m day 1 to day 5. On day 0, these rats were 7514038 divided into 2 subgroups and received either cycloheximide or vehicle into the bilateral hippocampal CA1 area. After the cycloheximide or vehicle injection, two more series of the Morris water maze test were performed on day 1 to day 5 and day 15 to day 19 to determine the effect of cycloheximide on the performance of the Morris water maze task. All behavioral tests were carried out during a similar time period 18998663 on each test day for all groups by an observer who was blinded to the group assignment. Statistical analysis The data from the nociceptive behavioral test was first analyzed using repeated measure two-way ANOVA to detect overall significance among groups using SPSS 16.0 for Windows. The post hoc Tukey test was performed to detect the source of differences among groups. For Western blot, the density of each band was measured using Photoshop and Quantity One and normalized against the corresponding loading control and the group difference was compared using one-way ANOVA. For the Morris water maze, one-way ANOVA Experimental design Experiment 1. To examine the effect of YM-155 web learning impairment on the development of nociceptive behavior, the A or ACSF solution was first injected into the bilateral CA1 area of the hippocampus. The Morris water maze test began on day 8 after the hippocampal injection to examine whether learning impairment was induced. The Morris 3 Nociceptive Behavior and Leaning Impairment doi: 10.1371/journal.pone.0074533.g001 or Student t-test was used to detect the group difference. The data are presented as mean SD. Results Impaired acquisition of spatial learning by the hippocampal A injection The effect of the hippocampal A injection on acquisition of spatial learning was tested by the Morris water maze. While no differences were detected among three groups in the hiddenplatform test on day 1 and day 2, rats in the A group showed a significantly longer escape latency than rats in ACSF group on day 3-5 and in ACSF and nave groups on day 4-5. Results from two additional tests indicated that the prolonged escape latencies in the hidden-platform test for rats in the A group were due to a learning impairment because 1) rats in the A group had moderately increased latencies to reach a target quadrant in the water pool during a probe test as compared with rats in the ACSF and naive groups, and 2) the escape latencies in the visible-platform test on day 7 and day 8 did not differ among rats in all three groups. In addition, rats in all three groups exhibited normal gait throughout the experimental period without any indication of locomotor dysfunction or poor swimming performance. Immunohistochemical examination of the hippocampal CA1 area showed a substantially increased reactivity of A1-40/1-42 in the A group when examined after the last Morris water maze test. In contrast, A immunoreactivity was barely detectable in rats from the nave and ACSF groups at the same hippocampal area. Western blot also showed a significant increase in the A expression on the contralateral hippocampus of rats receiving both A injection and ankle inflammation as compared with nave rats and rats with ACSF injection with or without inflammation. Moreover, the A expression was also significantly increased in the ipsilateral hippocampus in rats receiving both A and CFA injection as compared with nave rats. Collectively, the combined morphological and behavioral data indicate that the intra-hippocampal A