www.sciencesignaling.org/cgi/content/full/6/275/ra34/dc1 Supplementary Materials for An Increase in Synaptic NMDA Receptors in the Insular Cortex Contributes to Neuropathic Pain Shuang Qiu, Tao Chen, Kohei Koga, Yan-yan Guo, Hui Xu, Qian Song, Jie-jie Wang, Giannina Descalzi, Bong-Kiun Kaang, Jian-hong Luo, Min Zhuo,* Ming-gao Zhao* *Corresponding author. E-mail: min.zhuo@utoronto.ca (M.Z.); minggao@fmmu.edu.cn (M.-g.Z.) Published 14 May 2013, Sci. Signal. 6, ra34 (2013) DOI: 10.1126/scisignal.2003778 The PDF file includes: Fig. S1. Enhancement of behavioral sensitization (allodynia) to nerve injury in wildtype mice. Fig. S2. Subcellular fractionation of synaptosomes. Fig. S3. The amount of NMDARs in insular cortical homogenates from wild-type mice shows no change on day 7 after nerve injury. Fig. S4. Comparison of the amounts of GluN2B and GluN2A in insular cortical homogenates from wild-type, AC1 /, AC8 /, and AC1&AC8 / mice. Fig. S5. Elimination of behavioral sensitization to nerve injury in AC8 / mice but not in AC1 / mice. Fig. S6. Forskolin and rolipram treatment does not affect the amount of synaptic NMDARs in insular cortical slices from AC8 / mice. Fig. S7. ESI-09 does not affect the increase in the amount of synaptic GluN2B or GluN2A induced by forskolin and rolipram. Fig. S8. PP2 blocks the increase in the amount of synaptic GluN2B and GluN2A induced by forskolin and rolipram.
Fig. S1. Enhancement of behavioral sensitization (allodynia) to nerve injury in wild-type mice. Mechanical allodynia was tested in wild-type mice 3, 7, 14 and 28 days after nerve injury or sham surgery. The data were plotted as percentage-positive responses to the non-noxious mechanical stimulation of the ipsilateral hindpaw (n = 6 to 8 mice for each time points). *indicates p < 0.05 compared to sham group. 1
Fig. S2. Subcellular fractionation of synaptosomes. (A) The location of PSD95, synaptophysin, Rab5, and GAPDH indicated the successful separation of the PSD fraction from the non-psd fraction. (B) Most of the NMDAR subunits were located in the PSD-enriched fraction. 2
Fig. S3. The amount of NMDARs in insular cortical homogenates from wild-type mice shows no change on day 7 after nerve injury (n = 3 mice for each group). 3
Fig. S4. Comparison of the amounts of GluN2B and GluN2A in insular cortical homogenates from wild-type, AC1 -/-, AC8 -/-, and AC1&AC8 -/- mice. (n = 3 mice for each genotype). 4
Fig. S5. Elimination of behavioral sensitization to nerve injury in AC8 -/- mice but not in AC1 -/- mice. Mechanical allodynia was tested in AC1 -/- and AC8 -/- mice 7 days after nerve injury or sham surgery (n = 5 to 7 mice for each group). ** indicates p < 0.01 compared to sham group. 5
Fig. S6. Forskolin and rolipram treatment does not affect the amount of synaptic NMDARs in insular cortical slices from AC8 -/- mice. (A) When insular cortical slices from wild-type mice were treated with forskolin (FSK) and rolipram (Rol), the amount of GluN2B or GluN2A in the homogenates showed no change (n = 4 independent experiments). (B) Treatment of insular cortical slices from AC8 -/- mice with FSK and Rol significantly increased the amount of synaptic GluN2B and GluN2A subunits (n = 3 independent experiments). (C) Treatment with NB001 alone did not affect the amount of synaptic GluN2B or GluN2A in insular cortical slices from wild-type mice (n = 5 independent experiments). * indicates p < 0.05. 6
Fig. S7. ESI-09 does not affect the increase in the amount of synaptic GluN2B or GluN2A induced by forskolin and rolipram. (A) Pretreatment with the Epac inhibitor ESI-09 did not significantly affect the FSK and Rol-induced enhancement of synaptic GluN2A and GluN2B. * indicates p < 0.05 compared to control (n = 4 independent experiments). (B) Treatment with ESI-09 alone did not affect the amount of synaptic GluN2A or GluN2B (n = 5 independent experiments). 7
Fig. S8. PP2 blocks the increase in the amount of synaptic GluN2B and GluN2A induced by forskolin and rolipram. (A) Pretreatment with the SFK inhibitor PP2 significantly blocked the FSK and Rol-induced enhancement of synaptic GluN2B and GluN2A. * indicates p < 0.05 (n = 4 independent experiments). (B) Treatment with PP2 alone did not affect the amount of synaptic GluN2B or GluN2A (n = 4 independent experiments). 8