; 2 Polish Academy of Sciences, Department of Pharmacology, Krakow, Poland Recently the view that G protein-coupled receptors (GPCRs) can function as monomeric polypeptides has been broadened by the data indicating the existence of GPCRs dimers or higherordered oligomers [Milligan & Bouvier, FEBS J, 272, 2914]. The ability to differentially epitope-tag GPCRs has been central to widespread use of coimmunoprecipitation following coexpression of two forms of the same GPCR in heterologous expression systems. However, such methodology might reflect non-specific aggregation following detergent extraction of proteins from cell membrane. The biophysical methods, using the F¨orster resonance energy transfer (FRET) phenomenon seem more appropriate for studies of GPCRs oligomerization. The present study addressed the question whether human serotonin 5-HT1A or adenosine A2A receptors can form homodimers. Each pair of receptors was tagged with Cyan Fluorescence Protein (CFP, fluorescence donor) or Yellow Fluorescence Protein (YFP, fluorescence acceptor). HEK 293 cell line was transiently transfected with the fusion proteins, i.e. with 5-HT1A-CFP and 5-HT1A-YFP or A2A-CFP and A2AYFP. As a control we used the HEK 293 cells transfected with the plasmid encoding CFP-YFP hybrid mutant, in which both CFP and YFP were linked together by a short 15 amino acid chain. The fluorescence intensity ratio, measured at maximum of fluorescence emission of about 475 nm and 528 nm for CFP and YFP proteins, respectively, was equal to ca. 0.38. We employed steady-state fluorescence spectroscopy (Fluorolog 3, Jobin Yvon) as well as time resolved fluorescence lifetime measurements (picosecond diode pulse laser coupled to the inverted fluorescence microscope, Olympus platform IX 71) and confocal microscopy (Leica TCS SP2). The first two approaches were applied for measurements in cell suspension; for confocal microscopy measurements the transfected cells were grown on coverslips, and were fixed with 3.5% paraformaldehyde for 15 min. The cells were harvested at 48 h after transfection; specific receptor ligands were added to the culture medium for 30 min or 2 h prior to harvesting. Using three independent approaches we have observed that both types of receptors exist as oligomeric complexes in