for 1h in PBS containing 1% BSA, 0.3% Triton X-100 at room temperature, and then incubated for overnight at 4C with one of the following primary JW 55 antibodies: Beta-amyloid: 1:500, rabbit polyclonal; NR1 receptor NR1: 1:500, rabbit polyclonal; protein kinase CPKC: 1:500, rabbit polyclonal; choline acetyl transferase – ChAT: 1:100, rabbit polyclonal. Sections were incubated for 1h at room temperature with FITC- or Cy3-conjugated secondary antibody and then washed with PBS for 10min3. For controls, primary antibody was omitted. Sections were examined with a fluorescence microscope, and images were captured with a digital camera and analyzed with Adobe Photoshop version 7.0. Western Blot Rats were sacrificed by decapitation under sodium pentobarbital anesthesia. Brain and spinal cord samples, which were divided into ipsilateral and contralateral side, were removed and rapidly frozen on dry ice and stored at -80C until use. 2181489 Samples were homogenized in SDS buffer containing a mixture of protease inhibitors. Protein samples were prepared on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat dried milk for 1h at room temperature and incubated overnight at 4C with one of the following primary antibodies: Beta-amyloid: 1:500, rabbit polyclonal; NR1: 1:1000, rabbit polyclonal; PKC: 1:250, rabbit polyclonal; ChAT: 1:4000, rabbit polyclonal. Membranes were incubated for 1h at room temperature with HRP-conjugated secondary antibody. Blots were visualized in ECL solution for 2min and exposed to hyperfilms for 0.5-10min. Blots were again incubated in stripping solution for 30 min at 50C and reprobed with anti-actin antibody as loading control. All western blot analyses were made in triplicates. water maze test consisted of two paradigms: 1) the hiddenplatform test was carried out over 5 daily sessions from test day 1 to 5, and 2) the visible-platform test was performed in on test day 7 and 8 to rule out non-specific effects. On day 15 after the initial intra-hippocampal injection, either CFA or IFA was injected into the right ankle joint after obtaining the baseline thermal and mechanical nociceptive threshold. The test for nociceptive behavior was then made on day 1, 3, 5, 7, and 14 after the CFA or IFA injection. Accordingly, a total of five experimental groups were included: Nave group, ACSF /vehicle group, ACSF /CFA group, A /vehicle group, and A /CFA group. After the final behavioral test, the Morris water maze test was repeated to confirm the continuing 23584186 presence of induced learning impairment. Experiment 2. To examine the effect of learning impairment on the 
recovery of nociceptive behavior, cycloheximide was injected into the hippocampus according to the following experimental protocol. Rats first received the CFA injection into the right ankle joint and the nociceptive behavioral test was performed on day 0 and post-CFA injection day 1, 3, and 5. On day 0, these rats were divided into 2 subgroups and received either cycloheximide or vehicle into the bilateral hippocampal CA1 area. After the cycloheximide or vehicle injection, the nociceptive test was repeated on day 1, 3, 5, 9, 13, 17, 19 to compare the time course for the recovery of nociceptive behavior between the cycloheximide and vehicle group. To confirm the effect of cycloheximide on learning and memory, a group of nave rats without ankle joint inflammation was used. Rats in this group first underwent the Morris water maze test fro