Were analyzed using independent student’s t-test. In all statistical comparisons, the level of significances was set at p,0.05.Results Effects of NPS on olfactory functionsBuried food test. In comparison with vehicle-treated mice, i.c.v. administration of 0.1, 0.5 and 1 nmol of NPS significantly reduced the latency to find the buried food from 73.43611.77 s to 35.7465.37 (p,0.001), 12.7261.34 (p,0.001) and 24.6165.04 s (p,0.001), respectively (Fig. 2). Among the three doses, 0.5 nmol NPS reduced the latency most (p,0.001 and p,0.05 comparedwith vehicle and 0.1 nmol NPS, respectively; Fig. 2). In fact, high dose (1 nmol) of NPS insignificantly reduced the latency as compared to 0.1 nmol NPS (35.7465.37 s vs. 24.6165.04 s, p = 0.24; Fig. 2). Olfactory habituation and dishabituation test. Fig. 3A-D summarize the results from olfactory habitual and dishabitual behavior tests in mice intracerebroventricularly injected with vehicle or NPS (0.1, 0.5 or 1 nmol) to the same and different odors, respectively. Central administration of vehicle induced a habituation to water (p,0.05) and vanilla (p,0.001), and a dishabituation to vanilla (p,0.001, Fig. 3A). Mice administered NPS at 0.1 nmol were able to distinguish almond and vanilla as novel odors, but failed to Z-360 chemical information purchase CAL 120 habituate to the almond odor (Fig. 3B). Relative to the vehicle control, mice habituated and dishabituated all test odors following 0.5 or 1 nmol of NPS administration (Fig. 3C and D), indicating that NPS at these doses could facilitate mice to distinguish all of the same and different test odors. As shown in Fig. 3E, NPS dose-dependently increased the total sniffing time spent in olfactory habituation and dishabituation behavioral tasks.Effect of the NPS on olfactory behavior was blocked by [D-Val5]NPS. To identify whether NPSR antagonist blocks theeffect of NPS on olfactory abilities, [D-Val5]NPS, a selectiveNPS Facilitates Olfactory FunctionFigure 7. Effects of i.c.v. injection of NPS on Fos immunoreactivity in the AON and Pir in the mouse. A-D photomicrographs show Fos-ir neurons (black) in the AON and Pir in NPS- and vehicle-treated mice, respectively. E, F. Histograms show quantitative analysis of the number of Fos-ir neurons in the AON and Pir following NPS (n = 4 mice) and vehicle (n = 5 mice) i.c.v. injection. Values are means 6 SEM. * p,0.001. Data were analyzed by independent student’s t-test. Bar = 100 mm. Abbreviations: aci, anterior commissure, intrabulbar part; AON, anterior olfactory nucleus; lo, lateral olfactory tract; Pir, piriform cortex; OV, olfactory ventricle. doi:10.1371/journal.pone.0062089.gantagonist of NPSR [27], was injected with or without 0.5 nmol of NPS (i.c.v.) into mice. Our results indicated that 40 nmol of [D-Val5]NPS significantly antagonized the effect of 0.5 nmol of NPS on the latency to find the buried food (Fig. 4). However, when given alone, 40 nmol 
of [D-Val5]NPS did not affect the latency compared with vehicle (Fig. 4). Administration of 20 nmol [D-Val5]NPS significantly blocked the effects of 0.5 nmol NPS on olfactory differentiating ability (Fig. 3C) towards water and almond, but not vanilla (Fig. 5A). Further, 40 nmol [D-Val5]NPS completely inhibited the effect ofNPS on olfactory differentiating behavior (Fig. 5B) and markedly reversed NPS-induced increase in total sniffing time spent in olfactory habituation and dishabituation tasks (Fig. 5C).Inhibitory effects of NPS on food intakeFig. 6A and B summarize the effects of NPS (i.c.v.) on cumul.Were analyzed using independent student’s t-test. In all statistical comparisons, the level of significances was set at p,0.05.Results Effects of NPS on olfactory functionsBuried food test. In comparison with vehicle-treated mice, i.c.v. administration of 0.1, 0.5 and 1 nmol of NPS significantly reduced the latency to find the buried food from 73.43611.77 s to 35.7465.37 (p,0.001), 12.7261.34 (p,0.001) and 24.6165.04 s (p,0.001), respectively (Fig. 2). Among the three doses, 0.5 nmol NPS reduced the latency most (p,0.001 and p,0.05 comparedwith vehicle and 0.1 nmol NPS, respectively; Fig. 2). In fact, 
high dose (1 nmol) of NPS insignificantly reduced the latency as compared to 0.1 nmol NPS (35.7465.37 s vs. 24.6165.04 s, p = 0.24; Fig. 2). Olfactory habituation and dishabituation test. Fig. 3A-D summarize the results from olfactory habitual and dishabitual behavior tests in mice intracerebroventricularly injected with vehicle or NPS (0.1, 0.5 or 1 nmol) to the same and different odors, respectively. Central administration of vehicle induced a habituation to water (p,0.05) and vanilla (p,0.001), and a dishabituation to vanilla (p,0.001, Fig. 3A). Mice administered NPS at 0.1 nmol were able to distinguish almond and vanilla as novel odors, but failed to habituate to the almond odor (Fig. 3B). Relative to the vehicle control, mice habituated and dishabituated all test odors following 0.5 or 1 nmol of NPS administration (Fig. 3C and D), indicating that NPS at these doses could facilitate mice to distinguish all of the same and different test odors. As shown in Fig. 3E, NPS dose-dependently increased the total sniffing time spent in olfactory habituation and dishabituation behavioral tasks.Effect of the NPS on olfactory behavior was blocked by [D-Val5]NPS. To identify whether NPSR antagonist blocks theeffect of NPS on olfactory abilities, [D-Val5]NPS, a selectiveNPS Facilitates Olfactory FunctionFigure 7. Effects of i.c.v. injection of NPS on Fos immunoreactivity in the AON and Pir in the mouse. A-D photomicrographs show Fos-ir neurons (black) in the AON and Pir in NPS- and vehicle-treated mice, respectively. E, F. Histograms show quantitative analysis of the number of Fos-ir neurons in the AON and Pir following NPS (n = 4 mice) and vehicle (n = 5 mice) i.c.v. injection. Values are means 6 SEM. * p,0.001. Data were analyzed by independent student’s t-test. Bar = 100 mm. Abbreviations: aci, anterior commissure, intrabulbar part; AON, anterior olfactory nucleus; lo, lateral olfactory tract; Pir, piriform cortex; OV, olfactory ventricle. doi:10.1371/journal.pone.0062089.gantagonist of NPSR [27], was injected with or without 0.5 nmol of NPS (i.c.v.) into mice. Our results indicated that 40 nmol of [D-Val5]NPS significantly antagonized the effect of 0.5 nmol of NPS on the latency to find the buried food (Fig. 4). However, when given alone, 40 nmol of [D-Val5]NPS did not affect the latency compared with vehicle (Fig. 4). Administration of 20 nmol [D-Val5]NPS significantly blocked the effects of 0.5 nmol NPS on olfactory differentiating ability (Fig. 3C) towards water and almond, but not vanilla (Fig. 5A). Further, 40 nmol [D-Val5]NPS completely inhibited the effect ofNPS on olfactory differentiating behavior (Fig. 5B) and markedly reversed NPS-induced increase in total sniffing time spent in olfactory habituation and dishabituation tasks (Fig. 5C).Inhibitory effects of NPS on food intakeFig. 6A and B summarize the effects of NPS (i.c.v.) on cumul.
