Knockdown of voltage-gated potassium channel subunit 2 (Kv2) current in the cholera toxin subunit B (CT-B)+ anterior cingulate cortex (ACC) layer 5 pyramidal neurons (L5 PNs) partly attenuates the analgesic effect of metabotropic glutamate receptor subtype 1 (mGluR1) inhibition. (A) Magnified images showing CT-B and Kv2.1 (top) or CT-B and Kv2.2 (bottom) double labeling in the left ACC L5. The CT-B is injected into the right ACC. Scale bar = 50 μm. (B) Representative immunofluorescence images showing the efficiency of lentivirus transfection in the ACC on day 7 after the injection. Scale bar = 1,000 μm. (C) Western blots showing successful suppression of Kv2.2 in the ACC on days 7 and 14 after short hairpin RNA (shRNA)–lentivirus injection. The negative control shRNA (shNC; nontargeting shRNA) has no effect. One-way ANOVA and post hoc Bonferroni multiple comparison tests, all groups n = 6 rats; ** versus sham. (C, top) A schematic of the Western blotting experimental design. (D, top) A schematic of the behavioral testing protocol. Neither intra-ACC Kv2.2-shRNA nor shNC has a significant effect on chronic constriction injury (CCI)–induced thermal hyperalgesia (middle) and mechanical allodynia (bottom). Two-way repeated-measures ANOVA and post hoc Bonferroni multiple comparison tests, all groups n = 6 rats; ** versus CCI. (E) Intra-ACC microinjection of 9-(dimethylamino)-3-(hexahydro-1H-azepin-1-yl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3H)-one (A841720; day 7 post-CCI) produces significant analgesic effects. However, in the rats that had been infused with Kv2.2-shRNA before CCI surgery, A841720 partly loses its ability to alleviate thermal hyperalgesia (left) and nearly completely fails to alleviate mechanical allodynia (right). Preinfusion of shNC does not change the effect of A841720. Two-way repeated-measures ANOVA and post hoc Bonferroni multiple comparison tests, all groups n = 6 rats; *^,** versus CCI + vehicle. *P < 0.05, **P < 0.01.