Cannula (25 gauge) was stereotaxically implanted above the right lateral ventricle (AP 20.2 mm, ML +1.0 mm, DV 21.4 mm, according to the atlases of Paxinos and Franklin, 2001 [16]) for i.c.v.Olfactory behavior testsBuried food test. The buried food test 22948146 was performed as previously described by Yang and Crawley [18]. Briefly, after 7 days recovery following 
surgery, the mice were fasted for 32 hours starting from 9:00 h, with water available. On the test day, each mouse received an i.c.v. injection of vehicle, NPS or NPS + [DVal5]NPS and then was placed in a plexiglas test CAL-120 chamber (46 cm L623.5 cm W620 cm H) containing 3 cm deep of clean bedding made of freshly sterilized and deodorized wood chips. After acclimating to the environment for 15 min, the mouse was removed from the chamber and the mouse chow pellets (1.5 g, Beijing keaoxieli feedstuff Co. Ltd.) were randomly buried 1 cm beneath the surface of the bedding. Then, the mouse was placed back into the chamber and the latency to find the buried food was measured. The latency was defined as the time from the moment when a mouse was placed into the test chamber to the moment when it uncovered and grasped the food in its forepaws and/or teeth [19]. The test chambers were rinsed with distilled water and dried in air after each test. The bedding was changed before each test. The animals were video-recorded, and scored and analyzed by an investigator blind to the drugs administered.Figure 1. Schematic drawings show the localization of sections used for Fos-ir neurons counting. The grey zones represent the AON (Bregma 1.98 mm) and Pir (Bregma 0.62 mm). Abbreviations: AON, anterior olfactory nucleus; Pir, piriform cortex. doi:10.1371/journal.pone.0062089.gFigure 2. Latency to find the buried food following i.c.v. injection of vehicle or NPS in mice. Values are means 6 SEM (n = 10 mice in each group). * p,0.05, ** p,0.001. Data were analyzed by oneway ANOVA and followed by Fisher’s LSD test. doi:10.1371/journal.pone.0062089.gNPS Facilitates Olfactory FunctionFigure 3. Olfactory MedChemExpress 3PO habituation and dishabituation test following i.c.v. injection of vehicle or NPS in mice. A. Mice treated with vehicle exhibited significant habituation to water, dishabituation almond to vanilla, and habituation to vanilla. B. NPS at 0.1 nmol exhibited significant habituation to water, dishabituation water to almond, dishabituation almond to vanilla, and habituation to vanilla. C. NPS at 0.5 nmol exhibited significant habituation to water, dishabituation water to almond, habituation to almond, dishabituation almond to vanilla, and habituation to vanilla. D. NPS at 1 nmol exhibited significant habituation to water, dishabituation water to almond, habituation to almond, dishabituation almond to vanilla, and habituation to vanilla. E. NPS dose-dependently increased the total sniffing time spent in olfactory habituation and dishabituation tasks. Values are means 6 SEM (n = 10 mice in each group). * p,0.05, ** p,0.01, *** p,0.001 for habituation; # p,0.05, ## p,0.01, ### p,0.001 for dishabituation; data were analyzed using within-group Repeated Measures ANOVA and followed by the Newman-Keuls tests. p,0.01, p,0.001; data were analyzed by one-way ANOVA and followed by Fisher’s LSD test. doi:10.1371/journal.pone.0062089.gOlfactory habituation and dishabituation test. The olfactory habituation and dishabituation test was performed according to 
Yang and Crawley’s previous description [18]. On the test day, each mouse was.Cannula (25 gauge) was stereotaxically implanted above the right lateral ventricle (AP 20.2 mm, ML +1.0 mm, DV 21.4 mm, according to the atlases of Paxinos and Franklin, 2001 [16]) for i.c.v.Olfactory behavior testsBuried food test. The buried food test 22948146 was performed as previously described by Yang and Crawley [18]. Briefly, after 7 days recovery following surgery, the mice were fasted for 32 hours starting from 9:00 h, with water available. On the test day, each mouse received an i.c.v. injection of vehicle, NPS or NPS + [DVal5]NPS and then was placed in a plexiglas test chamber (46 cm L623.5 cm W620 cm H) containing 3 cm deep of clean bedding made of freshly sterilized and deodorized wood chips. After acclimating to the environment for 15 min, the mouse was removed from the chamber and the mouse chow pellets (1.5 g, Beijing keaoxieli feedstuff Co. Ltd.) were randomly buried 1 cm beneath the surface of the bedding. Then, the mouse was placed back into the chamber and the latency to find the buried food was measured. The latency was defined as the time from the moment when a mouse was placed into the test chamber to the moment when it uncovered and grasped the food in its forepaws and/or teeth [19]. The test chambers were rinsed with distilled water and dried in air after each test. The bedding was changed before each test. The animals were video-recorded, and scored and analyzed by an investigator blind to the drugs administered.Figure 1. Schematic drawings show the localization of sections used for Fos-ir neurons counting. The grey zones represent the AON (Bregma 1.98 mm) and Pir (Bregma 0.62 mm). Abbreviations: AON, anterior olfactory nucleus; Pir, piriform cortex. doi:10.1371/journal.pone.0062089.gFigure 2. Latency to find the buried food following i.c.v. injection of vehicle or NPS in mice. Values are means 6 SEM (n = 10 mice in each group). * p,0.05, ** p,0.001. Data were analyzed by oneway ANOVA and followed by Fisher’s LSD test. doi:10.1371/journal.pone.0062089.gNPS Facilitates Olfactory FunctionFigure 3. Olfactory habituation and dishabituation test following i.c.v. injection of vehicle or NPS in mice. A. Mice treated with vehicle exhibited significant habituation to water, dishabituation almond to vanilla, and habituation to vanilla. B. NPS at 0.1 nmol exhibited significant habituation to water, dishabituation water to almond, dishabituation almond to vanilla, and habituation to vanilla. C. NPS at 0.5 nmol exhibited significant habituation to water, dishabituation water to almond, habituation to almond, dishabituation almond to vanilla, and habituation to vanilla. D. NPS at 1 nmol exhibited significant habituation to water, dishabituation water to almond, habituation to almond, dishabituation almond to vanilla, and habituation to vanilla. E. NPS dose-dependently increased the total sniffing time spent in olfactory habituation and dishabituation tasks. Values are means 6 SEM (n = 10 mice in each group). * p,0.05, ** p,0.01, *** p,0.001 for habituation; # p,0.05, ## p,0.01, ### p,0.001 for dishabituation; data were analyzed using within-group Repeated Measures ANOVA and followed by the Newman-Keuls tests. p,0.01, p,0.001; data were analyzed by one-way ANOVA and followed by Fisher’s LSD test. doi:10.1371/journal.pone.0062089.gOlfactory habituation and dishabituation test. The olfactory habituation and dishabituation test was performed according to Yang and Crawley’s previous description [18]. On the test day, each mouse was.
